Bioscience Horizons Advance Access originally published online on April 19, 2009
Bioscience Horizons 2009 2(2):191-196; doi:10.1093/biohorizons/hzp022
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Characterizing chloroplast sensor kinase
School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End road, London E1 4NS, UK
* Corresponding author: Tel: +44 207 882 7010. Email: i.ibrahim{at}qmul.ac.uk
Supervisor: John F. Allen, Queen Mary, University of London, School of Biological and Chemical Science, London, UK.
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In higher plants and green algae, photosynthesis takes place within specialized sub-cellular organelles called chloroplasts. Chloroplasts were once prokaryotes and evolved by endosymbiosis from cyanobacteria. They contain a semi-autonomous genetic system that encodes for core proteins of photosynthetic reaction centres in the energy-transducing membrane known as the chloroplast thylakoid. The photosynthetic apparatus in the thylakoid membrane makes use of excitation energy from sunlight to remove four electrons and protons from two water molecules. The electrons transfer them to the electron acceptor ferredoxin and NADP+, respectively. In this system, plastoquinone acts as a mobile electron and proton carrier between Photosystem I and Photosystem II in reduction–oxidation or ‘redox’ reactions. A balanced redox state in the chloroplast is important for efficient energy conversion. However, the slightest error could lead to photo-inactivation as well as DNA mutation. Therefore, photosynthetic enzymes that are involved in photosynthesis are tightly regulated. In this study we analyse the mechanism of redox regulation involved in chloroplast gene expression that requires chloroplast sensor kinase (CSK). CSK is a bacterial-like histidine kinase that functions as a two-component system. Such simple but effective signalling transduction is abundant in prokaryotes, but found less widely in eukaryotic cells. CSK is encoded by the nuclear genomes of all higher plants examined, and the CSK proteins are targeted to chloroplasts where they function as a redox sensor. Through the cloning process, the result expressed the full-length CSK and the putative sensor domain (GAF domain) into a pGEX-6P-2 plasmid containing a GST tag. The construction was over-expressed into Escherichia coli cells. From bioinformatics study, it was found that in higher plants CSK is a modified histidine kinase, whereas in diatoms and red algae it is a typical histidine kinase.
Key words: Arabidopsis, chloroplasts gene regulation, histidine kinase
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Introduction |
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Photosynthesis is the mechanism of conversion of light energy into useful chemical energy. This important biological chemistry takes place within the chloroplasts of plants and algae as well as in some bacteria.
Chloroplasts have descended from a photosynthetic cyanobacterium and have been living as sub-cellular organelles ever since. Over the course of evolution, almost all of the original, cyanobacteria genomes had translocated to the host cell nuclear genetic system. However, from the Synechocystis genome, only 46 protein-coding genes are retained by all chloroplast genomes and 44 out of these encode proteins involved in the electron transport chain (ETC).1, 2 The chloroplast contains the light capturing photosystems that are integrated within the energy-transducing membrane, the thylakoid.3
The light-induced charge separations in oxygenic photosynthetic organisms are carried out by the two photosystems (PS), PSII and PSI. The transfer of stimulating energy from light harvesting complex (LHC) to PSII facilitates oxidation of a water molecule. The electrons are transferred from water molecule to PSII cofactors then to NADP+ via PSI. In the ETC, a lipid-soluble cofactor known as plastoquinone (PQ) acts as an intermediate electron carrier between PSII and PSI. PQ has the ability to gain electrons and protons (reduction) and also has the ability to lose electrons and protons (oxidation). The gain and loss of electrons and protons by PQ are redox reactions. Therefore, the ratio of PQH2 to PQ is the measure of redox state of the PQ pool.1
Photosynthesis is optimal when the environment meets species-specific requirements. However, environmental conditions fluctuate on the time-scale of minutes to days creating unwanted redox imbalance.4 Imbalance of redox state created from uneven light distribution has effects on the photosystems. For example, reduced PQ pool promotes the electron to transfer back to PSII. This will result in production of free radical superoxide. In living cells, oxygen radicals are harmful for their high rate of reactivity whereas in the chloroplast, the reactive free radical causes photo-inactivation and DNA damage with mutation that will result in incorrect functioning of enzymes. The chloroplast regulates redox imbalance in several ways; in the short term, pH changes in the lumen activates a mechanism that allows LHC to dump about 80% of the solar energy to be absorbed as heat and fluorescence which decreases the excitation of reaction centre. However, this dramatically reduces the efficiency of photosynthesis.
