Genetics - Theses

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    Studies on minor non-metrical skeletal variants in the mouse and man
    Kellock, Wendy Lorraine. (University of Melbourne, 1970)
    This thesis consists of papers presenting the results of studies on the genetical, developmental and anthropological aspects of minor non-metric al variants in man and the house mouse. The work is mainly on variants of the skeleton, particularly the cranium, but includes a limited discussion of published data on minor non-metrical variants of the muscular and vascular systems. Each study is based on a number of variants, and, where applicable, single measures have been obtained to express the overall difference in skeletal variability between populations or the overall effect on skeletal variability of certain environmental factors. Investigations into the role of genotype and environment in the determination of minor skeletal variants in mice and man indicate that most of them are under some genetic control but that maternal physiology and other non-genetic factors may influence the frequency of individual variants. Data presented here (Publication 1) on 25 minor skeletal variants in inbred strains of mice and their hybrids suggest that genotype is more important than environment in determining skeletal variability. Although the frequency of a few individual variants was found to be significantly affected by certain non-genetic factors, when many variants were considered together the environment had no overall significant effect. In contrast, large differences, due mainly to genetic factors, were observed between inbred strains and hybrids. Further studies on inbred strains of mice and hybrids (Publication 2) indicate that stabilizing mechanisms operate during the formation of the skeleton. For most of the 29 bilateral minor non-metrical variants studied , the frequency of asymmetrical mice (i.e., those with the variant present on only one side) was less than expected on the assumption that the number of mice with the variant present on both, one or neither sides depends solely on the frequency of the variant on each side. This tendency for the development of the skeleton to be canalized against asymmetry has been described as a form of morphogenetic homeostasis. The same phenomenon has been observed for bilateral minor non-metrical variants in man (Publication 3) for the skeletal, muscular and vascular systems (based on data published by Danforth in 1924) and for the skeletal system of Australian Aborigines. Studies on inbred strains of mice (e.g., Publication l) indicate that genotype plays the major role in determining the frequency of minor non-metrical variants. If these findings can be extrapolated to man, minor non-metrical variants may be of use in anthropological work. A general survey of skeletal variation, based on 30 such variants, was carried out on Aboriginal crania from many parts of Australia (Publication 4). Regional differences in the pattern of cranial morphology were observed which appear to culminate in two extreme populations: one in the north and north-west of the continent, the other in south-eastern Australia. These results were considered in relation to some current theories on the origin and ethnic composition of the Australian Aborigines.
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    The genetic and morphological characterisation of the zebrafish intestinal mutant setebos
    Badrock, Andrew Paul. (University of Melbourne, 2010)
    Colorectal cancer (CRC) arises by mutation of tumour suppressors and oncogenes that deregulate the genetic programmes that control intestinal turnover and homeostasis. The zebrafish intestinal epithelium shares extensive phenotypic and genetic homology with the mammalian intestine, providing a genetically tractable model organism for the discovery and characterisation of novel genes required for intestinal development. Increased knowledge of the genetic control of intestinal development may reveal new therapeutic avenues for the treatment of CRC. The Liverplus mutagenesis screen identified zebrafish embryos defective in endoderm organ morphogenesis, including the intestinal mutant setebos, described herein. This study reports the positional cloning of the setebos mutant locus, identifying a premature stop codon as the responsible mutation in the gene nucleolar protein 8 (nol8). Microinjection of wildtype nol8 mRNA rescued setebos developmental abnormalities whereas morpholino oligonucleotide-mediated knockdown of Nol8 phenocopied setebos morphology in wildtype embryos, providing functional proof that nol8 is the mutated locus underlying the setebos phenotype. This study demonstrated that Nol8 is essential for zebrafish survival and that nol8 mRNA is widely expressed in actively proliferating tissues of the zebrafish embryo at 72 hours post fertilisation (hpf), when developmental abnormalities in setebos mutants first become apparent. Northern blot analyses revealed that setebos mutants accumulate an intermediate ribosomal RNA (rRNA) orthologous to mammalian 36S at the expense of downstream 32S rRNA; defects predicted to result in reduced biogenesis of 28S rRNA. Bioanalyzer analyses confirm that setebos mutants are preferentially impaired for 28S biogenesis. 