DNA polymerase η (pol η), a crucial component of the cellular translesion synthesis pathway, allows cells to bypass and thereby temporarily tolerate DNA damage. Inherited deficiency of pol η, as reported in the variant form of xeroderma pigmentosum, predisposes to UV light-induced skin cancers. To date, pol η is the only DNA polymerase shown to exhibit a causal link to the formation of cancers in humans. However, the role of pol η in the cellular response to forms of DNA damage other than UV-induced lesions is largely unknown. In the first part of this thesis, it is shown that cells deficient in pol η are resistant to ionising radiation. Deficiency in the polymerase was associated with accumulation of cells in S phase of the cell cycle. Cells deficient in pol η demonstrated increased homologous recombination-directed repair of DNA double-strand breaks created by ionising radiation, and depletion of the homologous recombination protein X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells compared to pol η-complemented cells. These findings suggest that homologous recombination mediates S phase-dependent radioresistance associated with pol η-deficiency. In the second part of this thesis, it is shown that pol η-deficient cells have increased sensitivity to the chemotherapeutic compound, oxaliplatin, compared to pol η-deficient expressing cells, but not to the drug 5-fluorouracil that is usually administered in combination with oxaliplatin in the clinical setting. Despite the importance of pol η for cellular survival following exposure to oxaliplatin, the drug did not upregulate the enzyme after either short-term or long-term exposure. Inhibition of pol η activity by siRNA-mediated knockdown of the protein sensitised cells to oxaliplatin treatment, and partially reversed acquired resistance in oxaliplatin-resistant tumour cell lines. These data suggest that pol η is an interesting target whose function can potentially be interfered with to optimise oxaliplatin-based chemotherapy. In the third part of this thesis, clinical samples obtained from oesophageal cancer patients before and after treatment with oxaliplatin-containing chemotherapy were analysed for POLH mRNA levels encoding pol η protein. Malignant tissue specimens obtained before treatment demonstrated a significantly higher level of POLH mRNA than matched normal oesophageal tissue samples. Contrary to the preclinical data, high POLH mRNA expression before therapy was shown to correlate with increased overall and disease-free survival of the patient cohort in the clinical trial. Additionally, patients with high POLH mRNA-expressing cancers had better therapeutic responses (measured by PET-CT) to oxaliplatin-based treatment than those with low levels. These data suggest that POLH mRNA expression should be tested as a biomarker to predict survival and therapeutic responses in oesophageal cancer patients treated with oxaliplatin-containing chemotherapy.

A significant advance in understanding skeletal muscle adaptation to physical training has been the observation that nutrients and exercise work in synergy to enhance muscle protein synthesis. Physical activity triggers an adaptive response to which nutrition provides the necessary building blocks for an optimal response. The aim of the present work was to contribute to the understanding of the molecular events induced by exercise or nutrients (creatine and amino acids) to create the adaptive environment and to induce the cellular adaptation and growth, respectively. From a methodological point of view, two experimental models were used: muscle biopsies taken from the vastus lateralis of human volunteers and myotubes cultured from C2C12 cells. The transcription of a series of genes involved in muscle remodelling (MAFbx, MHCIIA, PGC-1á, PCNA and IL-6) was increased immediately after the completion of a resistance exercise session performed in the fasted state. The phosphorylation state of p38 and ERK1/2 was also increased, whereas the Akt/PKB pathway was negatively regulated. This contrasted with the high phosphorylation state observed on p70s6k and 4E-BP1 when subjects received a large amount of amino acids during the recovery period. Our results suggest that the MAPK pathway can be triggered by contractile activity alone, whereas the Akt/PKB pathway requires nutrients to be activated. Certain amino acids regulate the phosphorylation state of mTOR and its downstream targets, as demonstrated by one of our in vitro studies. However, that modulation did not lead to a systematic modification in the rate of protein synthesis. Amino acids were also able to influence the expression of muscle-specific genes, highlighting their importance in the control of muscle protein synthesis. Protein anabolism was largely enhanced and cell differentiation was accelerated by creatine in our in vitro model. We have identified the p38 and Akt/PKB pathways as mediators of these effects. Nevertheless, we were unable to confirm the existence of similar events in human skeletal muscle in vivo.