In the second pathway, light state transition balances electron flow between PSII and PSI. In the event of high light intensity, the rate of electron flow from PSII is faster and therefore PSI is saturated. The imbalance of redox state of PQ pool activates a specific membrane bound enzyme, a protein kinase (PK). The activation of PK leads to phosphorylation of LHC leading to dissociation of LHC from PSII.5 This increases PSI's ability to capture more light energy; hence, the light distribution between PSII and PSI is balanced. All of the light adaptations shown above are short term; however, if the redox imbalance continues for hours to days, then redox regulation of gene expression will be activated. The chloroplast genome encodes for the core proteins of the photosynthetic machinery.
Redox regulation of the chloroplast genomes rather than nuclear genomes has an advantage for this photosynthetic machinery. First, the chloroplast can react to environmental stress with a faster rate if the regulatory apparatus controlling gene expressions are located in the chloroplast. Secondly, environmental factors, in this case light, will have a direct effect on gene regulation. The nuclear genome and regulatory factors that are required for gene transcription are further away from where photosynthetic redox reactions are taking place; therefore, sensitivity to environmental change (redox state of reaction centre) will be slow. Even after redox signals reach the nuclear genome, their action is general as there are many chloroplasts within the photosynthetic eukaryotic cells.
In particular, the psbA and psaAB genes appeared to be affected directly by the redox state of PQ pool.6 Under PQ reducing conditions, the electron flow is counter-balanced by increasing the transcription rate for the psaAB gene; this balances the ratio of PSII to PSI. Under PQ oxidizing conditions, psbA gene transcription is increased. In cyanobacteria, the ancestors of chloroplasts, a two-component signal transduction system regulates the redox imbalance created during photosynthesis.6 Such systems in prokaryotes are abundant and are the major signal transduction systems to regulate environmental responses. These systems are composed of a sensor histidine kinase and a response regulator protein.
Cyanobacteria and chloroplasts contain a similar mechanism of photosynthesis in which electrons flow from PSII where they are finally transferred to NADP+ via PSI. Similarly, in cyanobacteria, a redox imbalance of the PQ pool was shown to affect gene expression of the two reaction centres and therefore they serve as a model for understanding redox effects on gene expression in chloroplasts.
In the genomic sequence of Arabidopsis thaliana, Puthiyaveetil et al.7 identified a bacterial-type sensor kinase termed CSK. The gene encoding for CSK is located in the nuclear genome of all higher plants and algae. The CSK protein is synthesized in the cytoplasm and targeted to the chloroplast stroma. It is found in the higher plants modified as histidine kinase that could be autophosphorylated on a tyrosine residue.7
In this study, the full-length sequence of CSK and its putative GAF domain were cloned into an E. coli expression system using a GST-tagged pGEX-6P2 vector.
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The full-length CSK and its GAF domain were cloned and expressed in bacterial cells. Fig. 1 illustrates the results of cloning. In Fig. 1A, the DNA bands at
5000 bp are for the plasmids and DNA bands at 2000 bp are for CSK; except for clones 5–7 all the other clones in Fig. 1A contain CSK at the right DNA band size. In Fig 1B, DNA bands at 600 bp are for GAF clones. Clone 1 of CSK and clone 5 of GAF were expressed by inducing with IPTG. Fig. 2A shows protein expression for CSK–GST and Fig. 2B shows protein expression for GAF–GST proteins. In both figures, the major protein band for both clones were present in the insoluble cells fraction but not in the soluble cells fraction.
