Synopsis Aims

Synopsis This piece of work is submitted as an ‘equivalency Report’ in lieu of a level #7 M qualification. The work, as detailed below, is a project designed and carried out entirely by me at the University of Edinburgh between 20010 and 2012; building on antecedent experiments/data, also performed exclusively by me between 2008 and 2010. The project in question, as elaborated below, was an attempt to design a high through put assay to identify antagonists of the vital Trypanosomatid enzyme, RNA Editing ligase 1 (REL 1), which is instrumental in a quixotic RNA transcription mechanism, found exclusively in Trypanosomatid parasites, including Trypanosome Brucei; this parasite is the etiological agent of Human African Trypansomiasis (HAT), or ‘sleeping sickness. Thus, REL 1 potentially constitutes a drug gable target for sleeping sickness. The object of this project work was therefore to express recombinant rREL 1 in a prokaryotic host (E Coli BL 21 DE3) and incorporate this enzyme into a high through put Fluorescent based assay, in order to screen compound libraries for putative inhibitors of REL 1 that might constitute compound families that can be subsequently refined for the purposes of therapy (‘Hit to lead’). The details of this project, provided below, pertain to the development of this assay in term of raw materials, sensitivity and specificity. All such work described and all data presented is exclusively mine. In accordance with this the work herein was incorporated into an Oxford University Publication Nucleic Acid Research (circa 2016), with myself as joint first author

Aims HAT or Sleeping sickness is endemic in 36 sub-saharan African countries associated with the Tsete fly, which transmits T Brucei. The disease derives it name from the untreated infection, which causes irregular sleep patterns, stupor and profound confusion and, without therapy, is nearly always fatal. Current chemotherapeutic regimes, e.g. Melarsoprol & Eflornithine are difficult to administer and based on outdated arsenicals which therefore prove toxic in about 10% of victims. There is an imperative to develop up to date smart target based therapies with high efficacy and low toxicity. RNA Editing ligase 1 was selected as such a target, since it orchestrates a type of RNA transcriptional editing not found in the human host and which, if ablated, is fatal to Trypanosome. Indeed a human homolog of REL 1 does not exist. Thus, a priori, it appears to constitute a specific and potent drug target. In particular: 

RNA editing by uridine insertion/deletion is essential for mitochondrial gene

    

expression in all trypanosomatids pathogens. Editing is catalyzed by multiprotein complexes, the editosomes. A key catalytic component of editosomes is RNA editing ligase 1 (REL1). REL1 has been validated as a drug target in T. brucei in vitro and in vivo (Schnaufer et al., Science 2001). The 1.2 A crystal structure of REL1 revealed a deep ATP binding pocket with numerous opportunities for specific binding of small molecule inhibitors (Deng, Schnaufer et al., J Mol Biol 2004). There are no close REL1 homologs in the mammalian host and thus this enzyme represents a target of potentially high specificity. Computational screening efforts based on molecular dynamics simulations resulted in the identification of several REL1 inhibitors with IC50 values in the single-digit μM range (Amaro, Schnaufer et al., PNAS 2008; Durrant, Hall et al PLoS NTD 2010)

.  

Here we describe the development of a high throughput screening (HTS) compatible, fluorescence-based REL1 assay. The assay was optimised with respect to reaction chemistry; fluorophores; rREL 1 production & enzyme kinetics culminating in higher sensitivity; better dynamic range & higher throughput

Outcome The principle underlying the assay is Fluorescent Resonance Energy Transfer or FRET. In essence, this involves photon transfer from a donor dye to an acceptor dye: subsequent excitation of this acceptor moiety results in emission at a slightly longer wavelength (the Stoke shift), detectable by photometry. In the context of this drug discovery assay, dye labelled oligo’s were placed in proximity to effect ligation and resultant FRET by ligation of the dye labelled oligo’s by REL 1 (L1). Thus, inhibition of REL 1 via library aptamers will prevent ligation and abrogate FRET. A reduction in FRET is therefore indicative of REL inhibition. To elaborate:



FRET (Fluorescence Resonance Energy Transfer) is achieved when two fluorophores with overlapping spectra are fixed in close proximity, such that virtual photons are transferred between a ‘donor’ (‘D’) and ‘acceptor’ fluorophore (‘A’), resulting in acceptor fluorescence when the donor is excited, which can be quantified by spectrophotometry



Annealing of fluorophore-labelled 5’ and 3’ RNA substrates to an RNA bridge (the “guide RNA”) anchors the donor and acceptor fluorophores in close proximity Consequently, transfer of virtual photons results in FRET After ligation and denaturation (to disrupt un ligated dsRNA), FRET emission is measured by spectrophotometry: In the presence of active REL1, ligation occurs, resulting in denaturationresistant FRET

  

 

In the absence of active REL1, the dsRNA dissociates with denaturation, abrogating FRET Thus, inhibitors of REL1 will abrogate FRET as compared with a control

This assay principle is illustrated schematically below:

6-FAM

Cy 5 = Fluorescence Resonance Energy Transfer

(3’ #2) (5’ #2) (gR #1) + 10 x ligation with Tris pH 6.0 + 10 x ligation with 1/5 molarity K/mg

Urea

In the schema depicted, FAM Labelled (donor) oligo (5’ #2) is brought into proximity with a Cy 5 labelled (acceptor) oligo (3’ #2) by an RNA guide (bridge). This is achieved by mixing these species in permissive buffer in vitro. Subsequently, the two proximal dye labelled oligo’s are ligated by addition of recombinant purified rREL (expressed in BL21 DE3; HPLC purified and activity checked by use of a 32P radio ligand assay, outside the scope of this report). They are then denatured - initially with heat and subsequently, by empirical investigation, urea (described later during ‘Development’) – and free dye labelled oligo’s sequestered by a molar excess of complementary oligo’s (#139). This excess ensures free oligo’s are removed from solution by mass action. Thus, FRET can only occur from the resulting double labelled ligated species and not free oligo’s in random proximity via simple kinetics

By optimising ligation buffer composition, donor substrate pairing, denaturation method and the kinetics of ligation time, substrate and enzyme concentration etc. the

assay was demonstrated to be specific, sensitive & effective in preliminary library screens. Consequently, a publication of this method was accepted in the Journal Nucleic Acid Research (NAR), Oxford University press (circa. 2016), with myself as joint first author, reflecting the lab based assay development detailed below

Development

1) Selection of Donor and acceptor dye for Maximum sensitivity: By comparing and contrasting FAM and Cy 5 it was ascertained that a donor oligo labelled with FAM (5’ #2) coupled with an acceptor oligo labelled with CY 5 (3’ #2) resulted in the highest signal to noise by photometric detection, employing FRET. Furthermore, to confirm that FRET output could only occur by bridging these 2 oligos with an RNA bridge (gR #1), heat denaturation, which eliminates bridging, likewise eliminated FRET; as did mixing the donor and acceptor dye labelled oligo’s in the absence of the guide bridge

FRET Output from 6-FAM-Cy5 & Cy3-Cy5 Dye linked oligo combinations: Signal modulation +/- g R #1 and/or +/- heat denaturation. FRET Spec. = 'Achim-FRET-Cy3-Cy5' (= Cy 3-Cy 5) 70000

 Highest signal yielded by Cy3 (5' #2)_Cy5 (3' #1) dye combination commensurate with 6-FAM_Cy5 Spec (= 'Achim-FRET-Cy3-Cy5' )

Spec = Cy 3_Cy 5 FRET: Ex = 544nm (550) Em = 670nm (670) 60000

In the absence of g R #1, no scaffold produced and signal

Cy 3_Cy 5

massively reduced 50000

Cy 3_Cy 5

S/B Cy3/5

6-FAM-Cy 5

+/- gR #1

In the presence of heat, signal intensity similar to 'no scaffold' in the absence of heat suggesting that denaturation is efficient

= 4.6

AFU

40000

In the absence of g R #1 signal intensity similar, irrespective of heat, implying that specific signal is attributable to scaffold

30000

6-FAM-Cy 5

 S/B associated with 6-FAM/Cy5 (oligo) dye combination 20000

higher than Cy3/Cy5

10000

0 gR #1 No heat

No gR #1 No heat

gR #1 plus heat

Test variable

‘Optimisation’ Assay #018_#019

No gR #1 plus heat

FRET Output from 6-FAM-Cy5 & Cy3-Cy5 Dye linked oligo combinations: Signal modulation +/- g R #1 and/or +/- heat denaturation. FRET Spec. = 'FRET 1-Achim' (= 6-FAM-Cy 5) 40000 Spec = 6-FAM_Cy 5 FRET: Ex = 490nm Em = 670nm

30000

6-FAM_Cy 5

35000

 Highest signal yielded by 6-FAM (5' #2)_Cy5 (3' #1) dye combination commensurate with 6-FAM_Cy5 Spec (= 'FRET 1-Achim' )

S/B 6-FAM/Cy5 +/- gR #1 = 23.1 !

25000

In the absence of g R #1, no scaffold produced and signal

In the presence of heat, signal intensity similar to 'no scaffold' in the absence of heat suggesting that denaturation is efficient

6-FAM_Cy 5 15000

10000

5000

CY 3_Cy 5

In the absence of g R #1 signal intensity similar, irrespective of heat, implying that specific signal is

CY 3_Cy 5

AFU

massively reduced 20000

attributable to scaffold

0 gR #1 No heat

No gR #1 No heat

gR #1 plus heat

No gR #1 plus heat

Test variable

‘Optimisation’ Assay #019_#019

2) Visualising the FRET scaffold by non denaturing PAGE To elucidate scaffold structure, dye labelled components and RNA guide, enabling hybridisation, were subjected to non denaturing PAGE. The resulting gel was fixed dried and scanned (Typhoon) for fluorescent output using a single FAM channel; a single Cy 5 channel and a double FAM Cy 5 FRET channel. The following was observed, clarifying that output is indeed a function of the expected hybridised scaffold:

Fluorescent output from reactions containing just FAM dye labelled oligo was only detected in the FAM channel; No signal was revealed in the CY 5 channel or indeed FRET channel Conversely, the CY5 dye labelled oligo exhibited signal from the CY 5 channel; no signal from the FAM channel and likewise no signal from the FRET channel A dual labelled FAM Cy 5 oligo, permissive to FRET, did indeed exhibit FRET In the presence of the RNA bridge and the absence of denaturation however un ligated dye labelled oligo’s initiated FRET In the absence of the RNA guide bridge they did not Similarly, in the presence of rREL 1 causing fragment ligation, and denaturation conditions FRET was also observed Finally, In the absence of REL 1 and therefore ligation, denaturation culminated in abrogation of FRET:

5’ #2 3’ #1

FRET 5’ #2

5’ #2

Double

FRET_Cy5_FAM

3’ #1

Double

#139 Scaffold

3’ #1 3’ #1

gR #1 Scaffold

Double 1µl Oligo @ 2µM

5’ #2

3) The effect of pH on S:N produced by FRET To investigate the effect of buffer type and pH on S:N associated with FRET, a mixture of the 2 dye labelled oligo species was subjected to ligation conditions in the presence or absence of purified rREL 1 and then heat denatured. This was done in the context of a buffer with a range of PH conditions and either Tris or HEPES. Specifically, for each buffer conditions, dual reactions were performed: one in the presence of REL leading to ligation and specific FRET and one in the absence of REL 1 wherein denaturation causes melting of the FRET scaffold and abrogation of specific FRET signal. Sensitivity was evaluated for each type of buffer by computing the ratio of +L1/-L1. As depicted below, it was established that Tris pH 6.0 – 8.0 & Hepes pH 8.0 were permissive to high S:N

The effect of Buffer composition and pH in ligation buffer & L1 dilution buffer versus magnitude of FRET and S:N: Asayed on Omega Plate reader 70000 7.3

Gain = 1531

6.4

+ L1/ - L1 60000

5.5 5.2 4.8

50000

3.7

AFU

40000

5.1

Plus 50ng #002-5 Minus L1 30000

20000

10000

0 Tris 3.5

Tris 5.0

Tris 5.5

Tris 6.0

Tris 6.5

Tris 7.0

Hepes 8.0

10 x Adenylation/ 10% Glycerol L1 diln buffer pH & composition

‘Optimisation’ Assay #067

The effect of buffer pH & composition on magnitude of FRET: 50ng #002_2 + 30 minutes ligation

100000

10.8 11.0

11.5

Gain =1484

10.9

+ #002_2

11.0 S/B = +L1/-L1

80000

+ #002_2

+ #002_2

+#002_2

+ #002_2

8.8 + #002_2

60000

AFU

6.4 + #002_2

40000

20000

- L1

- L1

- L1

- L1

- L1

- L1

- L1

0 Tris pH 6.0

Tris pH 6.5


4) Buffer composition

Tris pH 7.0

Tris pH 7.5

Tris pH 8.0

Buffer composition/pH: Annealing Buffer

Hepes 8.0

Tris pH 8.5

In the context of ligation buffer incorporating Tris pH 8.0, the effect of ionic composition, cf. {k+}, on S:N (+L1/-L1) was further investigated in buffer of standard pH.

Similarly, in the context of Hepes buffer pH 8.0 and Tris pH 6.0, the effect of alternative reducing agents in the ligation buffer, viz. β Mercaptethanol versus DTT, on resulting S:N was clarified

The combined effect of Titrating K & Mg ions in ligation buffer on magnitude of FRET: 50ng L1 # + 30 min ligation 70000 + L1

Gain = 1484 60000

S/B = +L1/-L1

+ L1

+ L1

10.5

+ L1 9.9

+ L1

9.7

50000 9.3 7.5

AFU

40000 Plus 50ng #002-5: #077 Minus L1: #077

30000

20000

10000

- L1

- L1

- L1

- L1

- L1

0 1mM Mg + 25mM K

5mM Mg + 25mM K

5mM Mg + 5mM K

1mM Mg + 5mM K

[K]/[Mg] in ligation buffer: mM


5mM Mg + 25mM K: Anneal T 8.0

The effect of substituting 0.2% (v/v) 2-ME for 2.5mM DTT in Reaction buffers of different composition & pH on Magnitude of FRET: 20 min Ligations using 50ng #002-5 per reaction 90000.0 + #002-5 + #002-5

80000.0

Gain = 1578

+ L1/ - L1 70000.0 5.7 6.7

AFU

60000.0

50000.0 + #002-5

40000.0

+ #002-5

30000.0 2.6

2.9

+ #002-5

20000.0 - L1

- L1

- L1

- L1

- L1

1.4

10000.0

0.0 Tris 6.0 + DTT

Tris 6.0 + βME

Hepes 8.0 + βME

Hepes 8.0 + DTT

Hepes 8.0 + 2% CHAPS

Composition & pH of 10 x ligation buffer & L1 diln buffer


Taken together, these two experiments sets lend themselves to an overhaul in buffer chemistry, culminating in significantly improved S:N: The original versus new buffer composition are summarised in the figure below:

The effect of changed ligation conditions on sensitivity and specificity of ligation adduced by FRET 100000

+ L1

90000

S/B = + L1/- L1

80000

14.4 70000

Improved

+ L1

 5mM Mg + 25mM K = 10x

 1mM Mg + 5mM K = 10x

AFU

60000 50000 40000 30000

Ligation buffer Tris pH 6.0 = 10x Lig buffer No BSA in Ligation rxn 2% Triton x-100 in ligation rxn 0.03% Brij 35 ® in ligation rxn L1 diluted in 10% Glycerol + Tris pH 6.0 Annealing performed @ Tris pH 8.0

Original

10.7

Ligation buffer Hepes pH 8.0 = 10x Lig buffer 1µg /ml BSA in Ligation rxn 2% Triton x-100 in ligation rxn No Brij 35 ® in ligation rxn L1 diluted in 10% Glycerol + Hepes pH 8.0 Annealing performed @ hepes pH 8.0

20000 10000

- L1

- L1

Improved conditions

Original conditions

0

Conditions of Ligation (10 min)

5) Denaturation conditions

At the inception of assay development, it was simple and expedient to denature the FRET scaffold by simple application of heat provided through a hot block. However, anticipating how the assay would actually be implemented in a high through put drug discovery compound library screen, it was hoped that reaction cessation and denaturation could be accomplished by simple addition of an additive to the reaction mix. Accordingly, a molarity titration of Urea was compared to prescribed heat denaturation and also DMSO. As depicted below, 4M urea for both a 5 minute and 30 minute ligation of dye labelled oligo’s culminated in S:N for FRET comparable to application of heat. I should add that urea at the concentrations advocated does not interfere with fluorescent signal (data not shown).

o #119: The Efficacy of different Denaturation conditions: 40% DMSO @ RT/40 C vs. Urea Titration @ o RT vs. Std heat (96 C) denaturation

200000

5.2 180000

10.3 160000 140000

S/B

1.3

11.7 1.3

AFU

120000

3.0

11.1

12.3 FRET FAM

100000

Cy5 80000 60000 40000 20000 0 #139 + RT

ND

40% DMSO 40% DMSO 1M Urea + + #139 + RT + #139 + #139 + RT 40oC


2M Urea + #139 + RT

4M Urea + #139 + RT

6M Urea + #139 + RT

8M Urea + #139 + 96oC #139 + RT

Denaturation Regimen

The object of the exercise is to discover an amount of urea that effectively denatures @ RT comparable to thermal denaturation 40% DMSO even at 40oC is not effective but >/= 4M urea is Moreover, 4 – 6M urea @ RT is comparable to thermal denaturation and does not perturb signal from the individual dyes