The accurate development of the wiring between the billions of neurons in our brain is fundamental to brain function. Development of this connectivity relies on activity-dependent modification of synapses similar to those that underlie learning and memory. Glutamate is the principal excitatory neurotransmitter in the mammalian brain and several brain disorders result from altered glutamatergic receptor signalling (Catania et al., 2007; Lau and Zukin, 2007). Genes encoding glutamate receptor associated proteins have a high incidence of mutation in cognitive disorders, especially X-linked mental retardation (MR)(Laumonnier et al., 2007). MR has long been associated with altered cortical connectivity, particularly dendritic spine dysgenesis. There is also an emerging view that aberrant local protein synthesis within dendrites and protein trafficking to dendrites underlies some forms of MR (Kelleher and Bear, 2008; Pfeiffer and Huber, 2006; Zalfa and Bagni, 2005). Most studies examining the role of glutamatergic receptors in MR have focused on adults. Little is known about how these MR genes regulate brain development despite their neurodevelopmental aetiology. Fragile X mental retardation (FXS) is the most common form of inherited MR and results from the loss of fragile X mental retardation protein (FMRP). FMRP is a RNA binding protein and is hypothesised to have a role in protein trafficking from nucleus to sites of synapses, and regulating local protein synthesis at sites of synapses (Bagni and Greenough, 2005). A prevalent theory of FXS causation is ‘metabotropic glutamate receptor (mGluR) theory of fragile X’, which postulates that all functional consequences of mGluR (predominantly mGluR5)-dependent protein synthesis maybe exaggerated in FXS (Bear et al., 2004). Primary somatosensory cortex (S1) of rodents provides an excellent model system to study the role of MR genes in development because of its stereotypic, glutamate receptor-dependent, anatomical development (Barnett et al., 2006b; Erzurumlu and Kind, 2001). Hannan et al., (2001) reported that genetic deletion of mGluR5 results in loss of ‘barrels’, the anatomical correlates of rodent whiskers in S1. Chapter 3 extends these findings to show that there is expression of mGluR5 as early as P4 in S1 prior to segregation of layer 4 cells into barrels suggesting a tropic role for glutamate in barrel formation. The expression of mGluR5 is postsynaptic during barrel formation and does not regulate tangential or radial cortical development. Its effects on barrel segregation are dose dependent and are not due to a developmental delay. During late S1 development, loss of mGluR5 results in decreased spine density suggesting a role in synaptogenesis. Supporting this hypothesis in mGluR5 mutant mice there is a general decrease in expression of synaptic markers in early S1 development. Chapter 4 explores the role of FMRP in cortical development. FMRP is expressed early in S1 development with peak expression prior to synaptogenesis at P14. It is expressed postsynaptically at P7 and pre and postsynaptically at P14. FMRP does not regulate cortical arealisation during barrel formation but results in decreased barrel segregation. In the absence of FMRP, biochemical studies show altered expression of glutamatergic receptors in the neocortex P7 and P14 suggesting altered glutamatergic receptor composition at synaptic sites. During late S1 development, loss of FMRP results in increased spine density in layer 4 spiny cells. Together these data indicate a role for FMRP during early and late S1 development. Chapter 5 directly tests the mGluR theory of FXS by examining whether genetic reduction of mGluR5 levels rescues anatomical phenotypes characterised in Fmr1-/y mice. The defect in barrel formation in Fmr1-/y mice is partially rescued by reducing mGluR5 levels. However, layer 4 spine density in Fmr1-/y mice does not appear to