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Sequence analysis in the SMART database using the At1g67840 gene product of A. thaliana encoding the fulllength CSK (Fig. 3) identified two conserved histidine kinase domains. The HisKA domain is the dimerization domain. A dimerization domain in a histidine kinase is formed through parallel association of homodimers that creates a four-helix bundle. The second domain identified was the ATPase domain. This domain catalyses the transfer of
-phosphate to the phosphate acceptor residue located in the dimerization domain. Further analysis of HisKA domains using the SMART database has revealed that CSK in A. thaliana is a modified histidine kinase as a result of the conserved phosphor-acceptor residue of typical histidine kinases (the histidine residue) in CSK being replaced by a glutamate residue7 (Fig. 4). Further study is required to explore the consequences of this amino acid substitution.
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A homology search for CSK in CYANOBASE, the cyanobacteria genome database, has recovered several histidine kinases. The highest probability score (with an E value of 8e–17) was for Nostoc punctiforme ATCC 29133 proteins, the GAF sensor signal transduction histidine kinase.
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In this study, I have cloned the CSK involved in chloroplast gene regulation. CSK is a redox sensor protein, which belongs to a family of signal transduction proteins known as sensor histidine kinase. CSK may function in two-component systems.
Both CSK–GST and GAF–GST constructs were expressed successfully in BL21-(DE3)-codonplus-RP cells but the majority proteins were insoluble and found in the pellet. This property is unexpected as CSK and GAF are soluble stromal proteins. The GST-tag fused with the clone is also soluble. When two soluble proteins are fused together, they should remain soluble and be recovered in soluble cell fractions. There are many reasons why these proteins may occur in the insoluble fraction. When proteins are over-expressed in E. coli, there is less chance for correcting the folding within the viscous environment of the cytoplasm and therefore the majority of the protein may form insoluble aggregates.8
The CSK belongs to a large family of signal transduction proteins known as histidine kinases. They form the major class of prokaryotic two-component signalling system. Protein–protein BLAST (Basic Local Alignment and Search Tool) searches in Microbial Protein Database and in Cyanobase recovered several bacterial two-component proteins. The highest homology was for a cyanobacterium protein, the GAF sensor signal transduction histidine. CSK contains a typical histidine kinase dimerization domain and modified H-box.
In higher plants, CSK is modified to histidine kinase because in the H-box, the histidine residue is replaced by a glutamate residue. However, in red algae, Cyanidioschyzon merolae, in the diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, CSK is a typical histidine kinase that contains the conserved histidine residue within the H-box.7
The initial signal transduction cascade in sensor histidine kinases involves autophosphorylation on their conserved histidine residue to elicit the physiological response. The catalytic ability of CSK has been demonstrated by Puthiyaveetil et al.7 using an in vitro radioactively labelled [32-P]-ADP phosphorylation assay. The interesting point these authors demonstrated that CSK has lost the ability to be phosphorylated on a histidine residue; instead CSK could be phosphorylated on the tyrosine residue. Plant ethylene response receptor 2 (ERT2) is another example of modified histidine kinase that is phosphorylated on serine residue.9, 10
Other mammalian kinases such as alpha-ketoacid dehydrogenase kinase (BCKDHK) and pyruvate dehydrogenase possess a greater degree of sequential similarity with prokaryotic histidine kinase. However, they have not been seen to be phosphorylated on their conserved histidine residues; instead, the catalytic domains are known to be phosphorylated on serine residues.11
Both ERT2 and BCKDHK use the conserved glutamate residue located in the ‘N-box’ (nucleotide binding site in the HATPase domain) as a general base catalysis to activate transfer of -phosphate to serine residue.9, 11 Clearly, there is evolutionary movement of the histidine kinase phosphorylation site where the histidine residue is no longer required for autophosphorylation. In prokaryotic cells, histidine kinase autophosphorylates on its conserved histidine residues but in eukaryotic cells phosphohistidine was replaced by other more stable residues such as phospho-tyrosine or serine/threonine in plants and phosphoserine in humans. Histidine kinases form unstable phosphoramidates and when this bond is hydrolysed, large negative energy is released. Therefore, the unphosphorylated protein is more favoured and consequently the phosphate transferred from histidine to the aspartate residue of the response regulator is passive.12
