The yellow fever mosquito Aedes aegypti is the main vector for several important arboviral diseases such as dengue, yellow fever, chikungunya and zika. With the advent of genetic control strategies, new species-specific tools have emerged for the control of Aedes aegypti. This thesis describes attempts at building different gene drive systems aiming for both population suppression or population replacement, as well as exploring the possibility of inserting exogenous sequences in the male locus of Aedes aegypti. An underdominance system, consisting of two mutually rescuing killers, was investigated in Drosophila melanogaster. It did not work as expected in the configuration tested. The chosen NIPP1 killer gene could not be upregulated by tTAV when under the control of hsp83, UAS and tetO. tetO and tTAV2 resulted in a lethal positive-feedback loop. Gal4Groucho and LexAGroucho fusion proteins, previously used as corepressors in the literature, were lethal when under the control of the tetO-tTAV system. Males showed the expected feminisation phenotypes, invovling male palp shortening and less feathery antennae, upon Nix knock-out using CRISPR-Cas9. However, subsequent homology directed repair into the Nix gene in the male locus of Aedes aegypti did not succeed even after reiterated injections. Setting out from the hypothesis of Act4 haploinsufficiency in Aedes aegypti, the building of two different gene drive systems was attempted; female-specific underdominance and RIDL with drive. A CRISPR-Cas9 driven act4 knock-in unexpectedly confirmed act4 haplosufficiency in Aedes aegypti. Whilst the initially devised gene drive systems could not function as such, act4 haplosufficiency marked the finding of a new female-specific recessive flightless target (effectively sterile) for use in future population suppression drive systems.

The insect Malpighian tubule is involved in osmoregulation, detoxification and immune function, physiological processes which are essential for insect development and survival. As the Malpighian tubules contain many ion channels and transporters, they could be an effective tissue for targeting with novel pesticides to control populations of Diptera. Many of the insecticide compounds used to control insect pest species are no longer suited to their task, and so new means of control must be found. The malarial mosquito, Anopheles gambiae, spreads the Plasmodium parasite which is responsible for over one million deaths each year, and is one of the species on which many current insecticides are no longer effective. Anopheles is notoriously difficult to study due to a lack of natural mutation stocks and transgenic capabilities, as well as the difficulties involved with maintaining a colony. The fruit-fly Drosophila melanogaster is a useful model organism for Anopheles, and previous studies suggest that the mechanisms of Malpighian tubule function are well conserved between the two species. Following microarray investigations to identify genes which were highly enriched in both the Anopheles and Drosophila Malpighian tubules, four homologous genepairs were selected, AGAP097752 and CG15406, AGAP012251 and Picot, AGAP009005 and ZnT35C, and AGAP002587 and CG8028. Analysis of the Anopheles Malpighian tubule microarray data-set showed ion channels and transporters to be highly expressed in the tubules, although similarly to Drosophila, very few of the renal up-regulated genes have been characterised. The gene-pairs chosen were all novel, but putatively predicted to be involved in sugar transport, phosphate transport, zinc transport and monocarboxylate transport respectively. These are functions which are likely to be essential, but so far remain unstudied in the insect renal system. The gene-pairs were chosen with two main purposes; to determine how closely expression of the genes was conserved between Anopheles and Drosophila, and also to determine which of the genes were essential, and could therefore be effective insecticide targets. The homologous gene-pair AGAP007752 and CG15406 have well-conserved expression in the Malpighian tubules, suggesting that they are functionally important genes. This was shown in Drosophila, where knockdown of CG15406 4 expression was lethal to the fly. A direct role in tubule fluid secretion was not found, and experiments to determine the sugars transported by CG15406 were inconclusive, possibly due to an abundance of highly-expressed sugar transporters in the tubules. The inorganic phosphate co-transporters AGAP012251 and Picot also show conservation of expression in the Malpighian tubules, and are likely to be involved in the transport of inorganic phosphate into the tubules for incorporation into metallo-organic concretions. In the Anopheles tubules the concretions are found in the main segment, in the Drosophila tubules they are located in the distal initial and transitional segments, where AGAP012251 and Picot are expressed. Picot is essential for Drosophila development through to adulthood, and for survival as an adult, although the transporter does not appear to be directly involved in fluid secretion. Expression of neither AGAP012251 nor Picot is confined to the tubules. The putative zinc transporters AGAP009005 and ZnT35C show a highly conserved expression pattern, and appear to be involved in the secretion of excess zinc from the Malpighian tubules. ZnT35C is essential early-on in Drosophila development, and for survival in the adult fly. Similarly to Picot and CG15406, there is no direct role for ZnT35C in fluid secretion from the tubules under normal zinc conditions. The putative monocarboxylate transporters AGAP002587 and CG8028 are not as well conserved, as AGAP002587 is highly upregulated in the tubules of female mosquitoes both before and after a bloodfeed, whereas CG8028 has no sex-specific up-regulation. CG8028 is not essential for Drosophila development or survival, and plays no discernable role in fluid secretion. The data collected during this investigation suggests that in general there is a high level of conservation of expression between homologous transport genes in the Anopheles and Drosophila Malpighian tubules. The three gene-pairs which show the greatest conservation of expression are also essential for development and survival in Drosophila. This suggests that cross-species studies are an effective way of finding essential and important genes. The data collected also suggests that Drosophila is a reliable model for Anopheles, and could be used as a high-throughput system of finding genes which could be effective insecticide targets in Diptera.