#121: Comparing 200x #139 + Heat denaturation with 0 x #139 + 5.8 - 6.0M Urea in the context of 5 min & 30 min Ligations (L1- #003-4): FRET

70000 Gain = 1453 60000

AFU FRET

50000

40000

30000

20000

10000

0 5 min Lig

30 min Lig


30 min Lig_Fresh A

Minus L1

Minus L1 + Fresh A

Denaturation conditions

For both 5 & 30 minute ligations denaturation associated with urea comparable to thermal denaturation Although magnitude of FRET signal higher for fresh annealing mix S/B comparable for both fresh & freeze thawed annealing mix

6) Reaction Kinetics: A consideration of Michaelis Menten kinetics to ascertain optimal substrate concentration etc. An integral component of the ligation reaction is ATP. This substrate, in concert with REL 1, is responsible for procuring ligation of the FAM labelled donor oligo to the Cy 5 labelled acceptor oligo, leading to FRET. However, apart from factors affecting sensitivity such as buffer chemistry etc., as already described, reaction dynamic range is also important. This is because the reaction, in a high through put situation and consequently subject to varying degrees of inhibition by multiple groups of compound libraries, must be able to distinguish specific low level inhibition from potent inhibition. A standard pre requisite for this, according to classical enzyme kinetics, provided in equations by Michaelis Menten, is to determine the so called Km of the substrate (cf. ATP). By definition, the Km is the substrate concentration at which reaction rate is exactly half the Max (Vmax), thus optimal for reaction rate increases and also decreases (produced by compounds screened by the assay). To determine Km, the initial reaction velocity is computed for a titration of ATP and by non linear regression (and calculus) a Km value is computed

Thus, separate reactions were established with a molarity titration of ATP and the resulting raw data, viz AFU FRET signal and these concentrations subjected to nonlinear regression in a statistical package called Graph Pad. The output from Graph Pad is shown below and the Km was computed as being 2.5µM [ATP]

Km = 2.40 +/- 1.20µM R2 = 0.9859 Linear order Rxn = 5 min

7) Determination of the quantity of recombinant ligation enzyme rREL 1 for incorporation into a standardised reaction for high through put library screens In the context of the Km substrate concentration, viz. 2.5µM [ATP], it was prudent to determine the optimal mass of rREL 1 in a standard drug discovery reaction. This figure pertains ideally to an expeditious reaction time (cf. 5min) to maximise throughput. Accordingly, a series of reactions were performed with Km substrate, but increasing amounts of rREL and progressively longer reaction times;To reiterate, I was looking to determine an amount of rREL 1 that with Km [ATP] would subtend toward maximum specific signal in a 5 minute ligation time period. As illustrated below, 100ng of rREL 1 satisfies this condition:

Time course Evaluation using a mass titration of L1 #002_1 in the context of 2.5uM [ATP] = Km 90000 Gain = 1484 80000 9.3

100ng # 002_1

= S/B

70000

AFU

60000

50000 5.7 50ng # 002_1

40000

30000 25ng # 002_1

3.5 20000

10ng # 002_1

2.2 10000

- L1

0 0

2

4

6

8

10

12 14 16

18 20


22 24 26

28 30

32 34

36 38 40

42 44

46 48 50

52 54

56 58

60 62

Ligation time (mins)

At the theoretical Km of ATP dynamic range and therefore assay sensitivity might be undermined when using < 100ng L1 Ideally we would want to use 100,000 reactions would prove extremely costly. Thus, we wondered if it might be possible to increase the [ATP] and concomitantly reduce the amount of rREL 1 per reaction. In terms of impact on dynamic range, we were not unduly concerned, as we proposed to increase the amount in incremental terms and not orders of magnitude and, furthermore, we were looking to identify compounds that antagonised REL 1 and reduced the reaction rate and not compounds that potentiate the reaction rate. Thus, the imperative to keep the Reaction midway on a classical Michaelis Menten curve did not apply. Accordingly, I set up reactions for incremental increases in ligation time, 50ng of rREL 1 and increasing concentrations of the ATP substrate (including the Km). As shown below, deploying 10µM ATP instead of 2.5µM allowed me to achieve significant S:N for a 5 minute ligation using 50ng of rREL 1 (instead of 100ng)

#098: Ligation time course using 50ng L1 #003_6 versus [ATP] titrants 80000 Gain = 1453

10uM ATP

70000

5uM ATP

60000 2.5uM ATP

AFU

50000

40000

30000

20000

0uM ATP

10000 No L1 0 5

0

10

15

20


25

30

35

40

45


As per previous assay @ 50ng L1 S/N comprimised using [ATP] = Km, but @ 5 & 10uM much better How much to ATP & L1 to use ? There again 5 minute ligation time represents mid linear order reaction point

To summarise:

#115: Verification of Ligation time course samples (by photometry) for analysis by Non denaturing PAGE : 50ng L1 #003-1 + 10µM ATP + 0 - 80 minutes ligation 70000 65000 Gain = 1453 60000 50ng #003-1 + 10µM ATP 55000 50000

AFU: FRET

45000 40000 35000 30000 25000 20000 15000 10000 5000

- L1 + 10 min

0 0

5

10

15

20

25

30

35

40

45

50

Ligation time: Mins

55

60

65

70

75

80

85

Evaluation In the first instance, I evaluated the reaction using a statistical parameter called the Z score, which quantifies how robust the reaction is, based on S:N for multiple reactions and the variance of AFU for control group (-L1) versus specific reactions (+L1): A specific, sensitive and robust reaction will obviously be characterised by bona fide ligation reactions (+L1) where the divergence in terms of increased signal over and above back ground (-L1) is very high and where data within the 2 groups is tightly compacted (i.e. low variance)

Accordingly, ~ 50 reaction with and without REL 1 were set up under the standardised conditions already described (i.e. 50ng L1 + 10µM [ATP] + 5 minutes ligation + 4M urea denaturation/reaction cessation)

Assessing QC of HTS (FRET assay ) using 'Screening window Co efficient' (or Z factor) 60000

#012 & #013

AFU_Individual data points

50000

+ L1 40000

30000

Plus L1_#013

Z Factor = 0.6 & 0.6: (1.0 > Z > 0.5) = 'Excellent'

Plus L1_#012 Minus L1_#012 Minus L1_#013 20000

10000

- L1 0 0

10

20

30

Sample #

40

50

60

#125A: Comparing Inter plate variable Z Scores between 2 x (+ L1) master mixes & 2 x (-L1) master mixes respectively (4 x 96 well plates): 50ng L1 + 5 minute ligation + 10uM [ATP] 50000 Gain = 1453 45000 + L1_# 003_5 40000

AFU: FRET

35000

S/B ~ 12.0

30000 25000 20000

Z = 1 - (3σs + 3σc) (µs - µc)

15000

Z = 1 - (8063.6 + 614.4) (41808.1 - 3383.6)

Z = 1 - 0.23 = 0.77!!

10000 5000

Minus L1

0 0

5

10

15

20

25

30

35


40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

Sample #

Variance should ideally be small; S/B = dynamic range large Thus, [variance]/[dynamic range] should yield a small fraction for a sensitive specific assay Thus, the ‘z score’ should subtend to 1.0 Infact z scores >/= ‘0.6’ designate ‘excellent assays’ !!!

To further evaluate this standardised drug discovery protocol, I compared FRET output (AFU) for progressively increasing reaction times (photometry on 96 well plates) with densitometry signal derived from fixed and dried non denaturing polyacrylamide gels (quantified using the Typhoon Scanner). As shown in the dual data derived figure below, there was complete concordance between signal magnitude derived from gel densitometry readings and FRET photometry readings: Specifically, as gel images of the FRET scaffold increased in intensity with increasing ligation times the quantified densitometry figures exactly matched after normalisation quantified FRET data derived from plate based reactions and spectrophotometry

Comparing the % activity relative to subtrate depleted signal @ 80 minutes ligation (= 100%) by FRET versus RNA densitometry (derived from non denaturing Glycerol- PAGE)

100.0

80.0

% Activity_Gel #13

% Activity

% Activity FRET assay #115 60.0

40.0

20.0

0.0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 84


% FRET activity relative to Signal Max derived from photometry mirrors signal exactly from gels !!

Finally, I decided to extend this analysis and look at the above in the context of real antagonist (of REL 1): The compound NSC 42067 had previously been found to inhibit REL 1 and was utilised as a positive control in low throughput radioactive ‘proof of principle assays’, preceding development of the FRET assay under scrutiny and published with myself as co first author (circa 2008).

To that end, a molarity titration of NSC 42067 was quantified by both (FRET) photometry in 96 well plate format and densitometry signals derived from non denaturing polyacrylamide gels

Comparing Efficacy of NSC #42067 on Inhibition of rREL1 Ligation by Image quant densitometry (Gel #12: Glycerol PAGE) versus # 114 (FRET) photometry 200

FRET

125

Cy5 #114_Photometry_FRET

150

Gel #12_% Activity_Cy5

100

75

50

25

0 30 min ND

5 min Den

30 min Den

30 min + 5µM

30 min + 10µM

30 min + 50µM

-L1

Ligation mix

% plate reader activity expressed as a % of ligated signal from 30 minutes (denatured) ligated product Thus, ‘30 min Den’ = 100% & ‘5 min Den’ ~ 50% Gel activity calculated by dividing [ligated product]/[Ligated product] + [ unincorporated primer] Remarkable concordance between these two data sets

Furthermore, when densitometry data & FRET data were both was subjected to non linear regression the computed efficacy of NSC 42067 were found to be identical and in accordance with previous published estimates: Non linear regression analysis of dose response curve for NSC #42067 versus % inhibition of FRET relative to 5% DMSO only

100

% Activity vs DMSO

% Activity

175

IC50 = 9.3µM +/- 0.673µM R2 = 0.9904

80 60 40 20 ‘Optimisation’ Assay #109_#110

0

0.5

1.0

1.5

Log [NSC # 42067] (µM)

2.0

#109 - #110: The relationship Between [NSC #42067] Titration series & putative inhibition of Ligation as evinced by FRET: 50ng L1 #003-3 + 10uM [ATP] + 5 min Ligation Data for Graph Pad 120

% activity (versus 5 % DMSO)

100

80

60

40 Replicates #1-3 20

0 -2

2

6

10

14

18

22

26

30

34

38

42

46

50

54

58

62

66

70

74

78

82

86

90

94

98 102 106 110 114 118

[NSC #42067] uM

To close this report, I will say that since completing this assay development and producing a standard protocol, drug discovery programs have been implemented with this FRET assay and ‘hits’ provisionally identified:

High-throughput screening collaborations:

Scripps Institute San Diego (LOPAC Collection), 1,280 cpds Dundee Drug Discovery Unit (DDU), ~23,000 cpds GlaxoSmithKline Tres Cantos Madrid (GSK), ~160,000 cpds Hit confirmation on going The Scripps Research Institute Florida (NIH/TSRI), ~90,000 cpds Medium-throughput

CHARTERED SCIENTIST – COMPETENCIES REPORT All evidence you submit for professional registration must be endorsed by your supervisor/manager PLEASE READ GUIDANCE NOTES FOR APPLICANTS BEFORE FILLING IN THIS FORM You should use more than one example for each competency and address all of the guidance notes. Name: A: Application of knowledge & understanding Competencies

Guidance

Evidence Chosen

A1: Use specialist experiential knowledge and broader scientific understanding to optimise the application of existing and emerging science and technology

You should provide sufficient detail here to show your specialist experiential knowledge and how you have applied it. Further to this, include any examples of where your broader scientific understanding is applied to your area of practice. Examples could include but are not limited to: • Writing and presenting internal papers, reports or standards • Conducting appropriate research to facilitate design and development of scientific processes

In this section you should describe why, what and how you use your specialist experiential knowledge and broader scientific understanding to optimise the application of existing and emerging science and technology to fulfil this competency.

Please return the form to [email protected]

1) In my present post, I have been responsible for the bench supervision of a particular Ph.D student & training of other students’ in the same research group and part of this practical remit was to devise methods to amplify and then measure expression of isoform specific genes of interest (linked to a separate cardiotoxicity project). This was necessary to elucidate whether cardio protection in our in vitro models was linked to general expression of particular genes or conferred by particular isoforms derived from alternative transcripts. Specifically, I devised an SOP for students which took them from mining transcripts in data bases like EBI/Ensembl to primer design, which includes general aspects of primer design, e.g. avoiding hair pin loops, formation of heterodimers and, crucially, how to select target sites for such optimal primers in order to amplify just one gene transcript and not another; viz. selection of exon-exon sites only found in that spliceform. Having compiled an extended SOP a

Document Ref:

2016/01

Page 1

CHARTERED SCIENTIST – COMPETENCIES REPORT

number of students were walked through it in a tutorial type discussion of the procedure 2) In my present role, I have also spent time writing up the materials and methods of manuscripts for publication pertaining to real time qPCR experiments in a prior position at the University of Nottingham (circa 2014-2015). At that time, I assisted and trained Ph.D students in (RT PCR and real time qPCR): See my extended CV for reference details, if required. In addition I communicated in person at the time with Ph. D students regarding such materials and data and provided a general theoretical basis and principles of such (gene expression) analysis. Since that time, and in my present post, I communicated with these students via e mail, clarifying data problems and requisite presentation requirements for publication, rooted in theoretical principles, which I explained. Thus far, this has resulted in one major publication as well as a series of both National & international conference poster presentations, with myself as a co author 3) Similarly, in a previous position at the University of Edinburgh – Made reference to throughout this application and linked to Reference #2 – I actively participated in a monthly Journal club where a group of us, including myself, were expected to critique a particular published paper 4) Also regarding my prior cited position at the University of Edinburgh, part of my remit was to implement a project to independently develop a high through put assay, to screen compound libraries to identify subsets of compounds cytotoxic to Trypanosome Brucei. Apart from requisite technical challenges in the lab, progress was facilitated by weekly Power Point presentations to fellow group members, to solicit comment on progress, practice and troubleshooting problems. Furthermore,


Document Ref:

2016/01

Page 2


with reference to presenting my project findings to the department as a whole, I actively participated in periodic departmental seminars where I summarised seminal findings in a more broad based narrative, delivered by Power Point. 5) Finally, in the context of this position at the University of Edinburgh & ‘ Writing and presenting internal papers, reports or standards’ I was responsible for authoring poster presentations on my project background, findings and future work at both national and international conferences, including Journal abstracts. Furthermore, I prepared summarised methods and findings by written report for our project collaborators in the USA and upon leaving this position (circa 2012) I prepared to detailed technical reports in word summarising key findings and presenting seminal pieces of data to include in any future publications. My efforts both in the laboratory and with reference to aforementioned presentations and technical reports did indeed culminate in 2 seminal peer reviewed publications in PLoS Neglected Tropical Diseases (circa 2010) & more recently (circa 2016) the Oxford University Journal Nucleic Acid Research; Both with myself as a joint first author I was able to complete these tasks proficiently and with autonomous status because specialist experiential knowledge and broad based experience, both technical and in with reference to technical training/teaching has been garnered over many years in similar prior positions; This knowledge and understanding is expounded in my extended CV


Document Ref:

2016/01

Page 3


A2: Exercise sound judgement in the absence of complete information and in complex or unpredictable situations.

This competence is asking you to identify and be aware of the limit of your own knowledge and professional competence, to demonstrate an ability to manage your own strengths and weaknesses and to recognise the level of risk attached to your actions. Examples could include but are not limited to: • Considering when you have approached a piece of work or project flexibly and in a novel or different way, or reacted to an unexpected outcome


In this section you should describe why, what and how you exercise sound judgement in the absence of complete information and in complex or unpredictable situations to fulfil this competency. 1) In my current position I am responsible for processing an experimental cohort of formalin fixed paraffin embedded (FFPE) tissue samples that are refractory to antibodies targeting CD31, a marker of new blood vessel formation. This experimental cohort is designed to investigate whether drugs delivered in the vicinity of a clot by stent can promote formation of new blood vessels as evinced by immunohistochemistry (IHC) using CD 31 inter alia. In the first instance I was expected to review current protocols & talk through experimental strategies and design and then implement pilot staining programs with pertinent antibodies. I then prepared figures in power point and reported back to my PI. In essence, the main this preliminary pilot study involved evaluating different methods of antigen retrieval (renaturation) in formalin fixed paraffin embedded archived experimental samples where preservation results in denaturation of epitopes. For one particular biomarker standard protocols evaluated by me for antigen retrieval, viz. Trypsin digestion and heat denaturation in low molarity citrate buffer both resulted in sufficient staining by IHC to score new blood vessel formation. However, the other principal biomarker utilised in this study failed to stain using standard antigen retrieval techniques. Having reviewed literature for FFPE materials I Conducted experiments where I subjected the tissue to proteinase K digestion and this yielded staining but of insufficient magnitude to score new blood vessel formation. I then improvised

Document Ref:

2016/01

Page 4


on a standard proteinase K antigen retrieval protocol based on some analogous optimisation experiments I had conducted many years before with similar FFPE materials. This more aggressive denaturation did indeed unmask the CD 31 epitope. Consequently I am now in a position to begin screening the experimental cohort and answer the question. ‘Do certain growth factors promote collateral blood formation in the vicinity of a simulated Atherosclerotic plaque (in experimental rat models’ )?’