28S, 5.8S and 18S rRNA molecules combine with ribosomal proteins to form functional ribosomes, which are essential for translation and cellular growth. Defects in ribosome biogenesis have been shown to activate the tumour suppressor Tp53, resulting in cell cycle arrest. Tp53 is activated in the setebos mutant, resulting in upregulation of pro-cell cycle arrest genes, including the N-terminally truncated isoform of Tp53, ?113tp53. While this provides a mechanism for the hypoplasia observed in the tissues of the setebos embryo, including the intestinal epithelium, craniofacial cartilages, eyes, liver and pancreas, inactivation of Tp53 signalling in the setebos mutant failed to obviously alter setebos morphology. Nevertheless, activation of Tp53 signalling was found to significantly extend survival of setebos embryos, identifying an important biological role for Tp53 in cells undergoing ribosomal stress. Multiple human syndromes have been linked to abnormal ribosome biogenesis (ribosomopathies), and several predispose to cancer. Zebrafish haploinsufficient for 17 different ribosomal proteins also display increased cancer susceptibility (Amsterdam et al. 2004; Lai et al. 2009). Tp53 is inactivated in these zebrafish tumours, suggesting that inactivation of Tp53 may be a key event in tumour formation in these animals. In summary, setebos provides an ideal in vivo model to further characterise the link between defective ribosome biogenesis, Tp53 signalling and cancer. Since disruption of Nol8 function preferentially impairs the growth of several highly proliferative embryonic zebrafish tissues in the absence of Tp53 signalling, inhibition of Nol8 may provide a therapeutic avenue for the treatment of Tp53 mutant tumours.
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    Investigating the mechanisms of zinc homeostasis in Arabidopsis thaliana
    Sinclair, Scott Aleksander. (University of Melbourne, 2010)
    Zn is an element essential for all forms of life, and either Zn-deficiency or excess Zn can have serious biological consequences. In plants, Zn levels are controlled by a number of physiological and cellular mechanisms to maintain appropriate physiological concentrations of Zn. This study further characterises the HMA2 and HMA4 transporters by developing a novel technique using the Zn fluorophore Zinpyr-1. A procedure for the use of Zinpyr-1 in Arabidopsis roots was developed and validated which allowed the visualisation and relative quantification of Zn. This technique showed that both hma4 and hma2/hma4 mutants differed in the amount of Zn and its distribution compared to wildtype or hma2. This correlates to previous data showing that hma4 and hma2/hma4 are Zn-deficient in shoots but accumulate Zn in roots. Thus we can say that this Zn- deficiency in shoot occurs due to an inability to load Zn into the xylem for transport to the shoot. To investigate the regulation of Zn-homeostasis, Zn-deficient and hma2/hma4 plants were compared to wildtype in micro-array experiments to test whether plants respond systemically to Zn- deficiency. hma2/hma4 plants are Zn-deficient in shoots but over-accumulate Zn in roots, so genes co-regulated in hma2/hma4 roots and shoots, and genes co-regulated in hma2/hma4 roots and Zn- deficient wildtype roots were identified. 52 and 27 genes were identified in these comparisons respectively as regulated >2-fold in the same direction. These were considered robust as their co-regulation was observed in four measurements, two technical replicates of two biological replicates. promoter
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    The roles of a novel group of plastid transporters in glutathione metabolism in arabidopsis
    Lim, Tingsen Benson. (University of Melbourne, 2010)