Human embryonic stem cells are derived from the pre-implantation blastocyst, residing in an extremely low oxygen environment. Application of this in vivo oxygen concentration to an in vitro setting has previously shown to be essential for driving the self-renewal and directed differentiation of human pluripotent stem cells. However, studies on hESCs at low oxygen have been frequently contra-indicatory. Here, comparative analysis of a hESC and iPSC line at a low oxygen concentration resulted in divergent effects across the two cell lines. Augmented TGFβ signalling was observed in conjunction with up-regulated transcription of pluripotency markers NANOG and POU5F1 in hESCs but not iPSCs. IPSCs also shifted to a state of increased proliferation whereas hESCs did not. It was also observed that exposure of hESCs and iPSCs to light throughout low oxygen culture induced large amounts of apoptosis, highlighting the requirement for careful selection of cell culture equipment for environmental oxygen control. Both the embryonic and adult brain retain tissue specific oxygen concentrations far below that at atmospheric oxygen concentrations. Previous studies showed that embryonic silencing of factors responsive to low oxygen caused a range of embryonic abnormalities, including deformation of the neural plate and tube. Here, differentiation of hESCs to an early neuroectodermal identity at low O2 did not definitively augment production of NPCs but did additively suppress BMP signalling above that at atmospheric O2. A concordant rise in apoptosis was also observed in a manner both independent of, and augmented by, exogenous BMP inhibition. Subsequently, neurogenesis at low oxygen produced terminal neurons with accelerated and augmented synaptic and induced excitability. This was characterised by a rise in the rate of membrane depolarisation, increased action potential overshoot and accelerated expression of pre-synaptic marker Synaptophysin. These results highlight a novel, critical role for low oxygen in augmenting the excitability of hESC derived neurons.

The circadian clock is the endogenous mechanism by which a wide variety of biological processes are regulated in anticipation of daily changes in the external environment. In Arabidopsis thaliana, the clock comprises a number of complex gene and protein interactions, involving multiple regulatory feedback loops. The clock gene LHY has a central role in these loops, activating and repressing morning- and evening- expressed genes respectively. These clock genes in turn sequentially repress the expression of LHY throughout the day and night, restricting it to a sharp transcriptional peak at dawn. However, the molecular mechanisms of these regulatory interactions with the LHY promoter were not known. Therefore, this project first aimed to determine which promoter motifs are responsible for mediating regulation of LHY circadian expression. This was achieved through luciferase assays with mutated pLHY:LUC reporter constructs, which identified the CT-rich region as responsible for rhythmic expression of LHY, and the G-box as mediating regulation by the clock protein TOC1. Since few regulators were known to target the LHY promoter, this project also aimed to identify transcription factors binding the promoter using a Yeast One-Hybrid assay. Transcription factors with roles in a wide variety of biological pathways were identified from this screen, with abiotic stress and plant defence pathways particularly well-represented. In addition, a number of antagonistic and synergistic regulatory interactions were established as occurring between stress factors and clock proteins at specific promoter motifs. We can therefore conclude that LHY is regulated by a complex network of transcription factor interactions, enabling the rapid integration of environmental stress signals into the clock.

A PCR strategy showed that a number of total mtDNA molecules was significantly decreased (~30%) in the striatum (no reduction in the cortex and cerebellum) of 24-month old HD mice, but not a 15 months of age, when compared to wild-type mice, suggesting mtDNA depletion is a progressive rather than a developmental phenomenon. In light of the ~30% reduction of total mtDNA in the striatum, expression levels of the mitochondrial DNA-encoded respiratory complex enzymes, cytochrome b(Cytb), cytochrome c oxidase I (COI) and cytochrome c oxidase II (COII) were investigated in different brain regions of HD mice. At ~25 months of age, there were no significant differences in mRNA levels of CoII and Cytb in any brain region (striatum, cortex and cerebellum) studied when compared to normal littermates. However, HD mice showed significantly decreased CO-I protein levels and marginally decreased CoI mRNA levels in the striatum. Reduced levels of mtDNA may be caused by decreased replication of mtDNA or increased oxidative damage of mtDNA. Increased levels of 8-OHdG, a marker of increased oxidative stress, were detected in the dorsomedial, dorsolateral and ventromedial striatum, but not in the cortex of 24-month old HD mice providing direct evidence that increased oxidative stress specifically occurs in the striatum of HD mice. As no alterations in the mitochondrial transcription factor (mtTFA) in the striatum of HD mice could be detected, it is likely that mtDNA depletion in the HD mice is caused by increased levels of oxidative stress rather than decreased replication. The results provide a basis for further studies investigating how mutant huntingtin causes increased levels of oxidative stress and for identifying novel therapeutic targets.