2) In my current position, over and above personal project work and training/mentoring of students, I am also responsible for the operation, maintenance, servicing and troubleshooting of multiple analytical platforms situated in the main molecular genomics lab I manage. Regarding one platform in particular, viz. the AB Viia 7 real time qPCR machine, when I first took up the post, we were experiencing a number of technical complications causing frequent cessation of operations. With regard to one particular incident, complying with this competence, it became evident that calibration for array cards, designed to screen Micro RNAs, had not been performed in more than 2 years: Specifically, our service warranty covers calibration for standard 96 and 384 well plates and not specialised array cards. Furthermore, and co incidentally, the halogen bulb used to excite fluorescent samples was about to expire (~2000 hrs of use) and with change of bulb you require new calibrations, as the bulb emits polychromatic light and thus the spectral profile of each bulb is unique, necessitating fresh calibrations to match this ‘fingerprint spectrum’. Since the original array card calibrations would inevitable be wiped to purchase kits in order to put back


Document Ref:

2016/01

Page 5


calibrations and render the machine operable for array cards going forward would cost approximately £3000. Liasing with my line manager and chief technician Dr Tim Barnes (cf. Ref #1), it because clear that ‘soft cash’ was not available in the short term to cover this expenditure. Moreover, having discussed the matter with the principal users’ of array card format it was also made clear to me that such monies would not be forthcoming from their budgets. It appeared we had reached an impasse. Co incidentally, our machine was due for an annual service under the terms and conditions of our fully comprehensive service contract. Accordingly, I persuaded the engineer to change the bub at ~ 1700 hours instead of the prescribed 2000 hours and because this would now fall under the aegis of ‘servicing’ Thermo Fisher themselves were prepared to provide the kits for array card calibration. This circumvented the above complication and rendered the machine operable with the new build for array card format (as well as standard 96 and 384 well plates). Coinciding bulb change (ahead of the official threshold) with the annual service and thus proving the calibration kits as part of the annual service covered by our service agreement was entirely my initiative (obviously with agreement from the service engineer) These aptitudes draw on prior experience in Manchester and New York, amounting to 10 years of my career, with reference to maintenance troubleshooting and improved performance of complex analytical platforms. In the case of troubleshooting IHC this has its practical inception in experiments conducted over 3 years at the University of Manchester


Document Ref:

2016/01

Page 6


A3: Demonstrate critical evaluation of relevant scientific information and concepts to propose solutions to problems

You should think of this competence in terms of selecting the best methodology, the subsequent data analysis and conclusions you draw and how you overcome any barriers or issues. Examples could include but are not limited to: • Engaging in experimental design and testing • Reviewing relevant literature, manuals or designs • Sharing your findings with others


In this section you should describe why, what and how you demonstrate critical evaluation of relevant scientific information and concepts to propose solutions to problems to fulfil this competency. 1) Another example in my current post of engaging in experimental design based on literature review and then passing on relevant protocols in the form of SOPs devised by myself was a project I participated in where my prescribed remit was to devise methods to amplify and then measure expression of isoform specific genes of interest (linked to a separate cardiotoxicity project). This was necessary to elucidate whether cardio protection in our in vitro models was linked to general expression of particular genes or conferred by particular isoforms derived from alternative transcripts. This required me to familiarise myself with identifying transcripts for particular cardio toxic protective genes in both EBI/Ensembl & Genbank to identify alternative transcripts common to both data bases. I then devised an SOP for students which took them from mining transcripts in data bases like EBI/Ensembl to primer design which includes general aspects of primer design, e.g. avoiding hair pin loops, formation of heterodimers and crucially how to select target sites for such optimal primers in order to amplify just one gene transcript and not another; viz. selection of exon-exon sites only found in that spliceform. Having compiled an extended SOP a number of students were walked through it in a tutorial type discussion of the procedure. In terms of subsequent experimental design and testing, this was trialled by me in the lab based on initial RT PCR. That allowed me to identify pertinent gene isoform specific transcripts actually expressed in our myocyte cell lines, which could then be taken forward by a Ph.D student by real time qPCR. The real time qPCR experiments were designed and explained by me to the Ph.D student and, having conducted the experiments, results were discussed with me to decide on the next

Document Ref:

2016/01

Page 7


experiment. Through this strategy of initial literature review, experimental design and testing of experimental strategies myself and the PhD student were able to successfully elucidate isoform specific gene expression in a number of cardiac cell lines and infer the relevance of such transcripts in protection against insult by foreign compounds 2) In my prior position at the University of Edinburgh, I was assigned to a project to Develop a simple, stream lined and cost effective assay for screening small compound libraries to identify putative lead compounds with antagonistic efficacy against a Trypanosome Target protein devoid of an ortholog in the human host (on account of a unique RNA editing process in Trypanosomatid parasites not found in humans: The first stage in this (drug discovery) assay development procedural pipeline was to find an optimal method for expressing and purifying a recombinant soluble target protein: In particular RNA Editing ligase 1 (REL 1) which has no equivalent ortholog in the human genome and thus potentially represents a parasite specific target: Having consulted the literature, experts in the field and trialled various pilot scale expression models, abundant soluble phase protein was achieved by expression in the E coli strain BL21 DE 3 by commencing induction with a simple heat shock and then expressing the recombinant protein over a protracted time period (20hrs) at a sub ambient temperature (18oC). These expression conditions were conducive to protein production with minimal mis folding and thus soluble active protein compatible with a functional assay. The next obstacle was to discover methods for long term storage of the protein @ -80oC without losing activity upon freeze thawing:


Document Ref:

2016/01

Page 8


This was achieved by Glycerol stabilisation @ 10% (v/v) coupled with particular additive known to help promote retention of nascent structure through a clathrate mode of action. The bench work was a combination of guidance from literature extolling ‘best practice’ coupled with empirical investigation Having produced soluble, stable target protein preliminary pilot scheme evaluated the effects of known inhibitors on REL1 based on a radio ligand assay followed by antagonism of REL 1 with substances predicted to have antagonistic efficacy towards REL 1 based on in silico modelling. This ‘proof of principle’ culminated in a major publication in PlOS with myself as joint first author and also first author poster presentations at National infectious diseases conferences. Joint first author status was conferred because all bench work and preliminary literature investigations were performed in dependently by myself The final stage of this procedural pipeline having demonstrated proof of principle was to develop a stream lined high through put assay to mass screen compound libraries. This required assay development in the context of (Michaelis Menten) enzyme kinetics & substrate, Fluorometric adducts & considerations about how best to quantify target hits in terms of ‘best practice’ viz. a statistical parameter called the Z score. All biochemical parameters were successfully and personally developed by me and the assay was subsequently used in drug discovery centres to identify ‘real hits’; Once again resulting an Oxford University press Journal publication (NAR), with myself as joint first author as well as joint first author on numerous posters @ national as well as international conferences


Document Ref:

2016/01

Page 9


3) In the context of my present position, I have been charged with developing an in vitro model of tumour metastasis, which simulates the interaction between tumour cells and endothelium, in the presence of modulating platelet factors. The first task was to develop a simple stream lined model of tumour endothelium interaction in culture: This has been achieved by seeding Boyden chambers with Huvec cells followed by additional seeding with a tumour cell line (HT 29) and then evaluating transmigration of the latter cells through the endothelial barrier. All seeding parameters have been personally and empirically investigated by myself, with proposals put at bi weekly meetings to my PI who then comments on my ideas. In addition, I have reached out to the scientific community at large via Research Gate to solicit best practice and also consulted current literature on transmigration to adopt and adapt such published precedents. The next stage, which will commence shortly, will be to set up the full transmigration assay, where purified platelets are added to the mix together with putative antagonists of platelet exosome factors to identify substances demonstrating putative inhibition of HT 29 transmigration Regarding prior experiences relating to this competence exome sequencing of QTLs over a period of 3 years (circa 2002-2005) directly informs methods and teaching relating to transcriptomics. Similarly, I spent 7 years in both the UK and Manchester devising and improving on sequencing methodologies, including a 1 year participation in the DNA sequencing committee of the ABRF: See my linked in profile and extended CV for details


Document Ref:

2016/01

Page 10


B: Personal responsibility Competencies

Guidance

Evidence Chosen

B1: Work autonomously and take responsibility for the work of self and others

It is important for this competence to ensure you describe your contribution, responsibility and impact on a certain task and make it clear what you personally have achieved i.e. “I” not “we”. In formulating your answers, you should consider the following:

In this section you should describe your level of autonomy and how you take responsibility for the work of self and others to fulfil this competency.

•

•

•

You will be expected to undertake much of your work without day-to-day supervision and so you should demonstrate that you are able to achieve this You should demonstrate your understanding of when you may need to seek guidance from others and how you would obtain this guidance If you are responsible for managing the work of others, you should clearly describe how you discharge those responsibilities


1) My current role as a ‘core research technician’ is a

centrally funded support role and my overall job scope is to offer technical support to individual end users’ particularly students with limited laboratory experience and also provide and maintain a fully functioning multi PI large molecular biology laboratory. I was hired into this position based on my abundant experience in both ambits and as such I am expected to organise my own time and interact with students, PIs admin and outside technical representatives with minimal input from my line manager. To that end, I manage my own time and set my own priorities; both with regard to project work and running a busy multi user molecular biology laboratory. In essence, this requires efficient time management in which one or more bench projects are intercalated with housekeeping duties to satisfy both sets of work objectives. Regarding lab management I discharge this aspect of my job description completely independently based on prior personal practice; I simply keep my line manager in the loop. Where bench project work is concerned I discharge technical duties independently but recognise the bigger picture informing why I do what I do is outside my own expertise

Document Ref:

2016/01

Page 11


(requiring theoretical expertise in the actual subject matter). Accordingly, I will try and trial particular technical solutions to problems associated with experiments of my own volition but the initial project scope is set by a PI who I will meet with on an intermittent basis. These progress meetings take the form of me presenting material(s)/results, usually in power point format as a personal presentation for comment and scrutiny. Technical details tend to be a 2 way discussion, based on my own findings and discussion elicited by me via forums like Research Gate but project priorities, vis-à-vis how they conform to the bigger picture and permission(s) to order relevant experimental consumables are given by the PI 2) With Reference within this varied remit to managing the

work of others, for about 6 months I bench supervised a Ph. D student: As already discussed, the object of managing this aspect of their project was to devise methods to identify and measure isoform specific gene transcripts of candidate genes with a putative role in conferring protection to toxic insults on cardiac cell lines ‘In vitro’. This management took the form •

•


Of training them in mining isoform specific gene transcripts from data bases, cf. Ensembl & Genbank. This in turn entailed me writing an SOP and then talking them through it Primer design which includes general aspects of primer design, e.g. avoiding hair pin loops, formation of heterodimers and crucially how to select target sites for such optimal primers in order to amplify just one gene transcript and not another; viz. selection of exon-exon sites only found in that spliceform

Document Ref:

2016/01

Page 12


• •

•

Having completed these preliminary design of genomic materials I was then responsible for training and demonstrating all aspects of RT PCR to the student This enabled the student with discussion and guidance from me to take a subset of the best performing genes/primer sets forward into actual real time qPCR in order to measure gene expression: Initial training in real time qPCR and design of gene expression experiments with discussions on how to proceed was managed exclusively be me

3) In my prior position at the University of Edinburgh

(Ref. #2) I was, as my PIs first employee in the UK, having come to the UK on an MRC fellowship from the USA, I was expected to assist with lab set up from its inception and also set up a drug discovery project, building on prior work in the USA. Specifically:

I spent the first 3 months in post: • • •

Procuring quotes for key pieces of equipment Purchasing such equipment and consumables with permission from my PI/line manager Organising the physical space in the lab including attendant organisation of fridges and freezers for perishable consumables for subsequent project work to be carried out by myself and other lab staff employed subsequently

Having set up the lab, I liased with my PI to identify bench work priorities and project scope with reference to the


Document Ref:

2016/01

Page 13


aforementioned drug discovery program I personally and independently performed in the lab. To autonomously perform this lab work, I versed myself with key achievements in the USA and then identified technical strategies to achieve key practical objectives as already outlined, viz. efficient production of soluble and stable recombinant target protein (rREL 1) and subsequent optimisation of the fluorescent assay utilising rREL 1 with reference to substrates, enzyme kinetics and statistical evaluation of preliminary hits. My principal role was to independently perform the wet science and inform my PI of progress and potential future experiments. He would ratify progress; confirm future objectives and also modify experimental objectives in line with MRC project milestones and finance. Progress meetings were weekly and took the form of one on one Power Point presentations of data, experimental design and future priorities. In addition, I was expected to present via Power Point to publicly to the group as a whole in order for my PI but also post doctoral fellows to comment on my data and plans Independent working and taking responsibility for training and mentoring others is something in the past I have done extensively; For example in my 3 years on a transcriptomic project (circa 2002-2005) I was expected to amplify and sequence whole exomic regions and duly train others in these methods. Similarly, for 10 years spanning the 90s I was responsible for training post graduate technicians and both train and supervise undergraduate B.Sc. project students. Similarly, between 1995-2000 I set up and independently ran a core DNA sequencing facility at the University of Manchester medical school and that experience culminated in me securing a position in the New York genome center in the summer of 2000


Document Ref:

2016/01

Page 14


B2: Promote and implement robust policies and protocols relating to health, safety and security

You should demonstrate that you understand the policies and protocols related to health, safety and security that apply to the work you are undertaking and describe any responsibilities that you have related to this. Security can include issues related to data, Intellectual Property, confidentiality, prevention of contamination, traceability of documents and information. In formulating your answers, you should consider the following: •

•

•

These policies and protocols will document how relevant aspects of your work must be carried out. Demonstrate that you know where these policies and protocols are documented, and that you are able to apply them in your practice What risks you are aware of related to the security aspects of the work you carry out, and how you seek to mitigate these risks How you “promote” the awareness and application of these policies and protocols


In this section you should describe why, what and how you promote and implement robust policies and protocols relating to health, safety and security to fulfil this competency. 1) In my current position, I am responsible for completing

COSHH forms for communal hazardous chemicals I order via smarter purchasing

2) In addition, I am responsible for training students in the

completion of such forms and making sure they understand and comply with GLP in terms of PPE and waste disposal: Training of novice students is an integral part of the students introduction/induction into the lab 3) Apart from this induction into health and safety, I provide an ad hoc service for anyone purchasing hazardous/toxic chemicals linked to a new procedure if they require help completing or complying with the COSHH form, e.g. interpretation of hazard statements; pictograms & risk phrases 4) In addition, safe working practice is inculcated by training, e.g. safe waste streaming by paying attention to such things as methods and routes of disposal: A seminal example would be making sure that cytotoxic chemicals are only handled in the fume hood and are disposed of in a cytotoxic bin, collected and eradicated by incineration; as opposed to working on the open bench and streaming such waste via standard orange waste bags, neutralised by microwaving and destined for land fill. An example of this would be use of Trizol for extraction of RNA 5) To ensure that such practices are continually adhered to, apart from initial training of students, we have monthly lab meetings where obvious transgressions are pointed out and GLP is clarified in such meetings and subsequent lab minutes.