In the model plant A. thaliana, the physiological role of CSK is to regulate the transcription of the psaA gene in response to the long-term redox imbalance of the PQ pool.7 In A. thaliana containing a mutant CSK gene, chloroplasts have lost the ability to regulate psaA gene transcription in a manner similar to the wild-type plants. The study by Puthiyaveetil et al. illustrated when the wild-type plants were exposed to a condition that favours PSII, after 26 h of exposure, the transcription of psaA increased by 11-folds.7 However, the mutant plants lacking CSK gene increased only by 5-folds. This is a 55% reduction when compared with wild-type plants. When the light condition favours PSI, in the wild-type plants the transcription of the psaA gene was decreased whereas in mutant plants it increased for the first 8 h.7
As proposed by Puthiyaveetil and Allen,13 PQ pool redox regulation of gene expression invokes a bacteria-like two-component system, where a redox sensor kinase senses the redox state of the PQ pool and activates/deactivates a response regulator up- or down-regulating chloroplast gene expression. One possible candidate response regulator could be TCP34 (Tetratricopeptide-Containing Chloroplast Protein of 34 kDa).14 In cyanobacteria a similar model has been proposed and the cyanobacterial redox regulation of gene expression works in a manner similar to that of chloroplasts, whereas transcription of genes for photosynthetic enzymes is influenced by light and also the redox state of the ETC. In the model organism Synechocystis PCC6803, the two-component system that regulates the redox state of the PQ pool contains a sensor histidine kinase termed RppB. The response regulator termed RppA acts as a transcription factor. Pfannschmidt et al.6 and Li and Sherman15 have demonstrated that mutation of RppA affects the level of mRNA encoding photosynthetic enzymes. Activation of RppA increases the transcription of genes encoding protein D1 of the PSI complex and reduces the transcription level of genes encoding PSII-related proteins. The gene coding for CSK originated from cyanobacteria and therefore the cyanobacterial mechanism of redox control of gene expression can be used as a model for chloroplasts.16
In conclusion, CSK is a nuclear-encoded protein that connects photosynthetic ETCs to chloroplast gene expression, therefore acting as a redox sensor.17 CSK is a modified bacterial-like sensor histidine kinase and it is indicated to work in a two-component system and regulate gene expression. The characteristics of CSK are those predicted by the ‘CoRR’ hypothesis for the function of the genetic systems of chloroplasts and mitochondria.18, 19
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Amplifying Full-length CSK Genes and Putative GAF Domain from CSK_GFP Plasmid
The CSK_GFP plasmid construct was kindly donated by T.A Kavanagh at Cambridge University and was used as a template to amplify the full-length CSK and putative GAF DNA using polymerase chain reaction. Primers used are shown in Table 1.
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A 3x PCR was prepared by adding together 15 µl of 10x pfu turbo buffer (Stratagene) (at a final concentration of 1x), deoxynucleoside triphosphates at a final concentration of 1 mM, 5 ng DNA template, a pair of primers (MWG) at a final concentration of 1 mM and pfu turbo DNA polymerase at a final concentration of 0.05 U (Stratagene). RNase-free water was added to a total reaction volume of 150 µl. PCR products were purified from the enzymatic reaction mixture using a GE Healthcare commercial PCR kit and eluted with 50 µl of RNase free water.
DNA Digestions
A 46 ng of insert (CSK/GAF) and 10 ng of plasmid (pGEX-6P2, Amersham Biosciences) were double digested using EcoRI and BamHI endonuclease. A total reaction volume of 50 µl containing the insert, 100 µg µl–1 BSA, 40 U EcoRI, 40 U BamHI, and RNase-free water was incubated at 37oC for 3 h. The restriction fragment was purified from the enzymatic reaction mixture using GE Healthcare gel purification kit.
Ligation
Two ligation reactions were prepared as follows:
- Reaction 1: contains 10 ng of double-digested insert and 2 ng of double-digested plasmid.