Malaria, caused by infection with intracellular protozoan parasites of the genus Plasmodium, is responsible for 300 to 600 million clinical cases annually (Snow et al., 2005), resulting in the deaths of up to three million people every year (Breman, 2001, Breman et al., 2004). There is a clear need for further research aimed at identifying novel drug targets (Ridley, 2002). Reversible phosphorylation of proteins is a major regulatory mechanism in most cellular processes, and protein kinases are considered promising drug targets, comprising as much as 30% of all protein targets under investigation (Cohen, 2002). The divergences between human and plasmodial protein kinases suggest that specific inhibition of the latter is an achievable goal (Doerig, 2004, Doerig and Meijer, 2007). This study investigates protein kinase CK2 of Plasmodium falciparum, seeking to establish by reverse genetics and biochemical approaches whether it represents a possible antimalarial drug target. Protein-kinase CK2, formerly known as Casein Kinase II, is a dual-specificity (Serine/Threonine and Tyrosine) protein kinase ubiquitously expressed in eukaryotes. It has over 300 cellular substrates catalogued to date (Meggio and Pinna, 2003). Consistent with its multiple substrates, the enzyme plays a crucial role in many cellular processes, and is essential to viability in yeast and slime mould (Padmanabha et al., 1990, Kikkawa et al., 1992). The human CK2 holoenzyme consists of two catalytic a or a’ subunits and two regulatory b subunits, and recent evidence indicates that the latter interact with several protein kinases in addition to CK2a (reviewed in (Bibby and Litchfield, 2005)), pointing to a likely role in the integration of numerous signalling pathways. A putative CK2a orthologue and two predicted CK2b subunits were identified in the P. falciparum genome (Ward et al., 2004, Anamika et al., 2005). Here we present the biochemical characterisation of the PfCK2a orthologue and both PfCK2b orthologues, and demonstrate by using a reverse genetics approach that each of the three subunits is essential for completion of the erythrocytic asexual cycle of the parasite, thereby validating the enzyme as a possible drug target. Recombinant PfCK2a possesses protein kinase activity, exhibits similar substrate and co-substrate preferences to those of CK2a subunits from other organisms, and interacts with both of the PfCK2b subunits in vitro. PfCK2a is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2a enzymes to a small molecule inhibitor. Taken together, the data indicate that PfCK2a is an attractive, validated target for antimalarial chemotherapeutic intervention.

The gene cluster encoding production of oxytetracycline (OTC) by Streptomyces rimosus (the commercial producer) has been studied in this laboratory. The topic of this thesis was the region of the otc cluster including and upstream of the OTC-resistance gene otrB, which has been shown previously to be responsible for reduced accumulation of the antibiotic. The otrB gene was sequenced. The sequence revealed some discrepancies with previously-published data on tet347 (Reynes et al., 1988; Journal of General Microbiology 134: 585-598), an OTC-resistant determinant from another strain of S.rimosus. The deduced gene product of the otrB showed considerable identity with efflux proteins from other Gram-negative and Gram-positive bacteria. These proteins contain conserved functional motifs, and OtrB was analysed in this context. The transcriptional start of otrB was identified. Several short investigations were undertaken into the physiology of antibiotic production. (1) A transcriptional fusion vector (pIJ2843) using catechol oxygenase as a reporter was used to monitor the response of transcription of various regions of the otc cluster (cloned from a high-producing strain) to changes in external phosphate concentration. These data were compared in relation to antibiotic production by the wild-type strain. (2) A transposon mutagenesis strategy was used to attempt to generate novel mutations within the otc cluster. (3) The presence of genetically-engineered haemoglobin cloned into S.rimosus production strains was investigated. The relationship between expression of the recombinant protein, antibiotic production and the aeration of the S.rimosus cultures is discussed.