Document Ref:

2016/01

Page 15


with others, especially peers and more junior colleagues

These monthly meetings are augmented by ad hoc e mails when mal practice is identified 6) Another example of safe working practice, compliant with COSHH, is storage of chemicals; This has been set up by me as part of my lab management remit: Specifically, acids and alkalis are separated from oxidising agents in separate metal fire proof cabinets and volatile cabinets are stored in ducted, ventilated solvent cabinets 7) To comply and implement the above I have attended Leicester specific COSHH and risk assessment courses 8) In my former position at the University of Edinburgh,

I was responsible for all aspects of health and safety including inter alia GLP, storage and safe handling of hazardous substances and waste streaming. Thus all of the above considerations and practices were similarly carried out 9) Furthermore, to gain an appreciation of such practices and how they are informed by HSE legislation I attended a 1 week Biosafety course with 1 full day of examinations and an oral presentation, culminating in the award of Bio safety practitioner (Foundation Level #1) Health and safety is an aspect of my technical bench work I have been expected to understand and adhere to since the early 1990's beginning in Manchester


Document Ref:

2016/01

Page 16


B3: Promote and ensure compliance with all relevant regulatory requirements and quality standards

You should demonstrate that you understand which regulatory requirements and quality standards apply to your area of work. In formulating your answers, you should consider the following: •

•

Describe what you do to ensure that these requirements and standards are being followed for those activities for which you are responsible Describe how you “promote” the awareness of regulatory requirements and quality standards amongst peers and more junior colleagues

In this section you should describe why, what and how you promote and ensure compliance with all relevant regulatory requirements and quality standards to fulfil this competency. In both my current role in the BHF cardiovascular centre and former role in Infectious diseases at the University of Edinburgh, part of my Lab management remit was/is to ensure safe and competent working practice through administering COSHH and paying due regard to laboratory organisation and GLP In that regard and to ‘promote and ensure compliance with all relevant regulatory requirements and quality standards’ I: • •

• •

•


Make sure that for all new chemicals that are communally ordered on behalf of the lab, COSHH forms for hazardous chemicals are completed These forms stored on a shared drive and in addition hard copies are archived in ring binders: Users are expected to read these forms and sign they have understood implicit risks before commencing procedures incorporating such hazardous substances Furthermore, these are made available via our departmental intranet and all new lab staff are aware of their location via an induction pack For new students (in particular) performing laboratory procedures with one or more of these chemicals they are instructed in implicit hazards and GLP by me before commencing such procedures In addition, for procedures utilising non communal but hazardous chemicals I make sure the students are

Document Ref:

2016/01

Page 17


•

aware of how to fill out relevant COSHH forms (by accessing and transposing MSDS information onto the departmental COSHH forms) and implied safe practice; In particular, storage & handling of such substances; disposal routes; PPE and the concept of risk phrases, hazard statements and pictograms Promotion of such safe COSHH compliant working practices as mentioned is reinforced through monthly lab housekeeping meetings which I contribute to in terms of housekeeping points and on occasions publicly conduct as well as devise minutes (for general circulation to lab staff)

Another example in my current position in Cardiovascular sciences of COSHH complaint practices is chemical storage. In particular, I am responsible for devising and implementing safe storage of chemicals in conjunction with the Chief Technician Dr Tim Barnes, who is also my direct line manager and (pertinent to this answer) health and safety officer (and in fact Referee #1 in this application): Specifically, Organic or generally Volatile solvents have recently been relocated by myself to a purpose vented solvent cabinet, liasing with the Chief Technician; Acids and alkalis are stored separately in a metal cabinet under the fume hood and Oxidising agents are segregated into a metal flame proof cabinet by themselves. To promote this delineation I have taken to placing laminated signs on the outside of respective containment facilities describing the type of storage, e.g. volatile solvents, viz. all organics and then listing the contents inside


Document Ref:

2016/01

Page 18


In my second cited position at the University of Edinburgh similar considerations applied. In addition, however, Because I arrived at the lab at the point of inception and assisted my PI in the actual set up of the laboratory I was also responsible for devising and implementing waste disposal policies in line with HSE, University wide policies and extant local regulations. These were enunciated in policies written by me and displayed in the laboratory as well as archived on local lab drives and also reinforced at our weekly lab meetings ad

hoc


Document Ref:

2016/01

Page 19


B4: Oversee the implementation of solutions with due regard to the wider environment and broader context.

You should demonstrate an understanding of the potential and actual impacts of your work on your organisation, on the profession, on the general public and on the physical environment. Examples could include but are not limited to: •

•

•

•

Indicating that you are aware of the sensitivity of your work and show how this understanding translates into the ways in which you carry out your work Showing an awareness of how your profession is portrayed and viewed by the public at large, and how you take responsibility for recognising this in the work you do Describing how you seek to avoid reputational damage related to the work you carry out Explaining how you set a good example to others in the way you discharge the responsibilities related to the work you undertake


In this section you should describe why, what and how you oversee the implementation of solutions with due regard to the wider environment and broader context.to fulfil this competency. In my current role and also second role at the University of Edinburgh, under scrutiny, in this document I am/was responsible for managing large molecular genomic laboratories, consisting of 20 or more lab based personnel, linked to multiple PI’s 1) One consequence of this many persons in terms of impact

on and due regard for the broader environment is waste streaming including ‘oversee and implementation’ of recycling provision

In particular, in my current role as a core (research) technician within cardiovascular Sciences at the Glenfield Hospital, I am responsible for recycling of empty tip boxes, accrued through the shire number of tips consumed. Recycling circumvents the original situation where empty tip boxes were disposed of in black standard refuse bins destined for landfill To accomplish effective and efficient recycling, I am expected to liase with the parent company who then make appropriate arrangements with the courier In addition, and at the outset of my post, users’ were mandated to place their empty tip boxes inside recycling polythene bags situated in a green wheelie bin. In the absence of collection problems linked to logistical problems with the courier, e.g. failing to go to the appropriate pick up point in the Document Ref:

2016/01

Page 20


hospital or failing to turn up for collection at all my primary communication with the parent company who outsource recycling to couriers has been to arrange a date for pick up and ensure polythene bags are sealed and situated in the designated pick up point. In addition, I am responsible for making sure we have requisite stocks of polythene bags and ties to continually provide recycling 2) In addition to environmental considerations and their impact

on the environment, in my current core role with lab manager responsibilities, I also oversee and implement waste disposal, which has to be COSHH complaint and in line with GLP. This in part is inculcated through information provided in COSHH forms and passed on in person to novice workers when I walk them through the implications of COSHH forms for particular hazardous chemicals. •

•

To take one specific example of waste hazard disposal and limiting environmental impact, We use Trizol and other phenol derivatives to extract DNA and RNA and these must be used in a fume hood and residual organic waste disposed of in cytotoxic bins in situ The use and disposal of such hazardous phenolic compounds is inculcated by COSHH – both in terms of initial training by myself for novices when it comes to nucleic acid purification – and also initial purification demonstration/Technical training by me for the benefit of new students

3) Another example in my current role of due regard to the

environment in terms of policy practice is making sure non


Document Ref:

2016/01

Page 21


hazardous but biological waste is segregated from general refuse. This is achieved by delineation of such waste streams into 2 types of physically distinct bins; Namely black bins for general landfill refuse and orange bins for general containment level 1 (non hazardous) biological material, which is microwaved prior to disposal in landfill. I ‘manage’ these routes od disposal by monitoring transgressions and either pointing them out by general e mail; speaking discreetly to the particular ‘offender’ or bringing up the issue in our monthly housekeeping lab meeting. Adding to point 2) I absolutely come down hard on anybody who mistakenly or casually disposes of toxic organics in orange bags instead of cytotoxic bins 4) In my current role and in terms of promoting cardiovascular

science in a good light I participate in a couple of outreach initiatives: •


The BHF centre relies primarily on program grants from the BHF to procure continuous funding. As part of that procurement I play a lead role in promoting and providing scientific tours for lay members of the BHF, e.g. volunteers and in house admin staff: These take the form of explanation in basic language of key themes underpinning our work, e.g. ‘what is DNA versus RNA versus protein ?; what is PCR ?’ and then explanation of what key pieces of equipment situated in the lab are used for to accomplish these aims. In conducting these tours I am mindful of professional and courteous conduct

Document Ref:

2016/01

Page 22


•


The second initiative I participate in to promote the work of general life scientists is participation in school tours; Thus far in my case this has taken the form of a personal professional profile compiled in Micro Soft Front page which was showcased on a board at Leicester Grammar school as part of their careers fair

Document Ref:

2016/01

Page 23


C: Interpersonal skills Competencies

Guidance


Evidence Chosen

Document Ref:

2016/01

Page 24


C1: Demonstrate the ability to communicate effectively with specialist and non- specialist audiences

A non-specialist audience is anyone working outside of your particular area of expertise, so it would not necessarily be a non-scientist. Your example(s) should indicate how you have communicated in a way that is effective to each type of audience. In formulating your answers, you should consider the following: • Not just the content of the message but also the mode or style of delivery that is adapted according to the audience • The feedback loop to gauge the understanding and improve future communications

In this section you should describe why, what and how you communicate effectively with specialist and non- specialist audiences.to fulfil this competency. In my current role, I am responsible for managing a multi user multi PI molecular genomics laboratory which includes at any one time approximately 20 lab based personnel. My remit includes interfacing on multiple projects; stock taking ordering and account auditing; assignations with Company equipment engineers and external sales specialists & training novice personnel in particular bench techniques. In addition I perform outreach duties engendering in house non specialist audiences and external (BHF) personnel. In consequence I interface with a plethora of specialist and non specialist audiences alike, including • • • • • • •


PIs in relation to managing bench based projects I personally participate in Students relating to technical training and health and safety training (COSHH) Technical specialists and equipment engineers from outside Medical Science companies Sales representatives from similar companies In house admin staff relating to smarter purchasing and account auditing In house financial personnel relating to account auditing Non specialist admin staff participating on scientific tours I organise and conduct

Document Ref:

2016/01

Page 25


•

•

•

Non specialist external personnel (BHF volunteers & in house London based staff) on similar scientific tours and latterly formal lectures on ‘measurement of gene expression’ (relating to a BHF open day, with 120 BHF staff and volunteers in attendance) A mixture of both specialist and non specialist personnel at a departmental away day where I delivered a 5 minute Power point presentation to approximately 150 personnel Finally I have also participated in one ‘Outreach program’ at Leicester grammar school, where I presented a career profile for display at a careers fair

In my prior role under scrutiny my remit was very similar. Consequently, the above relationships also pertained. In addition, However, in that role I was also expected to give public power point presentations to both my own group on a weekly basis and at departmental seminars. Finally, I was expected to communicate to a wider specialist audiences at both national and international conferences in the form of (1st) authorship of poster presentations and conference abstracts Regarding didactic style, when communicating with PIs in progress meetings, that takes the form of Power point figures, summarising data: Conversation is confined to data proclivities and experiments to do. The conversation in this regard is very much a dialogue, with myself leading discussions on methodology results and proposed future direction, with the PI


Document Ref:

2016/01

Page 26


commenting on data and confirming or altering the next experiments in sequence Regarding technical training delivered to specialist students, this takes the form of didactic instruction in the office, initially relating to an SOP that I have devised with explanation of practical principles underlying the procedure and then any safety implications if hazardous substances are incorporated. This is followed by didactic practical demonstration in the lab with, on one occasion bench supervision of follow on experiments: An example of this type of technical training with specialist students is extraction of RNA from mammalian cells, followed by reverse transcription, culminating in cDNA and then selective amplification of target genes using optimal primers designed to gene transcripts mined in EBI Ensembl & GenBank. SOPs have been respectively devised by me for RNA extraction, reverse transcription and selective amplification of genes of interest (GOI), using genomic data mined from Ensembl and GenBank databases & students are walked through these in person. One on one practical demonstration is then provided with emphasis on PPE, waste disposal and the procedure itself Regarding scientific tours to non Scientific non specialist audiences these require more thought but in many ways are more rewarding: In essence, I tend to commence such tours by providing background explanation of ‘what is DNA’ ‘what is RNA’ and ‘what is protein’ and the relationship between these 3 groups. I then talk about genes as bits of DNA and how mutations are like spelling mistakes in a book causing pathophysiology….etc. Crucially and for the purposes of this Please return the form to [email protected]

Document Ref:

2016/01

Page 27


competence, this material is presented with cartoon like depictions of each substrate and pitched at the level of a ‘Radio 4’ discussion, i.e. compatible with an educated lay person. Analogies are used as frequently as possible, e.g. the simile of mutations being like spelling mistakes in a book and the book itself constituting the whole genome sequence. Having provided this contextual background then and only then do I take the participants to see particular platforms in the lab, e.g. gel documentations systems for visualising DNA; PCR machines, which are like factories for making lots of the DNA for analysis, and real time PCR machines for measuring the RNA made from DNA. In presenting the lab tour, I always ensure that I select equipment/data that is tangible and engaging to the eye, e.g. ethidium bromide bands on agarose gels. On reflection, having given a number of these lab tours, I realise that key to engaging the participants is to keep dialogue as ‘open as possible’ since interest in medical science is often motivated by personal real world experiences. Thus, the ‘scripted tour’ might digress in to familial stories of heart disease in participant families. From that point of view and over time I have realised that the actual content of these tours is as much determined by lay participants as my rehearsed materials. Consequently, discussions tend to sound as much like a BBC news report as a didactic lesson in the genetics and genomics of Cardiovascular disease. In addition, over time, my presentations have become more replete with metaphors and analogies in order to make a point, e.g. when describing primer design for making lots of DNA by PCR (I often describe the actual PCR machines as ‘DNA production factories’) I talk about the fact that these little DNA strands find their intended target Please return the form to [email protected]

Document Ref:

2016/01

Page 28


in the same way as spell checker find a sentence in a word document. In addition, over time, I have resorted to recommending popular science narratives on genetics and genomics and even use their front covers as props, e.g. the ‘The deeper genome’ (John Parrington) uses the metaphor of an ice berg on the front cover to make the point that exposed and understood part of the genome (the protein coding exome) is quite literally the ‘tip of the iceberg’ where gene expression and development is concerned and most components orchestrating expression are the hidden bits or ‘dark matter ‘ of the genome (encoding non coding RNA species). I use this front cover to make this introductory preface when beginning my tour Another activity I regularly engage in is soliciting and providing answers to research questions on scientific forums; Principally Research Gate. I engage with this forum to find technical solutions to problems relating to a research project. For example, I am currently attempting to set up an in vitro model of tumour cell extravasation and the effects of platelets on this transmigration phenomenon. I have been guided by experts in the field by soliciting discussion on Research Gate Indeed, with regard to providing advice to external personnel via Research gate I was invited to become I review Editor for Frontiers in Medical Research (Infectious diseases and Microbiology)


Document Ref:

2016/01

Page 29


I duly accepted and have just completed my first editorial assignment, wherein the principal author took on board my caveats regard manuscript content and subsequently I recommended the manuscript be accepted for publication; Just today I have accepted my second editorial assignment with Frontiers in Microbiology : My recent editorial experiences have assisted with reinforcing my scientific critical reasoning; similar, in fact, to participating in a monthly Journal club in my prior cited position at the University of Edinburgh With reference to my previous Scientific post at the University of Edinburgh I regularly presented Power point updates to my group colleagues: These figures like informal presentations to PIs primarily stressed emerging data and future work. Concomitantly I also participated in departmental seminars. Delivered by power point these presentations tended to knit data together in order to reinforce a hypothesis or completion of a project milestone, e.g. optimisation of a fluorescent high through put drug discovery assay in order to implement actual drug discovery by screening small compound libraries Experience of and proficiency in teaching and public speaking to specialist audiences in particular has been an integral part of my job description since commencing my first molecular biology post at St Marys in 1988: In particular I have participated in group presentations since that time. Furthermore, in Manchester (1995-2000) I was not just expected to formally lecture M.Sc students but in addition design, organise and provide Sequencing workshops as a formal module on an M.Res course (then) offered by the University of Manchester. At both Manchester and in New York I also gave formal presentations at international conferences. In addition in more recent Consortia (2005-2008) I was expected to contribute to a project news letter to update collaborators in Ireland and the UK Please return the form to [email protected]

Document Ref:

2016/01

Page 30


C2: Demonstrate effective leadership through the ability to guide, influence, inspire and empathise with others

This competence is about understanding your leadership skills and is not reserved for those in management roles; it is applicable to all. Examples could include but are not limited to: • Experiences of mentoring or coaching you have had; you should consider how effective this was and the overall impact • Considering when you have managed change within your organisation or overseen the implementation of any new processes

In this section you should describe why, what and how you demonstrate effective leadership through the ability to guide, influence, inspire and empathise with others.to fulfil this competency. 1) With reference to my current position in Cardiovascular

Sciences and ‘mentoring, guidance, and influencing others’, I have been personally instrumental in effecting out comes in this regard in both scientific and non scientific contexts:

1)The first lab based/research project example, exemplifying leadership skills and influencing the behaviour of a student relates to a bench project I was responsible for implementing through a Ph. D student. The overarching objective of this project was to Devise a procedural pipeline for extraction of RNA from cardiac myocytes & consistently and robustly measure expression of pertinent genes of interest (GOI): •

• •


Using real qPCR to quantify such genes, do we elect to use customised & therefore expedient TaqMan assays or more technically involved but cheaper and flexible Sybr Green qPCR assays ? Having selected a real time qPCR protocol, how do we go about selecting stably expressed abundant housekeeping genes for normalisation of expression? Regarding IT considerations, what is the best method for quantifying expression between different sample groups (normalised to selected housekeeper genes)?