be rescued. Chapter 6 explores the expression patterns of three key synaptic MAGUKs (Membrane associated guanylate kinases) PSD95, SAP102 and PSD93, one of which (PSD95) is regulated by FMRP (Zalfa et al., 2007) and the others which have putative binding sites for FMRP. MAGUKs tether glutamatergic receptors to their associated signalling complexes at the postsynaptic membrane and also regulate glutamatergic receptor trafficking (Collins and Grant, 2007; Kim and Sheng, 2004). The immunohistochemical expression profiles of PSD95, SAP102 and PSD93 show dynamic regulation during S1 development that is unaffected by loss of FMRP (at P7), and biochemical data indicates that basal levels of these MAGUKs in neocortex are unaltered at P7 and P14 in Fmr1-/y mice. In Sap102-/y and Psd95-/- mice, there is altered expression of several synaptic proteins biochemically providing evidence for differential roles of SAP102 and PSD95 in regulating expression of glutamatergic receptors at synaptic sites during early S1 development. This thesis demonstrates that synaptic proteins associated with MR are expressed early in development and display regulatory roles in cellular processes governing S1 formation. An understanding of their role in early brain development would be critical in fully appreciating when and where they exert their regulatory effects, and this in turn would be beneficial in designing therapeutic interventions.

No description available.

The prevalence of type II diabetes is increasing at an alarming rate due to the western world lifestyle. Type II diabetes is characterized by an insulin resistance distinguished by impaired glucose uptake in adipose and muscle tissues. The molecular mechanisms behind the insulin recistance and also the knowledge considering normal insulin signalling in fat cells, especially in humans, are still unclear. Insulin receptor substrate (IRS) is known to be important for medating the insulin-induced signal from the insulin receptor into the cell. We developed and optimized a method for transfection of primary human adipocytes by electroporation. By recombinant expression of proteins, we found a proper IRS to be crucial for both mitogenic and metabolic signalling in human adipocytes. In human, but not rat, primary adipocytes we found IRS1 to be located at the plasma membrane in non-insulin stimulated cells. Insulin stimulation resulted in a two-fold increase of the amount of IRS1 at the plasma membrane in human cells, compared with a 12-fold increase in rat cells. By recombinant expression of IRS1 we found the species difference between human and rat IRS1 to depend on the IRS proteins and not on properties of the host cell. The adipocytes function as an energy store, critical for maintaining the energy balance, and obesity strongly correlates with insulin resistance. The insulin sensitivity of the adipocytes with regard to the size of the cells was examined by separating small and large cells from the same subject. We found no increase of the GLUT4 translocation to the plasma membrane following insulin stimulation in the large cells, whereas there was a two-fold increase in the small cells. This finding supports the idea of a causal relationship between the enlarged fat cells and reduced insulin sensitivity found in obese subjects. The insulin receptor is located and functional in a specific membrane structure, the caveola. The morphology of the caveola and the localization of the caveolar marker proteins caveolin-1 and -2 were examined. Caveolae were shown to be connected to the exterior by a narrow neck. Caveolin was found to be located at the neck region of caveolae, which imply importance of caveolin for maintaining and sequestering caveolae to the plasma membrane. In conclusion, the transfection technique proved to be highly useful for molecular biological studies of insulin signal transduction and morphology in primary adipocytes.