    The tripeptide glutathione (GSH) is an important compound with a broad spectrum of functions in plants, including stress responses and is a key element of the intracellular redox buffer that protects cells from oxidation. In Arabidopsis GSH is synthesized in two enzymatic steps by GSH1, found exclusively in the plastids, forming the pathway intermediate ?-glutamylcysteine (?-EC) and then by GSH2 which is located in both plastids and cytosol. This suggests a mechanism for ?-EC export from the plastids and, because the majority of GSH2 transcripts (90%) encode the cytosolic isoform, it is believed that the cytosol may be the main compartment for GSH biosynthesis. In addition, GSH may be efficiently transported between the two compartments to maintain proper homeostasis. However, comparatively little is understood about the mechanisms that control and interconnect these two subcellular GSH pools. The identification and characterization of a novel family of transporters, the CRT Like Transporters (CLTs), has provided further insights into glutathione metabolism in Arabidopsis. The CLT1 gene was originally identified in a screen for mutants resistant to L-Buthionine-SR-sulfoximine (BSO), a synthetic compound used to specifically inhibit GSH1 and GSH biosynthesis. Two paralogues in Arabidopsis, CLT2 and CLT3, were identified and all three CLTs are localized to the plastids. Previous studies have shown the clt1clt2clt3 triple mutant is GSH-deficient in roots and cadmium-sensitive suggesting a role for the CLTs in transporting ?-EC and/or GSH between the plastid and cytosol compartments. In this study redirecting GSH1 activity or GSH biosynthesis exclusively to the cytosol by complementing the Arabidopsis gsh1 and gsh2 mutants with E. coli GSHA and GSHB, respectively, has no significant impact on phenotypes or stress resistance suggesting efficient exchange of ?-EC and GSH between the plastid and cytosol compartments. The use of redox-sensitive roGFP2 reporters in combination with confocal imaging with monochlorobimane and measurements of cytosolic thiols has demonstrated that the clt1clt2clt3 mutant is GSH-deficient in the cytosol but not the plastid in both roots and shoots consistent with a predicted role for CLTs in ?-EC and/or GSH transport. The Arabidopsis gsh2 mutant accumulates high levels of ?-EC in the cytosol. However, the combination of the clt1clt2clt3 mutations with gsh2 did not decrease ?-EC levels suggesting CLTs may not contribute to ?-EC transport in vivo. To further study the roles of the CLTs separately on ?-EC and GSH transport in planta different clt mutations were introduced into transgenic lines expressing plastid- or cytosol-targeted GSH2. These observations suggest that de novo GSH biosynthesis in wildtype Arabidopsis occurs largely in the plastids and that CLT2 and CLT1/CLT3 are mainly involved in ?-EC and GSH export, respectively, in planta.
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    Structure
    Price, Gareth Robert. (University of Melbourne, 2010)
    MLK2 is a serine/threonine kinase with a N-terminal SH3 domain and a centrally located dual leucine zipper region that defines the mixed lineage kinase family. Motifs for interaction with the small Rho-like GTPases, 14-3-3 signalling adaptor proteins, as well as for nuclear import and export have been identified in MLK2 and other MLK family members, providing the capacity for a large number of diverse interactions. MLK2 extracted from over-expressing cells is catalytically active and long-term over-expression is lethal. A study of the contribution of the SH3 domain, catalytic domain and dual leucine zipper region to the overall activity of MLK2 was undertaken. This investigation utilised targeted mutagenesis to alter the coding sequence of conserved amino acids to produce non-functional and constitutively active protein domains. The SH3 domain contributes to the regulation of catalytic activity by binding within the protein C-terminal domain. Once released from auto-inhibition, the SH3 domain interacts with dynamin and is capable of activating dynamin I GTPase activity, thereby increasing the rates of dynamin mediated endocytosis. Both auto-inhibition and dynamin activation is ablated by substitution of a praline residue within the conserved binding cleft of the SH3 domain. Dim�risation via the dual leucine zipper region is required to achieve full catalytic activity. Substitution within the catalytic domain produced a kinase-inactive protein that was used throughout the study. In addition, this inactive variant also demonstrated the requirement for phosphorylation of the activation loop prior to an increase in catalytic activity. Membrane localisation of MLK2 was demonstrated to be independent of catalytic activity and SH3 domain or leucine zipper binding capacity. MLK2 is a member of the MAPKKK family capable of activating the c-jun N terminal kinases via MKK4 and MKK7. The use of kinase negative MKK4 confirmed the ability of MLK2 to signal to JNK through an alternative MKK protein. Kinase negative MLK2 was a target for JNK phosphorylation producing a pattern of phosphopeptide TLC-TLE spots with commonalities to autophosphorylated MLK2. Phosphorylation was concentrated within the C-terminal half of the protein where no large functional domains have been identified but a number of smaller motifs are found. Retrophosphorylation