Document Ref:

2016/01

Page 31


With reference to these 3 seminal questions my first task was to go away and prepare the ground by perusing current literature on best practice and engaging with the wider scientific community via Research Gate. Based on these initial endeavours it became obvious that: •

•

•


Sybr green assays although requiring design of primers to GOIs and working up the assay, would afford flexibility in terms of where in the cDNA sequence we were able to map the primer sequence: This seemed important in order elucidate if a particular protein isoform derived from a particular alternative transcript was responsible for conferring cardio protection to drugs. Moreover, Taqman probes, although off the shelf and therefore convenient are expensive compared to simple sybr dye labelling of cDNA and with inclusion of panels of housekeeping genes to select the best requisite 2 or 3 for expression analysis, it became evident that significant cost savings could be incurred by choosing to label with sybr green Based on current best practice and MIQE compliance it was also evident that we needed to screen a panel of housekeeping genes using programs like gene Norm to find the 3 optimal housekeepers in the context of our expression system Finally, from literature perusal, including discussion threads on Research Gate and also soliciting answers on Research gate to questions posed I realised that we needed to perform normalisation of gene expression using the geometric mean of 2-3 housekeepers and enumerate actual gene expression between different

Document Ref:

2016/01

Page 32


cardiac cells lines using the so called Delta Delta Ct method (or Livak method). This was carried out in Excel and students trained by me in the methodology Subsequently and crucially from the perspective of this competency, I communicated this information in the first instance to the student in question. Further, I devised SOPs to codify practice and initially I walked the student through the specifics of the procedural pipeline. Regarding implementation of the actual practical lab work, viz. preparation of materials such as gene specific primers; preparation of RNA from cardiac cell lines; production of cDNA and then preliminary RT PCR to screen for optimal primers/housekeeping genes selection of that subset demonstrating consistent and high level expression in the context of our cell lines; and finally actual measurement of gene expression in target proteins normalised against these housekeepers by real time qPCR I worked with the student in the lab at the outset to ensure competent practice. The student then took this project forward by themselves having been familiarised with pertinent concepts and received practical training from me. At this juncture the student and I interacted to monitor progress and troubleshoot practical issues and if necessary, whilst at this stage I did not personally engage in the lab work I did still design many of the students experiments In the final stages of this bench supervision, it was necessary to collate raw data and carry out a procedure to actually enumerate gene expression. This was done by the student using the so called Delta Delta Ct (Livak) method of analysis, incorporating calibration based on the geometric mean of 3


Document Ref:

2016/01

Page 33


housekeeping genes, selected with training and guidance from me, as described above. In terms of data analysis training, there again, I provided worked examples of the Livak method and computing the geometric mean in excel spreadsheets. Furthermore both housekeeping gene selection by use of software and subsequent Livak analysis were discussed in group meetings and also in specific one on one meetings with myself and the group head present. In conclusion through guidance and training the aforementioned student because confident and competent in measurement of gene expression 2) With reference to effecting change in my current position outside of my lab based technical duties, I have carried out reorganisation of our micro biology/ cloning lab. In particular, I now insist that • • •

Virgin LB agar plates are segregated from inoculated plates in a ‘clean fridge’ ( where reagents for plasmid preps etc. are also stored) Virgin plates are kept sealed by para film to minimise contamination by fungal spores Inoculated plates are segregated in a so called ‘Dirty fridge’: Such plates are sealed with Nesco film and dated

A. To prevent cross contamination B. To make sure that plates are disposed of within a 6 week time frame, after which time colonies are desiccated and not viable (for plasmid purification)


Document Ref:

2016/01

Page 34


Finally, spent LB agar plates must be disposed in separate bins (for incineration) and not general biological (Orange bag waste) as any agar from a breached orange bag that escapes during autoclaving can block the filters and put the autoclaves out of action All such competent practices are promoted by making sure that every new user of the cloning/Micro biology lab is subject to induction by me and also via laminated instructions on the fridges and bins within microbiology Regarding leadership, I have bench supervised in previous positions technicians, visiting graduate students (circa 2002-2005) and also undergraduate project students (circa 1994) Regarding management of change, in 1995 I set up a DNA sequencing facility from scratch with autonomy and also the laboratory for my 2nd referee (Circa 2008)


Document Ref:

2016/01

Page 35


C3: Demonstrate the ability to mediate, develop and maintain positive working relationships

You should describe or define the “working relationship” and provide at least one example which focuses on your handling of a challenging interpersonal situation and demonstrates your ability to mediate and achieve a positive outcome. You should consider how through your approach you have changed or modified the behaviour or attitudes of others to positive effect. Examples could include but are not limited to: • How you have managed the merger or integration of different teams • Managing working relationships across different departments or organisations • Interactions with committees, working groups or other professional body activities • How you have managed and resolved a difficult relationship situation between members of a team for which you are responsible.


In this section you should describe why, what and how you demonstrate the ability to mediate, develop and maintain positive working relationships.to fulfil this competency. 1) In both my previous role at the University of Edinburgh and my current role in BHF cardiovascular Sciences at the University of Leicester I deal with different constituent groups, e.g. scientific specialists (lab personnel), admin staff outside protagonists, each with their respective and different agendas. Productive & relationships are necessary to perform all aspects of my job description to the best of my ability. This is imperative in my support role to ensure smooth running of the labs on behalf of other lab users and perspicuous costings in relation to finance staff. Maintenance of positive working relationships keeps everything afloat 2) With specific regard to one type of working relationship and resolution of a problem, as described, part of my remit is management and organisation of lab resources and adherence to GLP. In general this is achieved by identifying transgressions and either communicating with the individual in question or sending out group wide e mails explaining the transgression and why this needs to change. In turn these ‘incidents’ are flagged and discussed by me in monthly lab housekeeping meetings. On one particular occasion however, there had been a recurring problem with agarose not being removed completely from conical flasks and problems with auto fluorescence when contaminated glass wear was employed in

Document Ref:

2016/01

Page 36


other types of experiments. This was recurring despite e mails from me informing users how to completely remove the agarose and re affirmation of these instructions in the monthly lab meetings. As a consequence of this I informed my line manager Dr Tim Barnes in his capacity as health and Safety officer. He then sent out separate correspondence. In addition, I asked the PI to raise the incident herself at our monthly gatherings. This duly happened and with independent cajoling the problem ceased. To cover all bases however and stop contamination of other types of fluorescent assays, I took the initiative and introduced specific and separate glassware for agarose gels vis-à-vis general glassware; In particular, I asked regular users’ of agarose gels to take a duran bottle, delineated by a label and red (as opposed to blue top) and keep for the specific purposes of running gels. This behaviour was successfully adopted With reference to positive relationships with multiple constituents, this was particularly important for example in setting up and running DNA sequencing activities in both Manchester (circa 1995-2000), New York (2000-2001) and Leicester (2002-2005), where I interfaced with technical specialists, equipment engineers and sales specialists, as well as technical personnel


Document Ref:

2016/01

Page 37


D: Professional practice Competencies D1: Scope, plan and manage multifaceted projects

Guidance Describe a project that you have managed and make it clear the level of autonomy you had while working on the project, especially if you were in a team. You should show how you contributed to determining the resulting courses of action. Examples could include but are not limited to: • An operational project utilising resources across several disciplines • A change management project aligning processes across sites • An industry-wide project establishing guidance on technical standards and requirements


Evidence Chosen In this section you should describe why, what and how you scope, plan and manage multifaceted projects.to fulfil this competency. My current position where I am expected to participate in multiple ad hoc research projects and manage laboratory resources for other laboratory users requires careful consideration with regard to my time and how I plan my activities With particular regard to lab based research projects I am currently participating in 3, cf. 1) IHC staining of experimental sections to elucidate new blood vessel formation in rat experimental models with artificial atheroma’s and proximal stents carrying growth factors in an effort to promote collateral blood formation, circumvent blockage and restore blood flow to the heart 2) Processing samples derived from patients undergoing cardiac surgery: Specifically, amplifying particular micro RNAs species in plasma and then measuring there levels by real time qPCR to make correlations between mIR levels and subsequent postoperative morbidity; In other words attempt to identify prognostic biomarkers in the form of mIR that might indicate postoperative complications 3) An attempt to model in vitro the interaction between tumour cells and blood vessels to evaluate diapedesis of

Document Ref:

2016/01

Page 38


tumour cells through these (model) vessels and how this trans migration is modulated by platelet (exosome) factors Because assignment to research projects is temporary and ad hoc, at the outset for all such projects and other projects going

forward, a discussion is initially had between myself and relevant PIs in order to identify project objectives. I have then gone away and thought about these objectives and based on a time commitment I can allocate to each of these projects (in conjunction with concomitant lab housekeeping duties already described), another discussion is had where we scope the anticipated time that each of these projects will take. Finally, a document is composed by the relevant PI; agreed to and commented on by me and then signed by all relevant parties including my line manager. To provide an example of project scope, with regard to IHC processing of experimental tissue sections the number of such archived sections was known. Accordingly, based on a commitment of 2 days per week with an ability to process 1020 sections in those 2 days requiring staining for 2 types of biomarker (CD 31 and actin), I deduced that my minimum commitment to that project would be 10 weeks.

In terms of how I then proceeded with each of the 3 projects and interfaced experimental duty that was left to me. To review project progress in each case I am expected to present key pieces of data to respective PIs in power point and make suggestions regarding future experiments (in the case of the in vitro model of tumours diapedesis) or comment on sections/samples processed for the other 2 projects (IHC and


Document Ref:

2016/01

Page 39


real time qPCR respectively|) and thus an anticipated completion date. In essence therefore projects are jointly scoped and managed by myself in concert with the PI and then both planned and managed exclusively by me Regarding material resources required for such projects, I am expected to stock take such resources e.g. antibody levels as experiments proceed and make recommendation to the PI when depleted stocks should be re ordered. Furthermore, although the PI is responsible for initial financial scoping and subsequent financial management of the project having made recommendations, when approved I am expected to order required materials and then charge them back to the PI (as an extension of my core laboratory stock taking and ordering duties)

Regarding prior experience of planning, scoping and managing multi faceted projects, my current position is particularly onerous in this regard but similar experiences have been adduced for example at the University of Manchester (1995-2000) where I ran a core sequencing facility necessitating interaction with perhaps > 100 clients at any given time participating in 2 or more genomic based sequencing projects


Document Ref:

2016/01

Page 40


D2: Demonstrate the achievement of desired outcomes with the effective management of resources and risks

Using the project you have discussed under D1, or another project with which you have been involved, you should describe your roles and responsibilities in managing the activities to achieve the desired outcomes. Examples could include but are not limited to: • Identifying the resources (people and/or money) needed to undertake the activities • Monitoring and surveillance of the progress of the activities • Identification, evaluation and implementation of changes that may be needed to ensure the activities are successfully completed • Identification and management of risks that could impact on the successful completion of the activities

In this section you should describe why, what and how you demonstrate the achievement of desired outcomes with the effective management of resources and risks.to fulfil this competency. An integral part of my responsibilities in my current role is providing material resources for other personnel – in a laboratory of 20 + personnel - to expediently conduct experiments. This entails • •

•

•


Stock taking and ordering of such materials Auditing the consumable stock & monitoring both expenditure on a consumables budget I am personally responsible for managing as well enumerating monies claimed through users signing for stock: This involves putting up charge sheets for users to sign as well as collecting such sheets; computing total expenditure by individual using formulas in Excel spreadsheets and then passing on these figures to personnel in accounts in addition to communicating with PIs to inform them of charges that would be levied against their grants (and put back into my consumables budget in order to balance the books) Furthermore, unclaimed stock also requires enumeration by myself to ensure that signed expenditure to claim back plus unclaimed stock approximately matches expenditure on the consumables budget I have been charged with managing

Document Ref:

2016/01

Page 41


All such accounting activities are carried out by me and at the outset of my position I quickly deduced that all such prescribed activities were not optimal, viz. Expenditure was not being monitored frequently and thoroughly enough and auditing to balance the books and thus to viably spend on consumables was not happening. Accordingly: 1) I introduced more detailed sign out sheets for users’ which included prices and grouped consumables into colour coded areas to simplify and render more transparent the actual process of signing out items, viz. tubes; gloves; tips etc. 2) In addition, I made sure these sheets were changed monthly instead of quarterly and enumerated claimed stock on a monthly basis, such that PIs could more efficiently monitor consumable expenditure on laboratory reagents 3) Furthermore I evaluated and periodically continue to evaluate stock usage such that we do not over stock and therefore overspend, bringing expenditure and recharges back into sync 4) In addition, with the advent of smarter purchasing about 18 months ago, I have been able to shop around and keep prices down. As part of this calculus, prices incorporated into excel spreadsheets are adjusted in real time to reduce expenditure and reclaims and also make sure that what we reclaim reflects the actual prices at that moment in time. This facilitates good book keeping 5) Finally, on annual basis I procure a detailed stock take, compute expenditure, add that to claimed expenses and then balance the net figure against actual expenditure. These calculations are carried out be me and presented to finance for approval: Apart from enumerating stock and ascertaining


Document Ref:

2016/01

Page 42


over stocked items and items in deficit this exercise serves to prove that the expenditure model is fit for purpose In terms of resource management, when I arrived it became evident that fridges and freezers in particular where chaotic rendering stock taking by me and finding particular items by end users difficult. Thus, I pushed via the chief technician to implement a whole scale clear out of fridges and about 10 20oC freezers. By doing this much space was liberated and as a result I was able to stream line freezer organisation in particular. In consequence, apart from assigning space to current lab users I was able to archive materials from personnel who had left in a different physical location and, crucially from the view point of this competency, dedicate 1 whole freezer to signed consumable resources, with stratification into reagent groups e.g. PCR reagents; Restriction enzymes, Sequencing reagents, antibiotics etc. and an attendant plan. This has greatly improved management of such stock 6) In terms of assisting with resource management, one such resource is capital expenditure on equipment and subsequent provision of service contracts. Regarding one type of platform, namely Qiagen Rotor gene real time PCR machines, when I took up the post, we were in a situation where capital expenditure linked to these platforms and ensuing service contracts were being taken from grant monies linked to one particular PI but were being used over and above her specific project work by other groups. In initial conversations with this PI, it was decided that I would record usage of platforms and then on a monthly basis devise histograms in Excel,


Document Ref:

2016/01

Page 43


enumerating total usage by machine, but also provide pie charts as well breaking down usage according to individual users. Every six months I have taken to pooling this data nd providing a longer term picture of usage versus groups: Moving forward, this should assist with building a legitimate case for other PIs contributing to annual service contract expenditure 7) With reference to risk management and resources, with the advent of our smarter purchasing system, which requires attachment of COSHH forms to chemical orders, I took that as an opportunity to ensure all requisite COSHH forms are present on line and as hard copies in the lab and encourage end users to clarify disposal, PPE and handling hazard issues in particular regarding new hazardous chemicals with me Stock taking and ordering has been an integral responsibility in multiple positions, going back to my first post in Manchester (1992). Furthermore, in terms of financial management, the core DNA sequencing facility at the University of Manchester (circa 1995-2000) was expected to be financially solvent, including provision of my salary. Accordingly, stock taking and ordering and auditing of accounts was paramount in this position in particular


Document Ref:

2016/01

Page 44


D3: Take responsibility for continuous performance improvement at both a personal level and in a wider organisational context

Your examples should indicate what actions you take to make improvements to your personal performance and to your organisation as a whole. This could be through encouraging the continuous development of junior staff or through improvements to processes within the organisation. Examples could include but are not limited to: • Identification of lessons learned from activities undertaken by yourself or by others for whom you are responsible, such as what went well, went badly or was lacking • Evaluation of the performance of specialists methods and tools used • Development of recommendations for future enhancements or modifications to procedures or working practices in order to achieve performance improvements Description of examples where your actions have led to performance improvement by

In this section you should describe why, what and how you take responsibility for continuous performance improvement at both a personal level and in a wider organisational context.to fulfil this competency In terms of fulfilling this competency my remit of engaging in personal research projects, training other personnel with reference to particular techniques and procedural practices and also managing the wider organisation of a large multi user/multi PI molecular genomics lab lends itself to a number of scenarios pertaining to performance improvement: 1) In terms of wider structural/organisation improvements I have just made reference to re organisation of personal and communal space in freezers in particular culminating in more efficient and accountable practice 2) As also mentioned previously (in another competence) a concomitant re organisation of the cloning/Micro biology laboratory was also implemented by me: This resulted in appropriate disposal of LB agar plates, a reduction in the incidence of cross contamination and better organisation of utilised communal stock 3) New practices were/are inculcated with the introduction of an induction by me for users’ of Microbiology and continued improved practice is ensured by regular monitoring of Micro biology and pointing out a departure from GLP in our monthly lab meetings and ad hoc group e mails, in line with inculcation of continued good practice in the large multi user laboratory I also manage Apart from communal resources in the form of lab reagents and space I am also responsible for up keep and optimal


Document Ref:

2016/01

Page 45


performance of key pieces of equipment in the laboratory. I have accordingly time tabled and declared on maintenance sheets next to said equipment any maintenance required by such equipment; in particular: • • • •

Acid cleaning of the pH electrode on a monthly basis; Acid cleaning of the pedestals on the Nanodrop; Weekly wiping of data on board the instrument Console linked to the viia 7 real time qPCR machine (to prevent memory saturation and cessation of running) Yearly calibration of the viia 7 ensuring optimal S;N from output data is sustained

Regarding one piece of equipment, I both personally use in my project work, but also manage on behalf of other end users’, viz. the Viia 7 Real time qPCR machine over the last 18 months in particular we have experienced a series of performance problems and outright cessation of running. This has required communication with laboratory staff to clarify the specifics of problems, e.g. Arrested runs owing to temperature sensor detector issues & faulty communication between the instrument console and PC linked running software inter alia and then communicating these issues to the engineer. Furthermore, subsequent to resolution of these problems, suggestions have been made by the engineer regarding better operational practices, which have then been incorporated into user SOPs by myself and inculcated to new and existing users’ via ‘group’ e mails and pertinent conversations, making reference to the updated SOPs. In addition, maintenance procedures such as self tests and calibrations are now timetabled and recorded on laminated sheets for perusal by


Document Ref:

2016/01

Page 46


myself, engineers and other end users. Further, I encourage users to record ‘error messages’ in a user log, inform me and take snap shots of the actual messages which are then archived into a desk top folder set up by me as a first aid tool for the engineer. As a result this systematic recording of problems, I have been able to collate a troubleshooting/diagnostic case and pass on to the engineer preceding any necessary visit. This has assisted the engineer in diagnosing the fault and expedited resolution of the problem Similar performance issues were identified with our bank of standard PCR machines cf. G Storms : On one particular occasion, it appeared that digital cycle programs were behaving erratically and LED displays were aberrant. This problem persisted, despite deleting and then re introducing such programs, implying the problem was not program corruption but rather faulty software/hardware linked to the PCR machine itself. Having had the machines investigated by the engineer, it became evident that the primary fault was asynchrony between cycle times in programs and the on board clock. This particular asynchrony, as it happens, can be diagnosed and corrected by running a series of ‘cycle tests’. In consequence, I learned how to run such tests from the service engineer and duly do as part of my maintenance schedule Finally, when I arrived provision of recycling for empty tip boxes was irregular and as it turned out provided by a company that we had since ceased to buy tips from so in effect the sporadic collection service they offered constituted a favour to our department!! I thus based on price and recycling provision transitioned this service to a new company and


Document Ref:

2016/01

Page 47


monitored collection of recycled tips bags outsourced by this company to a courier. Through these personal efforts initiated and carried out by me of my own volition we now experience a regular and reliable pick up of tip recycling bags In terms of improving my own practice regarding all aspects of my multifarious job description already described In view of my numerous and diverse responsibilities I am more fastidious about how I structure my day and how I keep all affected parties in the loop concerning scheduled activities and my whereabouts at all times A concomitant of ordering is product evaluation: Recently I have evaluated a new Taq from Appleton Woods for my genotyping PCR and the wider lab: I discovered that it isn't just cheaper but actually more sensitive. Indeed these evaluations were so convincing that Appleton Woods have placed the data on their web site and in promotion flyers for this product !! Improvement performances both with reference to equipment and bench based methods have been practiced and inculcated by me since 1992: Specifically, from 1992 to 1995 I was expected to maintain equipment as well as personally perform bench work and from 1992 to 2005 I was responsible in both UK and US positions for maintaining and improving the performance of multiple complex DNA sequencing platforms (ABI 377; ABI 3700 and Amersham Mega base 1000 instruments). Regarding improved performance yielded by method development that was an aspect of my aforementioned DNA sequencing positions in particular, culminating in contributions to books & co authorship on multiple posters and peer reviewed publications


Document Ref:

2016/01

Page 48


E: Professionalism Competencies E1: Demonstrate understanding and compliance with relevant codes of conduct

Guidance

Evidence Chosen

You should describe how the codes of conduct under which you practice relate to the work that you carry out and give examples of how they govern your professional practice. Within this, you should include any ethical considerations, both in terms of scientific and business practices. Examples you may wish to use may relate to: • Standards of professional practice in respect of your profession, employer, clients or patients • Standards of professional behaviour in respect of attitudes, respect and confidentiality • Standards of professional competence in respect of personal development and the development of others


In this section you should describe why, what and how you demonstrate understanding and compliance with relevant codes of conduct.to fulfil this competency In my current role I deal with a various constituent groups. When dealing with all such groups I am polite and courteous. Another dimension to my work place is the international nature of staff, particularly lab based staff, e.g. we have a number of Iranian, Iraq and Greek students in particular. One consequence of this multicultural element is being mindful of communications to ensure that practices and policies are understood. For example, when I send out group e mails regarding GLP (or more particularly transgression thereof) I tend to keep my language as simple as I can without diluting content. This tends to be true of personal spoken communications as well. With reference to one particular Iraq Ph. D student I played a part in training it became quickly obvious to me that whilst his spoken English was lacking his proficiency with the written word was better developed. Accordingly, when explaining a particular procedure, e.g. primer design, I would talk to him in conjunction with a written SOP which we discussed, but which he could read from during our dialogue. Moreover, I sent him papers pertaining to primer design; gene mining in Gen Bank etc. so he could peruse and reflect on this material in his own time and then get back to me with questions. In dealing with different ethnic groups/ Nationalities over the duration of my career I have come to realise that certain Asian groups in particular are not always

Document Ref:

2016/01

Page 49


inclined to speak up and say when they do not understand something. Accordingly, it is more efficacious and polite to clarify a procedure and then let them go away and ‘fill in the gaps’. In addition, I have found that my habit of training new students in general to comply with GLP particularly benefits foreign PhD students as demonstration of a technique, e.g. extraction of RNA then makes sense whereas prose might not Finally, to be mindful of and sensitive to cultural differences and accordingly best practice, we are obliged in our environment and working for a public body to submit to on line learning and self tests, in line with University wide prescribed

‘Equality and diversity’


Document Ref:

2016/01

Page 50


E2: Demonstrate a commitment to professional development through continuing advancement of own knowledge, understanding and competence

Your answer should provide specific examples of what you have already done in terms of continuing professional development (CPD) and your plans for the coming year. In your examples you should describe how your engagement in CPD has benefited your practice and the users of your work. Examples can be taken from any of the five categories of activity (work based learning, professional activity, formal/educational, selfdirected learning and other) defined and exemplified at: http://istonline.org.uk/wpcontent/uploads/2016/01/CSci-categoriesCPD-learning-activities.pdf

In this section you should describe why, what and how you demonstrate a commitment to professional development through continuing advancement of own knowledge, understanding and competence.to fulfil this competency An explanation of this competence is given in conjunction with the CPD form submitted for CSci and RSci status provided by IST when applying for professional registration directly through the IST Accordingly and as enunciated on that form my key CPD objectives are as follows: • • • • • • • •


Training courses: Both formal written courses and e learning format Elected membership of professional bodies Provision of technical training to others: Whether in person or on line (e.g. Research Gate community) Personal technical training linked to participation in lab project work Devising/optimising particular techniques, including formulation of new standard operating procedures (SOPs) Steering the course of project work based on data analysis, troubleshooting and networking with internal and external Technical specialist Attendance at external workshops, e.g. the Technicians summit Outreach activities, viz. Providing scientific tours to in house (non scientific) admin staff; External admin/volunteers linked to the BHF; Formal lectures on

Document Ref:

2016/01

Page 51


•

particular scientific themes provided to non scientific staff associated with the BHF Appraising papers as a review editor of specific Journals

With reference to actual competencies, these are specified in the IST prescribed PPD form below. I should add that this information has been transposed from an in house cardiovascular CPD form which is composed and then appraised by myself and line manager with goals achieved and future goals agreed upon: Activity 1 Higher Education Technicians Summit 2017 (HETS 2017) Points awarded1:

3

Category2:

2,3,4

Brief Description of activity: The main value of this meeting was to emphasise the importance of professional registration with and membership of external accrediting bodies as validation of CPD. In my case as a result I have become an elected member of RSB (MRSB) and also the Institute of Science & Technology (MIScT); A ‘Registered Practitioner’ with the IST & also a licentiate Value obtained Learning outcomes: Familiarisation with Registration and professional Society membership(s) Skills acquired: Registration procedure How it has benefited the quality of my practice:. As mentioned I have been elected to various professional Societies


Document Ref:

2016/01

Page 52


How it has benefited the users of my work: Reflections: This meeting was the prime motivator in enabling me to apply and successfully join a number of professional scientific outfits Activity 2 Awarded Membership of the Institute of Science & Technology & Registration as a ‘Practitioner (MIScT(Reg) Title: Points awarded1:

3

Category2:

2,3,4

Society membership Brief Description of activity: Gaining Elected membership of the IST acknowledges skills, experience, certifications and publications. Becoming elected as a ‘Registered practitioner’ reflects that my PPD aspirations are in line with IST standards. It was obtained by an extensive written application which obliges me to reflect on and codify my current job remit Value obtained Learning outcomes: Professional Qualification MIScT (Reg) and society membership Skills acquired: Ratification of current skills How it has benefited the quality of my practice:. How it has benefited the users of my work:


Document Ref:

2016/01

Page 53


Reflections: This qualification will motivate me to keep my CPD up to date and in doing so think carefully about structure work based responsibilities

Activity 3 Awarded membership of the Royal Society of Biology (MRSB) Title: Points awarded1: 3 Category2: 2,3,4 Brief Description of activity: Gaining Elected membership of the RSB acknowledges skills, experience, certifications and publications. It was obtained by an extensive written application which obliges me to reflect on and codify my current job remit Value obtained Learning outcomes: Professional qualification obtained together with Professional Society Membership Skills acquired: Ratification of current skills How it has benefited the quality of my practice:. How it has benefited the users of my work: Reflections: This qualification will motivate me to keep my CPD up to date and in doing so think carefully about structure work based responsibilities


Document Ref:

2016/01

Page 54


Activity 4 Elected membership of the Institute of Biomedical Sciences

(LIBMS)

Points awarded1:

3

Category2:

2,3,4

Brief Description of activity: Gaining Elected membership of the IBMS acknowledges skills, experience, certifications and publications. It was obtained by an extensive written application which obliges me to reflect on and codify my current job remit Value obtained Learning outcomes: Professional qualification and membership of a professional Scientific Society Skills acquired: Ratification of existing skills How it has benefited the quality of my practice:. How it has benefited the users of my work: Reflections: This qualification will motivate me to keep my CPD up to date and in doing so think carefully about structure work based responsibilities Activity 5 (Re) Learning ImmunohistoChemistry Title: Scientific Training


Document Ref:

Points awarded1:

2016/01

Page 55


Scientific Training 1,3,4

Points awarded1:

3

Category2:

Brief Description of activity: In house training with regard to practice of immunohistochemistry. This included devising a new protocol for a specific FFPE materials in conjunction with one primary antibody Value obtained Learning outcomes: This training enabled me to process an experimental sample cohort with proficiency and efficiency. Furthermore, the standard training then allowed me to improvise on the standard protocol to obtain scorable data for otherwise refractory samples/antigens. A specific SOP was then devised by myself Skills acquired: IHC How it has benefited the quality of my practice:. This has enabled me to perform competently and independently IHC on experimental tissue cohorts How it has benefited the users of my work: This training has allowed me to successfully complete IHC and thus scoring of biomarkers in tissue sections; This satisfies project milestones and thus benefits the PI/grant holder Reflections: This training led to productive scoring of an experimental tissue cohort contributing to successful completion of a cardiovascular experimental study


Document Ref:

2016/01

Page 56


Activity 6 Training in a primary tissue culture in vitro assay Category2: Scientific Training 1,3,4

Points awarded1:

3

Category2:

Brief Description of activity: In house training with regard to handling primary cells lines and establishing an in vitro model of tumour metastasis and its modulation by Platelets Value obtained Learning outcomes: This training has enabled me to begin to set up an in vitro assay modelling the metastasis of tumour lines from vasculature. In addition to practical training the process of standardising the protocol entailed visiting publications in this area and also networking & corresponding with pertinent experts via Research Gate Skills acquired: In vitro cell modelling/Tissue culture How it has benefited the quality of my practice:. This training has allowed me to successfully optimise an In vitro model of tumour transmigration How it has benefited the users of my work:


Document Ref:

2016/01

Page 57


The setup of this assay will enable screening of the effects of platelets exosome substances on tumour migration Reflections: Training expedited my ability to hit the ground run and optimise the basic in vitro model Activity 7 Scientific Training Title: Technical instruction/Project supervision Scientific Training 1,3,4

Points awarded1:

Points awarded1: 3

Category2:

Brief Description of activity: Provided principles, practice and bench supervision to a Ph. D student engaged in gene expression analysis by RT PCR and qPCR Value obtained Learning outcomes: Ph. D student under my instruction was enabled to independently and successfully perform these gene expression assays to evaluate candidate genes Skills acquired: Supervisory/Training skills How it has benefited the quality of my practice:.Reinforced my training and supervision skills How it has benefited the users of my work: The aforementioned Ph. D student can independently evaluate expression of candidate genes, contributing to her thesis


Document Ref:

2016/01

Page 58


Reflections: Productive and enabling training that conferred new skills to a student and reinforced my technical training and supervisory acumen Activity 8 Training in mining data bases & designing primers to quantify isoform specific gene expression Date undertaken: June-Present Title: Scientific Training Points awarded1: 1,3,4 Brief Description of activity:

3

Category2:

This training devised by me and based on an extended SOP I wrote for a number of Ph. D students was designed to enable them to independently mine transcripts from Genomic databases, e.g. EBI/Ensembl and then design primers to assay such genes by qPCR. I was responsible for devising all aspects of training from mining transcriptomic databases to analysis of gene expression data by qPCR

Value obtained Learning outcomes: This training reinforced my communication skills, both verbally and written and in addition provided an opportunity to update some of my own genomics based skills in the process of


Document Ref:

2016/01

Page 59


putting together training material(s). In addition, it contributed to Student GLP and facilitated productivity Skills acquired: Technical training/devising SOPs How it has benefited the quality of my practice: Reinforced my training and technical writing skills. How it has benefited the users of my work: Imparted technical methods that enabled them to investigate gene expression by real time qPCR Reflections: A Mutually beneficial training activity Activity 9 Scientific tours provided to in house admin personnel and external non scientific BHF personnel Date undertaken: Ongoing Title: Outreach tours Points awarded1: 3 Category2: 1,2,3,5 Brief Description of activity: This ‘outreach’ type activity is designed to familiarise both internal staff not engaging with research and also BHF personnel an over view of what actually goes on in a fully functioning genomics laboratory dedicated to cardiovascular research: Thus far this has involved informal tours of small parties with discussion & in the fall will also involve a formal lecture by myself (inter alia) to BHF personnel (~120 persons) on a particular specialist topic (‘measurement of Gene expression’)


Document Ref:

2016/01

Page 60


Value obtained Learning outcomes: These tours and associated material(s) allow me to practice and refine communicating key concepts in prosaic terms suitable for an educated lay audience. Skills engendered include finding and preparing relevant materials; preparation and structure on delivering of such materials and organisation of ‘key aids’ in the laboratory Skills acquired: Scientific communication How it has benefited the quality of my practice: These outreach activities provide niche opportunities to practice communicating complex scientific activity in lay persons language How it has benefited the users of my work: These tours have been received with enthusiasm and familiarise both in house non scientific personnel and similar persons linked to external funding bodies (BHF) what goes on in a modern laboratory and how the money gets spent !! Reflections: A Mutually beneficial training activity Activity 10 Presentation to departmental staff at away day Date undertaken: May 2017 Title:


Document Ref:

2016/01

Page 61


LecturePoints awarded1: 3 Brief Description of activity:

Category2:

1,2,3,5

Formal presentation to all staff within the department at an external ‘away day’ concerning my job description, responsibilities & how it illustrates the job family I am part of (‘Non Faculty support staff’) Value obtained Learning outcomes: This activity was designed to illustrate a particular job family within our school in the context of other job families: It involved a lecture to about 150 members of the department. Preparation & structuring of materials (‘Powerpoint’) and consideration of the level at which to pitch this presentation (vis-à-vis scientific and admin staff) were key attributes of this activity Skills acquired: Outreach communication skills How it has benefited the quality of my practice: Reinforced my presentation skills How it has benefited the users of my work: This talk hopefully familiarised CVS staff with what a typical core research technician does with their time!! Reflections: An enjoyable and worthwhile endeavour for myself as speaker and my captive audience


Document Ref:

2016/01

Page 62


Guidance notes for Applicants to the CSci scheme Competencies Form This form must demonstrate that you fulfil the competencies required for the scheme applied for. You must use specific examples to illustrate how you have fulfilled the competencies, simply stating that you fulfil the competencies is not enough. The examples must contain enough detail to allow the assessor to understand why, what and how you undertook the activity. The examples you use should paint a picture of what you undertook. Examples may be used to demonstrate more than one competency if relevant. The forms include illustrative examples of what you may have done to fulfil the competencies, you do not have to fulfil all the illustrative example evidence listed on the form. You should try to use examples of activities that you have undertaken recently, ideally any activity used as an example should have been undertaken within the last 2-3 years.