Calcium is a highly versatile intracellular signal molecule that can regulate numerous different cellular functions. In filamentous fungi there is evidence for it being involved in regulating various processes, including spore germination, hyphal tip growth, hyphal branching and conidiation. During colony initiation in the filamentous fungus Neurospora crassa, conidia form germ tubes which are involved in colony establishment, and conidial anastomosis tubes (CATs) which are involved in generating fused networks of conidial germlings. The primary research aim of this thesis was to analyze the role of Ca2+-signalling and homeostasis during colony initiation in N. crassa. Removal of Ca2+ from the growth medium showed that external Ca2+ was necessary for CAT fusion and, more specifically, was required for CAT chemoattraction. Two L-type Ca2+ channel blockers (verapamil and diltiazem) with different modes of action were found to inhibit both conidial germination and CAT fusion in wild type strains and CAT fusion was shown to be more sensitive to these two drugs. These channel blockers were additionally found to inhibit Ca2+ uptake by conidial germlings of the wild type expressing the aequorin Ca2+ reporter. However, the channel blockers also, unexpectedly, raised the cytosolic free Ca2+ ([Ca2+]c) resting level in these germlings suggesting that they did not just inhibit L-type Ca2+ activity. The morphological phenotypes (conidial germination, hyphal extension rate, conidiation and hyphal branching) of 22 mutants defective in different components of their Ca2+-signalling and homeostasis machinery were characterized in order to identify their possible roles of Ca2+ during colony initiation and development. The ∆cch-1 mutant lacking the CCH-1 L-type Ca2+ channel gene exhibited a reduction in CAT fusion. CAT fusion was decreased even further in a double mutant (∆cch-1∆mid-1) suggesting that that the CCH-1 and MID-1 proteins operate in combination during this process. Increased extracellular Ca2+ partially restored the phenotypes of the ∆cch-1, ∆mid-1 smco-1 and ∆cch-1∆mid-1 mutants which is consistent with CCH-1 and MID-1 being involved in Ca2+ uptake from the external medium. Calcium signatures following mechanical perturbation were successfully measured in populations of conidial germlings using aequorin expressed in the wild type and in deletion mutants (∆cch-1, ∆yvc-1, ∆fig-1) lacking different Ca2+ channels. The removal of external Ca2+ completely abolished the [Ca2+]c increase in response to mechanical perturbation and CCH-1 was found to partly contribute to this increase in [Ca2+]c. Various Ca2+-sensitive dyes (Oregon green 488, Fluo-4 and Calcium Green-1) were also tested to determine if they can be used to image [Ca2+]c at the single cell and subcellular levels. Only Fluo-4 allowed the measurement of [Ca2+]c in individual cells but the changes in dye fluorescence in response to changes in [Ca2+]c were too small to be useful for imaging [Ca2+]c dynamics at the subcellular level. The other two dyes underwent rapid compartmentalization in organelles when loaded into germlings. The plant antifungal proteins (defensins), MsDef1, MtDef4 and PAF were all found to disrupt Ca2+ signaling/homeostasis in conidial germlings of N. crassa. They all inhibited the [Ca2+]c increase and raised the resting level of [Ca2+]c in response to mechanical perturbation. Analysis of an aequorin expressing mutant that was defective in glucosylceramide synthase (∆gcs) showed that the effects of MsDef1 (but not MtDef4) on [Ca2+]c were mediated by the sphingolipid glucosylceramide. All of the defensins tested were found to exhibit different potencies with regard to their inhibitory effects on conidial germination and CAT fusion.