- Reaction 2: (negative control) contained only 2 ng of double-digested plasmid.
- Reaction 2: (negative control) contained only 2 ng of double-digested plasmid.
Recombinant Transformation
In a two pre-chilled 1.5 ml Eppendorf tube, 50 µl of thawed aliquot cells (E. coli strain MC1061, Invitrogen) and 0.5 ng of the ligation reaction was mixed. Eppendorfs were incubated in ice for 30 min before heat treatment in a 42°C water bath for 90 s and incubated in ice for a further 60 s. A super-optimal broth with catabolite repression medium containing 20% glucose and a super-optimal broth medium20 were used to dilute the aliquots to 1:10 ratio. It was also incubated at 37°C while agitating at 206 rpm for 1 h and 200 µl of cells were plated onto Agarose-Luria-Bertani broth (LB) medium containing 100 µg ml–1 ampicillin. This grew overnight at 37°C and, the following day, 12 colonies from each plate were inoculated with appropriate antibiotics (100 µg ml–1 ampicillin).
Recombinant Plasmid Extraction
Recombinant plasmids from the overnight cell culture mentioned previously were extracted using commercial mini-prep kit provided from Qiagen. The same enzymes as above were used to double digest the recombinant plasmids. The double digest plasmids were separated on a 1% agarose gel. Upon verification, clone 1 of CSK-GST and clone 4 of GAF-GST were transformed into cells E.coli strain (BL21-(DE3)-codonPlus-RP, Stratagene).
Small-scale Protein Expression
Starter cultures of the above clones (clone 1 of CSK and clone 4 of GAF) were inoculated and incubated overnight in 5 ml LB media containing20 100 µg ml–1 ampicillin and 50 µg ml–1 of chloramphenicol. The overnight culture was diluted 1:100 into 50 ml LB and incubated for 2 h with appropriate antibiotics while agitating at 250 rpm at 37°C. The cell cultures were induced with 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) and grew for a further 3 h at 30°C. The cells were harvested by centrifuging at 3000 rpm for 10 min and then the supernatant discarded. The pellet obtained was re-suspended in 5 ml 1x phosphate-buffered saline (PBS) (136.89 mM NaCl, 2.68 mM KCl, 1.47 mM KHPO4 and 8.10 mM NaHPO4 at pH 7.3) and centrifuged for 10 min. The pellet was re-suspended in 3 ml 1x PBS and sonicated at a maximum power for 10 s. Sonication was repeated five times with 30 s intervals, and 0.2% of Triton X-100 was added, then the solution was centrifuged at 18 000 rpm for 20 min. The supernatant (soluble fraction) was stored and the pellet (insoluble fraction) was re-suspended in 3 ml PBS and stored at +4°C.
Sequence Analysis
Sequence alignment was carried out for At1g67840 gene product of A. thaliana using the web-based BLAST (http://www.ncbi.nlm.nih.gov/genomes/prokhits.cgi), and SMART database (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1)21 was used tof predict the conserved domains. Cyanobase (http://genome.kazusa.or.jp/cyanobase/) was used to look for CSK homologues in cyanobacteria.
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I.M.I. thanks Queen Mary, University of London for a research studentship. J.F.A. thanks Wellcome Trust for a Value in People Award and The Royal Society for a Royal Society-Wolfson Research Merit Award.
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I thank Professor John F. Allen for giving me the opportunity to work on this project and for his help and guidance throughout. I would also like to thank Dr Sujith Puthiyaveetil and Dr Arefeh Seyedarabi for their help, encouragement and motivation throughout the project. Finally, I would extend my gratitude to Dr James Sullivan for kindly donating the competent cells and plasmids, as well as for his advice throughout the project.
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References |
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- Race HL, Herrmann RG, Martin W. Why have organelles retained genomes? Trends Genetics (1999) 15:364–370.[CrossRef][Web of Science][Medline]
- Martin W, Herrmann GR. Gene transfer from organelles to the nucleus: how much, what happens, and why? Plant Physiol (1998) 118:9–17.