Document Ref:

2016/01

Page 63

RECORD OF PROFESSIONAL AND PERSONAL DEVELOPMENT PLEASE READ THE GUIDANCE NOTES AT THE END OF THIS FORM Name:

Laurence Dawkins-Hall

Level of registration: C.Sci

Workplace: BHF Cardiovascular Research Centre, Cardiovascular Sciences, University of Leicester, Glenfield Hospital, Groby Road, LE3 9QP Job title: Core Research Technician E-mail: [email protected] Renewal due date: August 2018 Aims and plan for PPD over the next 12 months

The aim of this PPD is to record and ensure that certain professional development pre requisites are satisfied over a 2 year period In particular, the PPD in my case will seek to record, comply with and validate activities undertaken in the following areas: • • • • • • • • •

Training courses: Both formal written courses and e learning format Elected membership of professional bodies Provision of technical training to others: Whether in person or on line (e.g. Research Gate community) Personal technical training linked to participation in lab project work Devising/optimising particular techniques, including formulation of new standard operating procedures (SOPs) Steering the course of project work based on data analysis, troubleshooting and networking with internal and external Technical specialist Attendance at external workshops, e.g. the Technicians summit Outreach activities, viz. Providing scientific tours to in house (non scientific) admin staff; External admin/volunteers linked to the BHF; Formal lectures on particular scientific themes provided to non scientific staff associated with the BHF Appraising papers as a review editor of specific Journals

Please return the completed form to: [email protected]

Professional biography (please give us a short description of your professional career – a mini CV)

I have an HCPC accredited degree in Bio medical sciences and commenced my research career in 1987. I have about 15 years experience of molecular & cell biology and biochemistry and 10 years experience of genetics and genomics (plus transcriptomics), including 2 years in high through put genomics laboratories in the USA. I also have 5 years experience pertaining to drug discovery. Much of this time has been associated with infectious disease, genomic and cardiovascular environments. This has resulted in more than 40 publications in Peer reviewed papers; co authorship of a text book (chapter), EBI/ GenBank submissions (#L33779) and conference abstracts. I am a fully Elected of the Royal Society Biology (MRSB) and The Institute of Science & Technology (MIScT). I am an elected member of the Biochemical (FASEB) Society. I am a licentiate member of the Institute of Biomedical Sciences (IBMS). I am a review editor for 'Frontiers in Infectious diseases' & 'Frontiers in Microbiology' Consequently I have the standard panoply of molecular techniques . & gene expression skills including protein expression & purification, IHC P, immunofluorescence, ELISA, (35S) in situ hybridisation, RT PCR, real time qRT PCR & high throughput Affymetrix Microarray biochips. I have also been responsible for the development and optimisation of a high through put drug discovery assay I possess the standard panoply of TC skills; I have also generated primary cell lines from human tissue explants (thymus) as well as worked in specialist TC (Cat #3) facilities (for example) and maintained a Cat #2 TC facility, including cell line testing for Mycoplasma. I have set up and maintained organisation in numerous labs as well as routine equipment maintenance, stock taking and ordering and aspects of health and Safety including COSHH: I am formally qualified as a (BTI) Biosafety practitioner (SCQF Level #11) to discharge health and safety responsibilities. I also have extensive experience of training, supervising, mentoring, formally lecturing and organising practical workshops for both undergraduate students and postgraduate students/staff. In addition, I have managed projects, including full time supervision of other lab based staff. I have provided scientific tours & lectures on key genetic assays/lab operations for external visitors, e.g. British Heart Foundation (BHF) staff I have presented internal seminars, talks and posters at both national & international meetings; In addition I have presented at Industrial workshops (Anachem) & both collaborated and published industrial partners, e.g. Appleton Woods & Perkin Elmer (ABI). I have co authored & contributed to

Please return the completed form to: [email protected]

the writing of numerous (peer reviewed) articles, text book material & also contributed figures to (other) textbooks. This has resulted in ~400 citations including 4 non patent citations. I have participated in projects with international media coverage e.g. BBC world service; both BBC & channel 4 television, African news outlets, & US media.

Example of completed PPD record Example activity

Please complete all sections

Date undertaken: Title: Carriage of Points 3 Category2: 2,3,4 th 1 6 Jan 2016 (5 hr Dangerous Goods by awarded : course) Air Course Brief Description of activity: A course to get certification to be able to sign off packages for carriage by courier that contain dangerous substances Value obtained Learning outcomes: Ability to know how to assess and advise on how to label and package, packages for carriage by courier that contain dangerous substances. Skills acquired: Knowledge of how to use the relevant guides and other resources to identify and categorise different dangerous substances How it has benefited the quality of my practice: I now understand and am qualified to sign off packages that are to be couriered, which contain dangerous substances. Previously I was unable to do this and relied on persons in other departments to me to do this. This often meant waiting on them, and time wasted chasing them. Particularly frustrating when the packages had perishable goods in them. How it has benefited the users of my work: Less time between requesting packages getting approval for courier pick up and actual courier pick up. This means valuable perishable items are less likely to be damaged. It also means that we are complying with the law and will not have incorrectly packaged and labelled boxes returned to us when they fail to pass through customs etc.. Basically they get a better service with faster turn around and less returns. Reflections: Having done this course I believe that I am now the bottle neck for couriering packages. It is certainly more efficient than previously but the department would benefit from having more people certified, in different locations, so people don’t have to chase me down and so that there are alternatives when I am on leave/unavailable. To this end I am going to recommend to my line manager that we get some in house training for a cohort of technical staff. What is also clear is that when I am dealing with this I am having to tell people to go back and change the way in which they have labelled and packed their substances. I need to provide some written/online guidance for them to make this more efficient. 1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities

PLEASE READ THE ‘Guidance for the allocation of PPD points and Categories of Learning Activities’ BEFORE COMPLETING THIS FORM Activity 1 Higher Education Technicians Summit 2017 (HETS 2017) Date undertaken: Title: CPD Course Points 3 Category2: 2,3,4 1 awarded : May 17 Brief Description of activity: The main value of this meeting was to emphasise the importance of professional registration with and membership of external accrediting bodies as validation of CPD. In my case as a result I have become an elected member of RSB (MRSB) and also the Institute of Science & Technology (MIScT); A ‘registered Practitioner’ with the IST & also a licentiate Value obtained Learning outcomes: Familiarisation with Registration and professional Society membership(s) Skills acquired: Registration procedure How it has benefited the quality of my practice:. As mentioned I have been elected to various professional Societies How it has benefited the users of my work: Reflections: This meeting was the prime motivator in enabling me to apply and successfully join a number of professional scientific outfits 1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities Activity 2 Awarded Membership of the Institute of Science & Technology & Registration as a ‘Practitioner (MIScT(Reg) Date undertaken: Title: Points 3 Category2: 2,3,4 1 awarded : July 2017 Society membership Brief Description of activity: Gaining Elected membership of the IST acknowledges skills, experience, certifications and publications. Becoming elected as a ‘Registered practitioner’ reflects that my PPD aspirations are in line with IST standards. It was obtained by an extensive written application which obliges me to reflect on and codify my current job remit Value obtained Learning outcomes: Professional Qualification MIScT (Reg) and society membership Skills acquired: Ratification of current skills How it has benefited the quality of my practice:. How it has benefited the users of my work:

Reflections: This qualification will motivate me to keep my CPD up to date and in doing so think carefully about structure work based responsibilities 1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities

Activity 3 Awarded membership of the Royal Society of Biology (MRSB) Date undertaken: Title: Points 3 Category2: 2,3,4 1 awarded : July 2017 Society membership Brief Description of activity: Gaining Elected membership of the RSB acknowledges skills, experience, certifications and publications. It was obtained by an extensive written application which obliges me to reflect on and codify my current job remit Value obtained Learning outcomes: Professional qualification obtained together with Professional Society Membership Skills acquired: Ratification of current skills How it has benefited the quality of my practice:. How it has benefited the users of my work: Reflections: This qualification will motivate me to keep my CPD up to date and in doing so think carefully about structure work based responsibilities

1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities Activity 4 Elected membership of the Institute of Biomedical Sciences (LIBMS) Date undertaken: Title: Points 3 Category2: 2,3,4 1 awarded : July 2017 Society Applications Brief Description of activity: Gaining Elected membership of the IBMS acknowledges skills, experience, certifications and publications. It was obtained by an extensive written application which obliges me to reflect on and codify my current job remit Value obtained Learning outcomes: Professional qualification and membership of a professional Scientific Society Skills acquired: Ratification of existing skills

How it has benefited the quality of my practice:. How it has benefited the users of my work: Reflections: This qualification will motivate me to keep my CPD up to date and in doing so think carefully about structure work based responsibilities

1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities Activity 5 (Re) Learning ImmunohistoChemistry Date undertaken: Title: Points 3 Category2: 1,3,4 1 awarded : April-June 2017 Scientific Training Brief Description of activity: In house training with regard to practice of immunohistochemistry. This included devising a new protocol for a specific FFPE materials in conjunction with one primary antibody Value obtained Learning outcomes: This training enabled me to process an experimental sample cohort with proficiency and efficiency. Furthermore, the standard training then allowed me to improvise on the standard protocol to obtain scorable data for otherwise refractory samples/antigens. A specific SOP was then devised by myself Skills acquired: IHC How it has benefited the quality of my practice:. This has enabled me to perform competently and independently IHC on experimental tissue cohorts How it has benefited the users of my work: This training has allowed me to successfully complete IHC and thus scoring of biomarkers in tissue sections; This satisfies project milestones and thus benefits the PI/grant holder Reflections: This training led to productive scoring of an experimental tissue cohort contributing to successful completion of a cardiovascular experimental study 1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities Activity 6 Training in a primary tissue culture in vitro assay

Date undertaken: Title: June-Present Scientific Training Brief Description of activity:

Points awarded1:

3

Category2:

1,3,4

In house training with regard to handling primary cells lines and establishing an in vitro model of tumour metastasis and its modulation by Platelets Value obtained Learning outcomes: This training has enabled me to begin to set up an in vitro assay modelling the metastasis of tumour lines from vasculature. In addition to practical training the process of standardising the protocol entailed visiting publications in this area and also networking & corresponding with pertinent experts via Research Gate Skills acquired: In vitro cell modelling/Tissue culture How it has benefited the quality of my practice:. This training has allowed me to successfully optimise an In vitro model of tumour transmigration How it has benefited the users of my work: The setup of this assay will enable screening of the effects of platelets exosome substances on tumour migration Reflections: Training expedited my ability to hot the ground run and optimise the basic in vitro model 1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities Activity 7 Scientific Training Date undertaken: January-July 2016

Title: Technical instruction/Project supervision Brief Description of activity:

Points awarded1:

Category2:

1,2,4

3

Provided principles, practice and bench supervision to a Ph.D student engaged in gene expression analysis by RT PCR and qPCR Value obtained Learning outcomes: Ph.D student under my instruction was enabled to independently and successfully perform these gene expression assays to evaluate candidate genes Skills acquired: Supervisory/Training skills How it has benefited the quality of my practice:. Reinforced my training and supervision skills

How it has benefited the users of my work: The aforementioned Ph.D student can independently evaluate expression of candidate genes, contributing to her thesis Reflections: Productive and enabling training that conferred new skills to a student and reinforced my technical training and supervisory acumen Activity 8 Training in mining data bases & designing primers to quantify isoform specific gene expression Date undertaken: Title: June-Present Scientific Training Brief Description of activity:

Points awarded1:

3

Category2:

1,3,4

This training devised by me and based on an extended SOP I wrote for a number of Ph.D students was designed to enable them to independently mine transcripts from Genomic databases, e.g. EBI/Ensembl and then design primers to assay such genes by qPCR. I was responsible for devising all aspects of training from mining transcriptomic databases to analysis of gene expression data by qPCR

Value obtained Learning outcomes: This training reinforced my communication skills, both verbally and written and in addition provided an opportunity to update some of my own genomics based skills in the process of putting together training material(s). In addition, it contributed to Student GLP and facilitated productivity

Skills acquired: Technical training/devising SOPs How it has benefited the quality of my practice: Reinforced my training and technical writing skills. How it has benefited the users of my work: Imparted technical methods that ebaled them to investigate gene expression by real time qPCR Reflections: A Mutually beneficial training activity Activity 9 Scientific tours to non specialist staff: Internal admin staff and external BHF staff Date undertaken: Title: Points 3 Category2: 1,2,3,5 1 awarded : Ongoing Outreach tours Brief Description of activity: This ‘outreach’ type activity is designed to familiarise both internal staff not engaging with research and also BHF personnel an over view of what actually goes on in a fully functioning genomics laboratory dedicated to cardiovascular research: Thus far this has involved informal tours of small parties with discussion & in the fall will also involve a formal lecture by myself (inter alia) to BHF personnel (~120 persons) on a particular specialist topic (‘measurement of Gene expression’)

Value obtained Learning outcomes: These tours and associated material(s) allow me to practice and refine communicating key concepts in prosaic terms suitable for an educated lay audience. Skills engendered include finding and preparing relevant materials; preparation and structure on delivering of such materials and organisation of ‘key aids’ in the laboratory Skills acquired: Scientific communication How it has benefited the quality of my practice: These oureach activities provide niche opportunities to practice communicating complex scientific activity in lay persons language How it has benefited the users of my work: These tours have been received with enthusiasm and familiarise both in house non scientific personnel and similar persons linked to external funding bodies (BHF) what goes on in a modern laboratory and how the money gets spent !! Reflections: A Mutually beneficial training activity Activity 10 Presentation to departmental staff at away day Date undertaken: Title: May 2017 Lecture Brief Description of activity:

Points awarded1:

3

Category2:

1,2,3,5

Formal presentation to all staff within the department at an external ‘away day’ concerning my job description, responsibilities & how it illustrates the job family I am part of (‘Non Faculty support staff’)

Value obtained Learning outcomes: This activity was designed to illustrate a particular job family within our school in the context of other job families: It involved a lecture to about 150 members of the department. Preparation & structuring of materials (‘powerpoint’) and consideration of the level at which to pitch this presentation (vis-à-vis scientific and admin staff) were key attributes of this activity Skills acquired: Outreach communication skills How it has benefited the quality of my practice: Reinforced my presentation skills How it has benefited the users of my work: This talk hopefully familiarised CVS staff with what a typical core research technician does with their time !!

Reflections: An enjoyable and worthwhile endeavour for myself as speaker and my captive audience

1. A total of not less than 15 points required on a balanced portfolio of development activities for each year as described in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Tables 1, 2 and 3. 2. Development should include activities in at least 3 of the 5 categories listed in ‘Guidance for the allocation of PPD points and Categories of Learning Activities’, Table 4 ‘Examples of Learning Activities’, and any one type of activity should not comprise more than 40% of total activities If you have undertaken more than 7 activities, copy and paste the activity table to create more. Guidance for the Allocation of PPD points No more than 3 points can be given for any one activity. If the activity spans more than one day you can only award more than 3 points if you can clearly demonstrate that there were different learning outcomes and skills acquired on different days. Duration of activity

Work-related*

Non-work related

30 mins – 1hr

Up to 1 point (see table 1)

0 point

2 - 4 hrs

Up to 2 points (see table 2)

1 point

5 - 8 hrs

Up to 3 points (see table 3)

2 points

*must include a measure of the usefulness of the event Table 1 Work-related activity

30 mins - 1hr

Value to individual

Criteria

0

No significant benefit

1 point

Refreshing existing skills and knowledge of introduced new skills and/or concepts and their use

Table 2 Work-related activity 2 - 4 hrs

Value to individual 0

Criteria No significant benefit

1 point


2 points

Extremely useful for developing new skills for work activities and inspired development activity

Table 3 Work-related activity

5+ hrs

Value to individual

Criteria

0

No significant benefit

2 points


3 points

Extremely useful for developing new skills for work activities and inspired development activity

Table 4 Examples of development PPD should be a mixture of activities relevant to current or future practice and should include activities in at least three (exceptionally two) of the following categories (no one type of activity should be used for more than 40% of all activities):

1 2 3 4 5

Work based (e.g. acquiring new skills, refining existing skills, devising/delivering training programmes, writing articles/papers, reflective practice) Professional activity (e.g. involvement in a professional body, mentoring) Formal / Educational (e.g. attending conferences, obtaining qualifications) Self-directed learning (e.g. reading journals, reviewing books / articles, researching topics) Other (e.g. voluntary work, public service, non-work-related studies)

This is not an exhaustive list of the types of activities that can count as PPD activities. The best type of learning activity is one which allows the individual to interact with other professionals, but it is impossible to provide guidelines for all types of activities. Diversity is encouraged as, without it, PPD activities would be less effective. More details and examples of these types of development activity are available on the IST website.

Guidance notes for Applicants to the CSci, RSci and RSciTech schemes PPD form This form must demonstrate that you have undertaken Professional and Personal Development and contain enough detail to allow the assessor to understand what you have done. The form should demonstrate that: 1. You maintain a continuous, up-to-date and accurate record of your PPD activities; 2. Your PPD activities are a mixture of learning activities relevant to current or future practice; 3. You seek to ensure that your PPD has benefited the quality of your practice; 4. You seek to ensure that your PPD has benefited the users of your work (employee, customer, student etc); 5. You can present a written profile containing evidence of your PPD You must engage in PPD, this is an initial undertaking of all Registrants, and you should keep continuous, up-to-date and accurate records as you undertake PPD. You must record your activities in the accepted format defined by the IST.