was proposed to have a positive effect on MLK2, sustaining activity and signalling that ultimately leads to apoptosis. Stable expression of GFP tagged MLK2 was only achieved with kinase inactive or truncated protein coding constructs, confirming the lethal effect of over-expressing active MLK2. Kinase negative MLK2 expression decreased cell doubling rates, increased the number of fine membrane projections and trafficked to the nucleus in a cell-cycle dependent manner. The CRIB domain allowed for interaction between Cdc42, Rad and MLK2. MLK2 was proposed to influence cytoskeletal remodelling in response to Cdc42 and Rad activation leading to stimulation of the JNK pathway. Finally, MLK2 was has been shown to have a role in the progression of the neurodegenerative pathology of Alzheimer�s disease. Stimulation of the JNK pathway, coupled with the ability to mediate receptor activation by modulation of rates of endocytosis theoretically places MLK2 in a key position in the response to excitory amino acid excitotoxicity, in Huntington�s disease, and to apoER2 activation in Alzheimer s disease. The characterisation of the contributions made by SH3 domain, catalytic domain and dual leucine zipper region to the overall activity of MLK2 will allow for the design and optimisation of novel therapeutic targets aimed at reducing MLK2 activity, opening up new scope for disease management.
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    Population genetics and ecology of the lucerne flea, sminthurus viridis, with applications for control
    Roberts, John Michael Kenneth. (University of Melbourne, 2010)
    The lucerne flea, Sminthurus viridis (Collembola: Sminthuridae), is a major pest of broadacre grains and pasture throughout southern Australia. Current control methods for this pest rely heavily on the prophylactic use of broad spectrum pesticides and are unlikely to be sustainable. Development of an integrated pest management (IPM) strategy is needed. However, this requires a greater understanding of the biology, ecology and genetics of S. viridis. This thesis aims to address this by examining the population genetics and life-cycle of this pest, as well as investigating chemical and biological control methods. Laboratory pesticide bioassays were used to compare the response of S. viridis with the redlegged earth mite (Halotydeus destructor) to six broad spectrum pesticides. Sminthurus viridis displayed significantly higher pesticides tolerance than H. destructor and the tolerance differences were greater for the synthetic pyrethroids than the organophosphorous pesticides. This indicates that application rates for S. viridis should be greater than H. destructor and that organophosphates are likely to provide the most effective control of this pest. Seasonal abundance patterns and summer diapause response of S. viridis in south-eastern Australia were investigated using field and shade-house experiments. The seasonal activity agreed with previous studies and parameters predicting autumn emergence were defined. A summer-diapausing egg stage was also confirmed, with diapause eggs accumulating across the season. These results suggest autumn emergence of S. viridis can be predicted for target control strategies, but late-season spraying strategies may be difficult to implement. The species status, mode of reproduction and population genetics of S. viridis were examined using microsatellite markers I developed, together with allozymes and a mitochondrial DNA marker. A single, sexually reproducing species was found with limited gene flow between Australian populations. Genetic similarity between some distant populations was also shown, indicating that wind or human-mediated long-distance dispersal occurs. These findings suggest that management strategies may be more effective when locally developed and need to consider the effects of human- mediated dispersal. The impact of the predatory mite, Bdellodes lapidaria, for controlling S. viridis and its susceptibility to several pesticides were assessed using laboratory and field experiments. Bdellodes lapidaria was more susceptible to the synthetic pyrethroids in the laboratory, but was not affected by pesticide applications in the field. In addition, no detectable predator-prey relationship was found between these species. These results indicate that B. lapidaria is not strongly susceptible to pesticides in the field and may not be an effective predator of S. viridis. Overall, this thesis contributes important knowledge of the pesticide tolerance, life- cycle, population genetics and biological control of S. viridis. These findings highlight ways to improve the effectiveness and sustainability of control methods and bring closer the development of an integrated pest management strategy for S. viridis.