Oxytetracyline, (OTC), is a secondary metabolite antibiotic produced by the actinomycete Streptomyces rimosus. All of the structural genes for OTC synthesis are clustered on the S. rimosus chromosome and flanked by resistance determinants, otrA and otrB. OtrA mediates resistance through non-covalent modification of the bacterial ribosome whilst otrB, the subject of this thesis, acts by efflux of the drug from the cell. At the outset of this thesis otrB had previously been cloned and sequenced. During the course of the work some discrepancies with previously published data were found, parts of the gene were then re-sequenced and a revised otrB sequence submitted to the NCBI database. The OtrB protein is a transmembrane protein belonging the Major facilitator Superfamily (MFS). Sequence comparison with other members of the family shows OtrB to be part of a subfamily of antibiotic transporters and multi drug resistance proteins made up from 14 transmembrane helices (6+2+6) arrangement. OtrB contains many conserved motifs typical of the subfamily and of the MFS. otrB was cloned into E. coli and expressed. The functional activity of the cloned gene was assessed by growth on various concentrations of OTC. Substrate specificity was investigated using TET and CTC in the growth media. Isolation of OtrB from E. coli as a polyhistidine fusion was attempted, reasons why this was not successful are discussed. Tetracycline exporters from Gram-negative bacteria generally contain 12 transmembrane helices (6+6). The relevance of the two "extra" helices present in OtrB was investigated by the construction and expression in E. coli, of a deletion mutant in which the two putative central helices were absent.

Plasmodium and Toxoplasma nuclear genomes possess an ORF for a putative protein which contains domains homologous to the T7 bacteriophage primase-helicase like Twinkle enzyme and the prokaryotic family A polymerase enzyme. It has been hypothesised that this nuclear encoded protein may be responsible for the replication and repair of the apicoplast genome. Thus it was named PREX (Plastidic DNA Replication/Repair Enzyme complex). In Plasmodium falciparum the ORF (Pfprex) is 6,051 bp with no introns. RT-PCR data revealed that prex is present as a single transcript but western blot analysis of Plasmodium falciparum asexual parasite extracts revealed smaller size proteins indicating post-translational cleavage of the protein. Gene knock out studies have shown that the Pfprex genome locus is recombinogenic although parasites with a disrupted Pfprex locus appear to be unable to survive in culture. The analysis of the recombinant polymerase domain confirmed the polymerase property of the protein. In Toxoplasma gondii, the gene (Tgprex) is 7,740 bp long and interrupted by 19 introns as identified by RT-PCR. The polymerase functionality of the PREX protein was also confirmed by a study on recombinant protein from Toxoplasma gondii. The recombinant protein can be inhibited by known family A polymerase inhibitors. Other related apicomplexan parasites including Theileria, Babesia and Eimeria also possess this prex homologous gene in their nuclear genome. This PREX protein, apparently an amalgamation of functions derived from viral and bacterial origins, is probably important for maintenance of genomic integrity of the apicoplast. The recombinant protein and the assay system may provide the platform for screening of compounds for future drug search against the apicomplexan parasites.

Adult articular cartilage has a limited repair capacity. This leads to an increasing demand for optimised repair techniques. Furthermore, current procedures to regenerate articular cartilage fail to achieve sufficient results. Previous work within our group suggested that combination of functional tissue engineering and gene transfer represents a promising alternative approach. In this thesis, different viral gene transfer methods were investigated and optimised. A clinically relevant three dimensional transduction model was developed. These results were directly implemented in further work aiming to investigate the combined effect of multiaxial mechanical stimulation and adenoviral-mediated over-expression of bone morphogenetic protein 2 on human chondroprogenitor cell chondrogenesis and progression towards hypertrophy. Two cell sources were investigated, namely human mesenchymal stem cells and human articular cartilage progenitor cells. The combined approached enhanced human mesenchymal stem cell chondrogenesis. Yet, it was not possible to completely prevent progression towards hypertrophy. For human articular cartilage progenitor cells, over-expression of bone morphogenetic protein 2 did enhance their chondrogenic differentiation potential. However, mechanical stimulation alone, in the absence of exogenous growth factors, led to stable chondrogenic induction without signs of hypertrophic differentiation. This suggests these cells should be further investigated. Additionally, the potential of Dorsomorphin, as possible agent to block hypertrophic differentiation by inhibition of bone morphogenetic protein signalling, was investigated in a fibrin polyurethane composite system, using human mesenchymal stem cells. As opposed to the pellet culture model, application of Dorsomorphin led to a cytotoxic effect which decreased the general differentiation potential. Finally, the chondrogenic potential of the two cell types was directly compared, using the pellet culture model. Under serum-free conditions, human articular cartilage progenitor cells were not able to undergo chondrogenesis. The reasons for this remain to be elucidated. The combined results of the thesis can help to develop a novel one-step procedure to treat articular cartilage defects.