SUMMARY

I have in Bio medical sciences (University of Bradford) and commenced my laboratory career in 1987. I have about 15 years experience of molecular/cell biology and biochemistry and 10 years experience of genetics and genomics (plus transcriptomics), including 2 years in high through put genomics laboratories in the USA. I also have 5 years experience pertaining to drug discovery. In that time I have worked in infectious disease, Cancer research and cardiovascular environments Consequently I have the standard panoply of molecular techniques & tissue culture skills. In addition I have skills pertaining to gene expression work, including protein expression & purification, immuno fluorescence, IHC, ELISA, in situ hybridisation, RT PCR & real time PCR. I also have applied/high through put molecular skills such as antibody panel screening using tissue microarrays, (BAC clone derived), shot gun sequencing & Affymetrix Gene expression Microarrays With reference to data analysis & IT I possess standard Microsoft office skills, including data graphing and use of formulas in Excel, e.g. ΔΔCt analysis; and power point. I am familiar with statistical packages, e.g. Graph Pad I have set up and maintained organisation in numerous labs as well as routine equipment maintenance, stock taking and ordering, financial auditing and aspects of health and Safety including COSHH: I am formally qualified as a (BTI) Biosafety practioner (SCQF Level #11) I have extensive experience of training, supervising, mentoring, formally lecturing and organising practical workshops for both undergraduate students and postgraduate students/staff. I have participated in outreach activities, including scientific tours and specialists lectures to volunteers and admin staff linked to the British Heart Foundation (BHF) I have presented at internal seminars & both national & international meetings; In addition I have presented at Industrial workshops (Anachem). I have coauthored in excess of 40 peer reviewed poster presentations & publications and also text book

materials, resulting in 300 Citations, including 4 non patent citations I have participated in projects with international media coverage e.g. BBC world service; both BBC & channel 4 television, Africa & US media. I am a review Editor (Infectious diseases) for 'Frontiers in Microbiology'

CURRENT POSITION: CORE RESEARCH TECHNICIAN BHF Cardiovascular Sciences Research Centre, Glenfield Hospital, University of Leicester [email protected] - +44 (0)116 204 4747

https://www.linkedin.com/in/laurence-dawkins-hall-a0856074/ https://www.indeed.com/r/121fa3f4d719943d?sp=0

Post Graduate Work Experience (2017 – 2002): February 2015 – Present (Permanent): Research Core technician, BHF Cardiovascular Research Centre, Cardiovascular Sciences, Glenfield Hospital, University of Leicester LE3 9QP. Manage the day by day running of a post graduate (BHF) cardiovascular research lab involved in characterising genetic biomarkers linked to cardiovascular disease. This involves: • Stock taking and smarter purchasing; financial auditing of consumables, including consumables budget management • Equipment maintenance and repair (including different automated platforms), including liasing with Medical technology representatives and engineers • Laboratory management, including assisting with setting up/improving and maintaining organization within molecular biology & microbiology laboratories; Waste stream procurement & management, including provision of recycling by external couriers; Planning and occasionally leading monthly lab management/housekeeping meetings • Enforcing good laboratory practice, including devising COSHH forms and training students in their ‘best practice’ & use • Linked to the above, liasing with medical sales and technical representatives, including product evaluation • Assisting students with & autonomous participation in lab based project work, including optimisation of procedures/Techniques e.g. IHC for evaluating gene expression in cardiovascular in vivo models, Cardiovascular ‘In Vitro’ modelling by tissue culture (investigation of platelet modulation of tumour metastasis & compound which inhibit this pehnomenon; Real time qPCR of candidate gene expression in clinical samples linked to longitudinal cardiovascular prognostic studies, in order to identify biomarkers and stratify patient samples & in separate studies identify compounds that modulate cardiotoxicity • Bench supervision, technical advice & technical instruction/training of Ph.D student(s) ad hoc, e.g. primer design using Primer Blast & BLAST; mapping primers to cDNA using Ref Seq derived from Ensembl/NCBI GenBank & training students in

RT PCR and real time qPCR; The latter includes aspects of data analysis such as gene expression evaluation (by algorithms such as ∆∆Ct/Livak analysis) • Devising standard operating procedures (SOPs) to standardise and implement ‘best practice’ • In addition, I provide scientific tours and formal lectures to external lay visitors, e.g. BHF personnel & similarly internal (non scientific) administrative staff, as part of my ‘outreach participation’ • Providing lectures in job responsibilities to all staff at departmental ‘away days’

April 2014 – February 2015: Research Technician (TS #2), Immunology, Life

Sciences, QMC, University of Nottingham, Nottingham NG7 2UH. Research Technician in a laboratory investigating macrophage ontogeny (M1/M2), antigen display & associated clinical pathologies in the context of simulated ex vivo (organotypic) models of skin & prosthetic inserts: • • • • • •

Set up and trained (Ph.D.) students in Gen bank transcript retrieval, targeted & optimal primer design and implementation and data analysis linked to RT PCR and qPCR assays (including ∆∆ Ct analysis) of pertinent (bio marker) transcripts Contributed to published manuscripts & conference posters Extract macrophages and monocytes from blood and quantify biomarker transcripts in RNA extracted from cytokine polarized ‘In vitro’ cultures cultures by qPCR Immuno fluorescent staining of biomarkers on cytokine polarized macrophages cultured ‘In vitro’ on (trans well) prosthetic surfaces and image enhancement using Adobe photoshop Set up & trained students in Western blotting to examine candidate protein levels derived from aforementioned model systems Stock taking, ordering (Agresso) and laboratory organization and GLP

August 2013 – April 2014 (Fixed term): Research Technician (TS #3), Veterinary Medicine & Science, Sutton Bonnington, University of Nottingham, Nottingham LE12 5RD.

Technical project manager on a Novartis funded drug discovery project, designed to identify compounds that inhibit Acanthamoeba Castellanii. Compounds identified from (small molecule) libraries (~1000) that inhibit encystation; Job Scope: • • • • • • •

Maintenance of In vitro parasite stocks (Category II tissue culture) In vitro interrogation of parasites with library compounds (cytotoxic assay) Adoption and adaption of existing protocols to high through put screening context Continued development & standardization of drug screening (counting based cytotoxic) assay Secondary screening of statistical lead compounds to substantiate efficacy (i.e. IC50) Communicating & co ordinating project logistics & results to lab based assistants and project partners (Novartis & Managed Chemical compound Facility, School of Pharmacy) Responsibility for generating, interpreting, troubleshooting & archiving data (Access)

• •

Statistical, QC (Z score; t analysis; 99% CI), & pharmacological (IC50) data analysis Training, mentoring & full time supervision of three other project personnel

October 2012 – August 2013 (fixed term): Research Technician (TS #3), Clinical Oncology, University of Nottingham, City Hospital, Nottingham NG5 1PB Characterization of key biomarkers associated tumour biochemistry/tissue re modeling, orchestrated by Cysteine proteases (Calpains), and potential consequences for pathogenesis, prognosis, metastasis and diagnosis of breast cancers: • • • • • • • • •

Screening panels of biomarkers against protein lysates derived from breast carcinoma tissue by Western blotting (including use of blocking peptides to verify specificity) Histo pathological characterization of markers by IHC using tissue micro arrays derived from biopsies of graded tumour specimens Serological characterization of biomarkers by ELISA In situ characterization of biomarker RNA (CISH; RNAscope®) In Vitro modeling of (carcinoma) cell motility, including pharmacological manipulation of Calpains and biochemical(fluorescent) analysis Mentoring and training students in particular techniques Supervising/instructing M.Sc. & B.Med.Sci. students’ Stock taking and ordering General housekeeping activities for (PI) research group

November 2008 – October 2012 (Fixed term): Research Technician/Lab manager (Band #5 to 6), Centre for Immunology, Infection & Evolution Research, University of Edinburgh, Edinburgh EH9 3JT. Drug discovery program (NIH/MRC/Wellcome funded) to identify antagonists of a Trypanosome respiratory complex associated with mitochondrial (Kinetoplast DNA) encoded proteins. In particular, small scale ‘proof of concept’ and large scale drug screen programs were implemented based on rational drug design to a key respiratory component of Trypanosome • • • • • • • • •

Development of a high throughput (FRET based) ligase assay for identifying (small compound) antagonists of an enzyme target (REL1) in T. Brucei: Optimized with respect to recombinant enzyme (rREL1) expression; dye labeled oligo design; enzyme activity; reaction chemistry; reaction kinetics (including Km); denaturation conditions; Enzyme stability (excipient considerations) & throughput Identification of ligase antagonists (IC50) using a preliminary radioactive assay for limited compound screens ‘In vivo’ determination of cytotoxicity & efficacy (EC50) using Alamar blue (Cat III parasite culture) Cloning, transformation, expression & purification (ACTA ® LC) of target enzyme Confirmation (Western blotting) & characterization (PAGE & FRET) of expression Real time PCR interrogation of key complex component transcripts Data analysis linked to above utilizing MS Excel; Graph Pad & Sigma plot In house progress reports & lab presentations

• • • •

Contribution to published manuscripts & conference posters Active communication/collaboration with partners in the UK & USA regarding assay set up and implementation of high through put drug screens Departmental seminars & external (first author) poster presentations Training and mentoring of hotel and resident staff (students & post docs)

Assisted with the laboratory set up; Oversaw stock taking and ordering (Syquest), liasing with medical technology representatives; assisted with equipment evaluation and purchase, including procuring quotations; and all aspects of health & safety, including COSHH, as a proxy for the PI. Formally qualified to discharge health & safety responsibilities as a (BTI) Biosafety Practioner Level #1 (foundation). November 2005 - November 2008 (Fixed Term): Research Assistant (Band #6), EBRC, Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG Genetic characterization (host parasite interaction) of Trypanosome infection in cattle; Functional genomics consortium: RNA extracted from African clinical samples; data base archived (Access); QC assayed (Agilent chip & Nanodrop); amplified and derived cRNA used to interrogate Affymetrix Micro array (3’ EST & exome) chips & Cy3/Cy5 (Amersham) spotted arrays. Preliminary QC metrics (GCOS, Expression Console and dChip) performed. Data disseminated by FTP. Transcripts interrogated by real time (RT) PCR. Contributed to departmental seminars & in house lab meetings. Participated in electro elution preparation & sequencing of BAC end library fragments for transgenic micro injection. Trained/mentored project students & contributed to equipment evaluation (Gel Documentation systems). Contributed reports to consortium news letter. Contributed to manuscripts, conference posters & text book materials October 2002 - October 2005 (Fixed term) Technician (Band #5), Cardiovascular sciences, University of Leicester, Glenfield hospital, Leicester LE3 9QP.

Genetic aspects contributing to hypertension investigated. Functional genomics approach utilizing normotensive & hypertensive congenic rats, which were mined for candidate genes (& associated causal SNPS's) mapping to defined QTL's: • • • • • • • •

Maintenance, troubleshooting & method development associated with AB 3700 sequencing Interrogation/validation of data base transcript models (Ensembl & NCBI GenBank) linked to genes within introgressed (QTL) regions by targeted Exome RT PCR & Sanger sequencing Contig builds (Sequencher 4.2) to identify (putative) causal SNP's Western blotting of candidate genes Contributed to published manuscripts & Conference posters Training and mentoring of staff in any and all of the above Full time supervision of a genomic technician (~ 6 months) Presentations at lab meetings and participation at external meetings

• •

Marker assisted breeding of transgenic mice based on tail chop DNA sample genotyping Contributed to miscellaneous housekeeping duties, viz. Laboratory organization, implementation of GLP, stock taking and ordering, equipment maintenance

Post Graduate Work Experience summary (1985 – 2002):

1) 2001 – 2002: Research Associate, Rubicon Genomics, 4370 Varsity Drive, Ann Arbor, Michigan, USA: Targeted genomic sequencing projects 2) 2000 – 2001: Research Assistant, Einstein Genome Center, AECOM, 1695 Poplar Street, New York 10461, USA: Shotgun genome sequencing of mouse libraries (public consortium participant) 3) 1995 – 2000: Senior Technician (TS #4))/Experimental Officer (TS #5)), School of Medicine, Manchester University, Oxford Road, Manchester M13 9PT. 4) 1992-1995: (Fixed term) Technician (TS #3)), School of Biological Sciences, Manchester University, Oxford Road, Manchester M13 9PT. 5) July 1992-Dec. 1992: (Fixed term) Technician (TS #2), Faculty of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham. 6) 1988-1992: Research Technician, Ludwig Institute for Cancer Research, London. 7) 1987-1988: Technician/Research Officer, Cancer Research UK, London. 8) 1985: Intercalated year, Immunology Division, BBSRC Institute of Animal Physiology, Babraham, Cambridge. http://orcid.org/0000-0002-6138-6715

Summarised laboratory skills DNA:

RNA:

Sequencing:

Cloning; DNA extraction (genomic, plasmid, BAC, PCR, ); gel electrophoresis (Agarose/Urea-Acrylamide); Dye labeled oligo PAGE (genotyping/ Genome scans); PCR; Electro elution; Transfection; Southern blotting; Primer design Extraction; RT PCR; Real time qPCR; Taq Man allelic discrimination (real time) PCR Northern blotting; In vitro transcription & In situ hybridization; CISH (RNAscope ®); Native PAGE; Exome & whole transcript Affymetrix Micro array chip analysis; RNA/cDNA amplification; Agilent Nanochip. Cross platform automated (Sanger) DNA sequencing (ABI; Licor; Amersham); Phred (Q20/40) analysis; Contig building (Sequencher); primer design/primer walking (Oligo 3.0); Gap filling/high throughput sequencing of shot gun cloned (BAC insert derived) PUC libraries. Cycle sequencing method development. Targeted EXOME sequencing

Protein Chemistry: Histo chemistry: Tissue culture:

Drug/Assay development

Protein extraction & quantitation (Bradford assay); Protein expression (E Coli); Coomassie SDS PAGE; Western blotting; Immuno precipitation; Affinity (AKTA® FPLC) purification; ELISA. Immuno histochemistry (FFPE); immune cyto chemistry. TMA staining Mycoplasma screening; Mammalian culture; primary culture; Cat II/III parasite culture; Viability assays (viz. Alamar blue/Trypan blue). Cytotoxic assays. Migration assays High throughput assay optimizations; radio ligand (mimetic) assay Enzyme kinetics, e.g. Km evaluation; IC50; EC50 (Graph Pad Prism® & Sigma Plot® & SPSS) & ‘Hit to lead’ compound library screens

Computing Skills 1.

General: Microsoft office (Word; Outlook; Power Point; Excel; Access); Adobe

Acrobat; Illustrator; Photoshop; Web building; Agresso; Syquest 2. Bioinformatics: ENSEMBL; NCBI; TrypDB; Sequencher; SeqScape; Vector NTI; Trace tuner (PHRED); Oligo 3.0; Primer 3.0; Oligo Calculator; Net primer; GeneScan & Gene Mapper 4.0; Chromas; Bioedit; SeqEd; ABI view; GCOS/Expression Console (Affymetrix); d Chip; Blue fuse; Freezerworks Data base 3. Statistics: Gen stat; SPSS; Graph Pad; Sigma Plot

Education B.Sc. Biomedical Sciences (2.2) Bradford University (1987): NVQ Level #5 City & Guilds, Information Technology & Computing (1990): NVQ Level #4 (Former) Personal animal project license holder (#60/12113) (BTI) Biosafety Practitioner Level #1 (foundation): SCQF Level #11. NVQ Level #4

Inter personnel/management skills Preparation, delivery and organization of sequencing course module for M. Res. students Formal lecturing of M.Sc. students relating to DNA technologies Technical straining of students and staff Project management & Bench supervision Financial auditing & resource allocation Participation in interview panels (UoE) Intellectual & data input into the design & content of published manuscripts Management of budgets including financial auditing

Outreach activities, including provision of lectures and scientific lab tours to both in house admin staff and external protagonists linked to the British Heart Foundation. Presentations to all staff at departmental ‘away days’

Achievements & Awards Speaker at ABI DNA sequencing core facility conference (Sanger Centre 1997) Invited speaker at an Anachem workshop (1997) Speaker at 2001 ABRF conference (DSRG 2001, in San Diego) Bonus re numeration for ‘exceptional contribution’ (2010) Promotion (Band #5 to band #6) (2011) Discretionary point above the standard (band #5) pay grade (2003) Band #5 to band #6 promotion at (1997) and then to ‘Experimental Officer (2000)

Society Member ship(s) Full member of the Royal Society of Biology (MRSB). Elected 1st July 2017 (#P0128699) Full member of The Institute of Science & Technology (MIScT). Elected 27th June 2017 (# T15859). ‘Registered Practitioner’ (13th July 2017) Full member of the Biochemical society. Elected 30th June 2017 (#01078615) Elected ‘Licentiate’ member (LIBMS) of the Institute of Biomedical Sciences (IBMS): July 2017 Former member of the ABRF DNA Sequencing Research group (DSRG) Licentiate membership (LIBMS) of the Institute of Biomedical Sciences (IBMS). Elected July 2017 (membership # = 412464)

References 1. Professor Sir Nilesh Samani BHF Chair of Cardiovascular Sciences Leicester NIHR Biomedical Research Unit Glenfield Hospital LE3 9QP Tel. 0116 204 4758 [email protected] Current Employer/Department Head http://www2.le.ac.uk/departments/cardiov ascular-sciences/people/samani

2. Professor Amir Ghaemmaghami Immunology, Life Sciences University of Nottingham (QMC) Nottingham NG7 2UH Tel. 0115 823 0730 mailto:[email protected] Former Employer/PI http://www.nottingham.ac.uk/lifesciences/people/amir.ghaemmaghami

3.Dr Achim Schnaufer Centre for Infection, Immunity & Evolution University of Edinburgh Edinburgh EH9 3JT Tel. 0131 650 5548 [email protected] Former Employer/PI http://schnauferlab.bio.ed.ac.uk/index.html

4.Professor Alan L. Archibald FRSE The Roslin Institute, R(D)SVS, University of Edinburgh Midlothian EH25 9RG Tel. +44(0)131 651 9249 [email protected] Former Employer/PI/ Department Head http://www.roslin.ed.ac.uk/alan-archibald/

Publications

https://independent.academia.edu/LaurenceHall1 http://www.researcherid.com/rid/L-8773-2016 https://www.mendeley.com/profiles/--------laurence--------hall/