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    The mammalian stonin proteins : from translation to degradation
    Wall, Adam Alexander. (University of Melbourne, 2009)
    Recent work has centred around the role of stonin 2 in clathrin-mediated endocytosis (CME) of the integral synaptic vesicle protein synaptotagmin I. Without a mode of internalisation of this protein, synaptic vesicles that are formed from the plasma membrane are fusion-incompetent and therefore no neurotransmitter can be released (Mohrmann, 2008). Stonin 2 was first identified on the basis of homology with the Stoned B gene of Drosophila melanogaster. The stoned B protein is encoded in the second open reading frame (ORF) of a dicistronic transcript that encodes another protein involved at the synapse, named stoned A. It was in 1973 that the locus was first identified in a screen for temperature sensitive paralytic mutants. These mutants were shown to have defective nervous systems and had aberrant synaptic vesicle retrieval at the plasma membrane of the neuromuscular junction (NMJ). Like stonin 2, stoned B interacts with synaptotagmin I through its C-terminal domain that shows significant homology to the ?2 subunit of the clathrin adaptor protein complex AP-2. Stonin 2 is not the only stoned B homologue in mammalian genomes, with another protein named stonin 1 showing equal protein similarity. In this report, the stonin 1 antibodies that had previously been utilised (Arnott 2004) were re-characterised and it was concluded that they were only useful to detect expressed recombinant protein. Affinity purified stonin 2 antibodies, created in this project, were successfully utilised to detect both endogenous stonin 2 and recombinant expressed-tagged stonin 2 for both Western blot and immunocytochemistry, which required a novel antibody purification method to be created to achieve a greater resolution. After analysis of expressed tagged stonin proteins in the neuroendocrine PC12 cell line, it was suspected that both proteins were being rapidly degraded. This led to the discovery that both stonin 1 and 2 were being targeted for degradation by the ubiquitin proteasome system that appears to be neuroendocrine specific. Stonin 2 is additionally cleaved by an unknown protease to produce C-terminal fragments that are the predominant protein species in PC12 cells. The analysis of the stonin 2 protein fragmentation pattern compared to the endogenous banding pattern led to the discovery that protein variation is also achieved through initiation of translation at internal start sites. Although the specific methionine residues were not characterised, at least two protein species were identified that were the result of internal initiation. These protein species may be regulated through the use of a short upstream open reading frame that overlaps the predicted stonin 2 ORF start codon. The internal start sites for translation initiation would give rise to N-terminally truncated protein species that would lack one or two critical AP-2 binding motifs that may modulate the function of stonin 2. The final series of experiments presented in this thesis analyse the localisation and protein-protein interactions of the stonin proteins through immunocytochemical analysis of exogenously expressed-tagged stonin proteins during inhibition of the ubiquitin proteasome system. This lead to the finding that when stonin 1 is bound to internal membranes it is predominantly bound to internal structures that can be motile. Under these conditions, it was also shown that stonin 1 could bind synaptotagmin I, although it is unlikely to occur at the plasma membrane. Membrane-bound Stonin 2 is found predominantly sub-cortically or at the plasma membrane. The sub-cortical localisation may be at sites that vesicles are, or will be, attached to the plasma membrane. Stonin 2 can be part of a protein complex that involves AP-2, synaptotagmin I and the fission molecule dynamin I. These results provide new insight into the role that both stonin 1 and 2 play in the cellular trafficking network. The protein variation identified in Stonin 2 adds further complexity to the myriad of regulatory events that can occur to modulate synaptic plasticity.