Variant GnRH ligand and receptor subtypes have been identified in a number of non-mammalian vertebrate species, however research into avian species GnRH systems is lacking. Two isoforms of GnRH are present in the domestic chicken, the evolutionarily conserved GnRH-II and diverged cGnRH-I. The expression of two GnRH ligands parallels the expression of two chicken GnRH receptor subtypes; cGnRH-R-I and the novel cGnRH-R-III. The occurrence of two isoforms of the receptor in the chicken raises questions about their specific biological functions and interactions with the two ligands. Differential roles for these molecules in regulating gonadotrophin secretion or other functions are currently unclear. To investigate this, cGnRH-R-III cDNA was cloned from a broiler chicken anterior pituitary gland and its structure and expression was compared with cGnRH-R-I. Expression profiling of cGnRH-R-III cDNA showed that it is predominantly expressed in the anterior pituitary, approximately 1400 times more abundantly than cGnRH-R-I suggesting that cGnRH-R-III is the predominant regulator of chicken gonadotrophin synthesis and secretion. Additionally, pronounced sex and age differences existed, with higher pituitary cGnRH-R-III mRNA levels in sexually mature females versus juvenile females. In contrast, higher mRNA expression levels occurred in juvenile males compared to sexually mature males. Determination of ligand-binding selectivity and the level of cGnRH-R-III activation in response to the endogenous ligands, cGnRH-I and GnRH-II, was anticipated as facilitating the elucidation of the physiological roles of the receptor subtypes. Additionally, the development of analogs that differentially promote or inhibit activation of the receptor subtypes may be valuable tools for determining the role of receptor types in the regulation of gonadotrophin production. To investigate this, pharmacological profiling of cGnRH-R-III in terms of ligand-binding selectivity and inositol phosphate production in response to GnRH analogs was determined in comparison with the pharmacological profile of cGnRH-R-I. Functional studies in COS-7 cells indicated that cGnRH-R-III has a higher binding affinity for GnRH-II than cGnRH-I (IC50: 0.57 v 19.8 nM) and more potent stimulation of inositol phosphate production (EC50: 0.8 v 4.38 nM). Similar results were found for cGnRH-R-I, (IC50: 0.51 v 10.8 nM) and (EC50: 0.7 v 2.8 nM). Mammalian receptor antagonist 27 distinguished between cGnRH-R-I and cGnRH-R-III (IC50: 2.3 v 351 nM), and application of this synthetic peptide may facilitate delineation of receptor subtype function either in-vitro or in-vivo. The length of the C-terminal tail of cGnRH-R-III is 8 residues longer than that of cGnRH-R-I and this observation stimulated investigation of differences in ligand-induced internalisation between the two receptor subtypes. The initial rate of receptor internalisation was faster for cGnRH-R-III than for cGnRH-R-I (26%.min-1 v 15.8%.min-1). Although proteins encoded by cGnRH-R-III splice variants do not bind GnRH ligands independently and mRNAs were not detectable by Northern blot analysis, cGnRH-R-III_SV2 significantly reduced maximum ligand-binding of cGnRH-R-III, suggesting that it may impair the function of the full-length type III cGnRH receptor. It was anticipated that the two cGnRH-R subtypes may have differential roles in the regulation of luteinising hormone (LH) and follicle stimulating hormone (FSH) gene transcription through the activation of differential second messenger pathways. Three putative Src homology domain 3 (SH3) binding motifs were identified in the type III cGnRH receptor cytoplasmic C-terminal tail domain which are not present in the type I cGnRH-R and suggested the potential for differential coupling to the Mitogen Activated Protein Kinase (MAPK) cascade. To investigate this possibility, activation of the MAPK cascade via cGnRH-R-III and cGnRH-R-I was determined by quantifying elevation of phosphorylated ERK (pERK 1/2) in response to GnRH. Studies performed in COS-7 cells showed a 4-6 fold increase in ERK 1/2 phosphorylation via the type I and type III receptors within 10 minutes of GnRH-I or GnRH-II stimulation, indicating that both receptors signal through the ERK 1/2 pathway in response to cGnRH-I or GnRH-II. The responses were dose-dependent at cGnRH-R-I and cGnRH-R-III. Effects of pre-treatment with PLC and c-Src inhibitors showed that both cGnRH-Rs may activate pERK 1/2 independently of PLC but dependently upon c-Src. However, it must be noted that 100% of the PLC activity was not inhibited by PLC inhibitor as measured by inositol phosphate production at 60 minutes, and the PLC inhibitor has not been shown to inhibit PLC in the same time frame used for the pERK experiments. Mutagenesis of the individual SH3 binding motifs of cGnRH-R-III were performed and the effects on pERK 1/2 levels quantified. The results indicated that the SH3 binding motifs of cGnRH-R-III do not contribute to additional MAPK activation when compared to the native cGnRH-R-III. Both cGnRH-R-I and cGnRH-R-III were HA epitope-tagged (HA-cGnRH-R-I and HA-cGnRH-R-III) and the methodology was optimised for HA-cGnRH-R-III immuno-precipitation. Several size forms of HA-cGnRH-R-III were detectable by immuno-precipitation, facilitating characterisation of the composition of the receptor protein-protein complexes formed using a western blot approach. In summary, the abundance of cGnRH-R-III expression compared to cGnRH-R-I suggests it is probably the major mediator of pituitary gonadotroph function, and that tissue-specific recruitment of cGnRH-R-isoforms has occurred in the avian pituitary during evolution. Pharmacological profiling demonstrated that cGnRH-R-III, like cGnRH-R-I, has a higher ligand-binding selectivity and induction of inositol phosphate production in response to GnRH-II than with cGnRH-I, although cGnRH-I is established as the physiological regulator of gonadotroph function. These results suggest that evolutionary recruitment of ligand-receptor pairing for particular physiological processes does not correlate with in-vitro properties such as highest ligand-binding affinity or efficacy of inositol phosphate production. Therefore evolutionary plasticity has occurred in the tissue-specific adoption of GnRH ligand and receptor subtypes for regulation of particular physiological functions in birds.