[Free Full Text] - Allen JF. Photosynthesis of ATP—electrons, proton pumps, rotors, and poise. Cell (2002) 110:273–276.[CrossRef][Web of Science][Medline]
- Pfannschmidt T. Chloroplast redox signals: how photosynthesis controls its own genes. Trends Plant Sci (2003) 8:33–41.[CrossRef][Web of Science][Medline]
- Allen JF. State Transitions—a question of balance. Science (2003) 299:1530–1532.
[Abstract/Free Full Text] - Pfannschmidt T, Nilsson A, Allen JF. Photosynthetic control of chloroplast gene expression. Nature (1999) 397:625–628.[CrossRef][Web of Science]
- Puthiyaveetil S, Kavanagh TA, Cain P, et al. The ancestral symbiont sensor kinase CSK links photosynthesis with gene expression in chloroplasts. Proc Natl Acad Sci USA (2008) 105:10061–10066.
[Abstract/Free Full Text] - Thomas JG, Baneyx F. Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing heat-shock proteins. J Biol Chem (1996) 271:11141–11147.
[Abstract/Free Full Text] - Moussatche P, Klee HJ. Autophosphorylation activity of the Arabidopsis ethylene receptor multigene family. J Biol Chem (2004) 279:48734–48741.
[Abstract/Free Full Text] - Saito H. Histidine phosphorylation and two-component signaling in eukaryotic cells. Chem Rev (2001) 101:2497–2509.[CrossRef][Web of Science][Medline]
- Lasker MV, Thai P, Besant PG, et al. Branched-chain alpha-ketoacid dehydrogenase kinase: a mammalian enzyme with histidine kinase activity. J Biomol Tech (2002) 13:238–245.
- Wolanin PM, Thomason PA, Stock JB. Histidine protein kinases: key signal transducers outside the animal kingdom. Genome Biol (2002) 10:3013.3011–3013.3018.
- Puthiyaveetil S, Allen JF. Transients in chloroplast gene transcription. Biochem Biophys Res Commun (2008) 368:871–874.[Web of Science][Medline]
- Weber P, Fulgosi H, Piven I, et al. TCP34, a nuclear-encoded response regulator-like TPR protein of higher plant chloroplasts. J Mol Biol (2006) 357:535–549.[CrossRef][Web of Science][Medline]
- Li H, Sherman LA. A Redox-responsive regulator of photosynthesis gene expression in the cyanobacterium Synechocystis sp. Strain PCC 6803. J Bacteriol (2000) 182:4268–4277.
[Abstract/Free Full Text] - Buchanan BB, Balmer Y. Redox regulation: a broadening horizon. Annu Rev Plant Biol (2005) 56:187–220.[CrossRef][Medline]
- Allen JF. Redox control of transcription: sensors, response regulators, activators and repressors. FEBS Lett (1993) 332:203–207.[CrossRef][Web of Science][Medline]
- Allen JF. Control of gene-expression by redox potential and the requirement for chloroplast and mitochondria genomes. J Theor Biol (1993) 165:609–631.[CrossRef][Web of Science][Medline]
- Allen JF. The function of genomes in bioenergetic organelles. Philos Trans R Soc Lond B Biol Sci (2003) 358:19–37.
[Abstract/Free Full Text] - Sambrook J, Fritsch FE, Maniatis T. Molecular Cloning: A Laboratory Manual (1989) New York: Cold Spring Harbor Laboratory Press.
- Schultz J, Milpetz F, Bork P, et al. SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci USA (1998) 95:5857–5864.
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Iskander Ibrahim graduated in 2008 from Queen Mary, University of London with a first-class Honours degree in Biochemistry. The undergraduate degree modules covered were Biochemistry, Genetics, Molecular Neuroscience and Pharmacology. These modules lead him to undertake a final year project in molecular Biochemistry. Currently, Iskander is reading for a PhD at Queen Mary, University of London under the supervision of Professor John F. Allen and Dr Robin Maytum. Upon successful completion of his research degree, Iskander's objective is to pursue a career in academia.
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