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    Characterisation of putative fatty acyl-CoA synthetases and genes involved in �-oxidation in Aspergillus nidulans
    Reiser, Kathrin. (University of Melbourne, 2009)
    Aspergillus nidulans is able to use short and long chain fatty acids as sole carbon and energy sources via the 13-oxidation pathway. This pathway occurs in both peroxisomes and mitochondria of A. nidulans. While various genes encoding the mitochondrial 13- oxidation enzymes are known, the genes encoding the peroxisomal ?-oxidation proteins have not been fully described. To investigate the first step of peroxisomal 13-oxidation two putative fatty acyl-CoA dehydrogenases, AcdA and AcdB, and two putative fatty acyl-CoA oxidases, AoxA and AoxB, were identified in the genome by blast search using the Saccharomyces cerevisiae fatty acyl-CoA oxidase FoxA and a Neurospora crassa fatty acyl-CoA dehydrogenase. The localisation of these proteins, the induction of the genes and the gene deletion phenotypes have been characterised to assess their possible involvement in ?-oxidation. The results have shown that AoxA is the major fatty acyl-CoA oxidase for peroxisomal long chain fatty acid utilisation, however, the leaky loss-of-growth phenotype of the aoxA? strain implies that there are additional peroxisomal ?-oxidation pathways. The induction of the four putative fatty acyl-CoA dehydrogenases and fatty acyl-CoA oxidases in response to fatty acids was shown to be dependent on the transcriptional regulators FarA required for the induction by short and long chain fatty acids and FarB and ScfA required for short chain fatty acid induction. This pattern of induction was observed for aoxA, while acdA, acdB and aoxB show a previously undescribed induction pattern with FarB and ScfA being required for short chain and long chain fatty acid induction and FarA for short chain fatty acid induction only. The occurrence of two induction patterns implies a more complex regulation by the three regulatory proteins. Six putative fatty acyl-CoA synthetases, FatA, FatB, FatC, FatD, FaaA and FaaB, have been identified using the S. cerevisiae fatty acyl-CoA transporters and synthetases Fat1, Fat2, Faa1, Faa2, Faa3 and Faa4. Investigation of the localisation of these proteins and phenotypes associated with the deletion of these genes showed that FaaB is likely to be the major peroxisomal fatty acyl-CoA synthetase and activates fatty acids with a wide range of chain lengths. The effect of deletion of the genes encoding these putative fatty acyl-CoA synthetases on the regulation of aoxA and acuJ was investigated to determine whether any of the putative fatty acyl-CoA synthetases were required to generate activated fatty acids for induction. It is clear that there are multiple genes contributing to fatty acid utilisation and that there is considerable redundancy. Additional genome and phylogenetics analyses have identified a variety of additional proteins, which may be involved in peroxisomal ?-oxidation pathways, as well as a potential mitochondrial fatty acyl-CoA synthetase (AN4659.3). This protein is predicted to localise to mitochondria, contains a short chain fatty acyl-CoA synthetase motif and is likely to be a synthetase activating short chain fatty acids, which are utilised in mitochondria.