GnRH neurons represent the final common pathway for the regulation of the reproductive axis and they are modulated by multiple signals. It has recently been shown that a potent effector of GnRH neuron function is an afferent network of kisspeptin-producing neurons. Kisspeptin released from these neurons acts upon a specific receptor (gpr-54) expressed on GnRH neurons, and increases the secretion of GnRH from the hypothalamus. The kisspeptin system has since been implicated as a downstream mediator for regulation of the Hypothalamic-Pituitary-Gonadal (HPG) axis by steroid hormones, metabolic signals and photoperiod, potentially placing it at the centre of reproductive physiology. However, the supporting evidence to date has been indirect, relying on interpretation of changes in mRNA levels and immuno-histochemical staining to infer the actions of kisspeptin upon the central control of reproduction. The detailed mechanisms of kisspeptin action are yet to be fully elucidated. The research within this thesis elucidates the effect of kisspeptin on the HPG axis via the development of kisspeptin-10 (kp-10) analogues with antagonistic properties. Functionally important residues within the peptide were delineated. Structure-activity studies of kp-10 analogues indicated that residues Asn2, Trp3, Phe6, Arg9 and Phe10 interact with gpr-54 to facilitate receptor binding. Two other residues, Tyr1 and Leu8 were shown to be critical for receptor activation by kisspeptin. Four synthetic peptide antagonists were selected according to a consensus sequence for good antagonism: X1-N-W-N-X5-F-G-X8-R-F-NH2 where X1 = D-Ala or D-Tyr, X5 = Gly or D-Ser and X8 = D-Trp or D-Leu. One of the antagonists, peptide 234, was used in in vivo studies, where it inhibited the amplitude of GnRH and LH pulses without affecting basal secretion of GnRH or LH. These results indicate for the first time that basal and pulsatile secretion of these factors is regulated by separate pathways. Use of the antagonist also demonstrated the direct involvement of endogenous kisspeptin in steroid hormone negative feedback, positive regulation of the pre-ovulatory LH surge and in regulating the onset of puberty in rodents, as had been suggested via indirect methods. Although a major role of the kisspeptin system is in the regulation of the HPG axis, the system may also be important in the inhibition of cancer cell metastasis and in placental development (trophoblast cell invasion) but little is known about the mechanisms involving kisspeptin in these processes. This thesis describes novel signalling mechanisms for the regulation cell migration by kisspeptin, involving the MAPK and GSK3β signalling pathways. Using a stably transfected CHO cell line, kisspeptin-gpr-54 signalling can activate all members of the MAPK pathway, the β- catenin/GSK3β pathway, NFκB and FAK. These factors are involved in inhibiting the migration of these cells via an ERK1/2-p90rsk-GSK3β-β catenin pathway to potentially up- regulate formation of adherens junctions at the plasma membrane. This pathway was also shown to be involved in the inhibition of migration within an immortalised human first trimester placental trophoblast cell line and in human umbilical vein endothelial cells. Some of these pathways were also active within a mouse GnRH neuronal cell line, where ERK1/2, NFκB and GSK3β were activated by kisspeptin with no effect on migration. However, the role of these pathways in the GnRH neuronal cells requires further investigation. In summary, the research presented within this thesis defines receptor-binding and activating residues within kisspeptin-10, which should enable more details of ligand-receptor binding interactions to be fully elucidated. Novel gpr-54 antagonists have been identified and used in in vivo studies. The