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    Regulation of gluconeogenesis in aspergillus nidulans
    Suzuki, Yumi. (University of Melbourne, 2009)
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    Novel roles of cytochrome P450 genes in insect development
    Sztal, Tamar Esther. (University of Melbourne, 2009)
    Cytochrome P450s form one of the largest enzyme superfamilies. They are involved in the catalysis of a number of different chemical reactions and are involved in a wide range of biological processes. In the vinegar fly, Drosophila melanogaster, 85 P450s have been identified. This thesis uses genetic approaches to investigate the roles of specific P450s in different aspects of D. melanogaster development and reproduction. The expression patterns of all 85 P450s in the D. melanogaster y ; cn, bw; sp strain were analysed by RT-PCR and in situ hybridisation during embryogenesis. In situ hybridisation patterns were obtained from 50 out of 65 P450s expressed during embryogenesis. Many novel expression patterns were identified; Cyp28c1 was detected in the salivary glands, Cyp49a1 was detected in the hindgut and Cyp18a1 was detected in the epidermis. These results were compared with larval in situ hybridisation patterns of P450s previously characterised, to identify P450s with conserved expression throughout the life-stage. An RNAi screen was also performed. Of the 60 P450s targeted by RNAi, nine were shown to be lethal (Cyp18a1, Cyp28c1, Cyp306a1, Cyp309a1, Cyp311a1, Cyp314a1, Cyp4c3, Cyp4d2, Cyp4g1, Cyp6g2) and three showed reduced survival (Cyp318a1, Cyp4ac2, Cyp4s3). The function of Cyp301a1 was also investigated using in situ hybridisation and RNAi. Cyp301a1 was detected in the hindgut and epidermis in D. melanogaster y; cn, bw; sp embryonic and adult stages. RNAi knockdown of Cyp301a1 as well as analysis of the Cyp301a1f02301 mutant flies showed disrupted cuticle phenotypes, suggesting that Cyp301a1 has a role in cuticle formation. Cyp6g2 was the only P450 detected in the corpus allatum, the site of juvenile hormone synthesis. RNAi knockdown of Cyp6g2 is lethal at pupal stages and lethality can be rescued by over-expression of D. willistoni Cyp6g2. Cyp6g2 appears to be unique to the Drosophila genus and is highly conserved in all twelve sequenced Drosophila genomes. Cyp6g2 expression fluctuates dynamically during larval and pupal development and is roughly correlated with high JH titres. Knockdown of Cyp6g2 disrupts the expression of other genes known to be regulated by JH, suggesting that Cyp6g2 may have an essential role in juvenile hormone regulation. Interestingly, Cyp6g2 is also expressed in the adult male ejaculatory bulb and was differentially expressed during mating, indicating an additional reproductive role. The evolution of the Cyp307a genes in Drosophila was investigated. The Cyp307a genes are involved the synthesis of 20-hydroxyecdysone, an essential insect moulting hormone. In D. melanogaster, there are two Cyp307a genes; Cyp307a1, which was detected in the yolk cells of the early embryo and in the follicle cells of the female ovary and Cyp307a2, which was detected in the prothoracic cells of the ring gland. Phylogenetic and microsynteny analyses in Drosophila showed a complicated evolutionary scenario for the Cyp307a genes, invoking multiple gene duplications (and subsequent losses) in different lineages. The varied temporal and spatial expression of the Cyp307a genes in Drosophila suggested that they have undergone independent evolution and sub-functionalisation within the Drosophila genus. Finally, this thesis investigated roles of cytochrome P450s in reproduction. Using in situ hybridisation, several P450s were detected in the adult female reproductive tissues. Cyp6a19 and Cyp6a22 were highly expressed in the nurse cells whereas Cyp18a1 was detected in the follicle cells of the developing oocyte. Cyp305a1 was detected in the follicle cells covering the late stage oocyte and in the dorsal region specifying the appendage. Ubiquitous knockdown of Cyp305a1 by RNAi was lethal, suggesting it plays an essential role during development. RNAi of Cyp305a1 in the follicle cells, showed defects in nurse cell dumping and dorsal appendage formation. Several P450s were also detected in the adult male reproductive system, with Cyp312a1 expressed highly in the developing spermatocytes of the male testes. RNAi knockdown of Cyp312a1 was viable and Cyp312a1 RNAi progeny showed normal testes development. However Cyp312a1 RNAi males were less fertile, which may be due to defects in sperm maturation or protection in the female reproductive tract.