thesis demonstrates the direct involvement of endogenous kisspeptin in the regulation of GnRH/LH secretion at the onset of puberty and throughout the reproductive cycle in mature animals. The antagonists developed within this thesis represent useful tools to further delineate mechanisms of kisspeptin action within the HPG axis and peripheral tissues. Other findings describe kisspeptin signalling mechanisms for the inhibition of cell migration, potentially important in a variety of normal and pathological processes, including for the first time a description of the regulation of GSK3β and β-catenin signalling factors by kisspeptin and gpr-54.

Poly is a novel, essential protein in Drosophila melanogaster, loss of function of which results in late larval lethality. Importantly, Poly is evolutionarily conserved with a human homologue. poly mutation was isolated in a P-element mutagenesis screen that aimed to generate a larger collection of single P-element induced mutants. Mutant poly larvae are characterized by extreme larval longevity without pupation, formation of melanotic masses, smaller imaginal discs and brains, and abnormal nuclear morphology in neuroblasts. During the course of my project, I attempted to identify cellular processes and pathways that Poly might be involved in. Interestingly, my data suggest that Poly is a novel interactor and regulator of Insulin receptor/target of rapamycin (InR/TOR) signalling in Drosophila. Linking environmental cues to cell growth and metabolism is an essential process that multicellular organisms need to accomplish successfully for normal development. InR/TOR signalling is a highly conserved pathway that mediates the link between the environment and cellular processes such as growth, metabolism and ageing. My analysis in Drosophila suggests that Poly interacts physically with the InR and mutation of Poly leads to an overall down-regulation of InR/TOR signalling in Drosophila as revealed by decreases in the phosphorylation levels of Akt, S6K and 4E-BP - all downstream effectors of this pathway. In addition, loss of poly results in constitutive activation of autophagy in Drosophila fat body and a decrease in stored triglyceride levels. Furthermore, I show that localisation and levels of Poly protein are dependent on insulin action in both Drosophila and human cells. Together, these data suggest that Poly is a novel mediator of InR signalling that promotes an increase in cell growth and metabolism. Taking into consideration the observed poly mutant phenotype, I also investigated the potential involvement of Poly during cell cycle progression and the Drosophila innate immune response. While my analysis suggests that poly loss of function does not have a direct effect on cell cycle progression, alteration of Poly has consequences on various aspects of the Drosophila innate immune response. Therefore, I conclude that the Drosophila innate immune response is a cellular process in which Poly plays a crucial role.

The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex, which is required for the maintenance of genome stability. The rmi1Δ deletion mutant has proven difficult to study because it exhibits very poor growth, and rapidly accumulates second site suppressor mutations. Furthermore, deletion of the putative HJ resolvase genes, MUS81-MUS81 or SLX1-SLX4 in rmi1Δ mutants causes synthetic lethality. In order to study phenotypes caused by loss of functional Rmi1, and to explore the genetic interactions between RMI1 and the MUS81, MUS81, SLX1 and SLX4 genes, a temperature sensitive mutant of RMI1 was isolated, named rmi1-1. Similar to rmi1Δ deletion mutants, rmi1-1 cells are highly sensitive to the DNA damaging agent, MMS and the replication inhibitor, HU. In addition, rmi1-1 mutants accumulate replication-associated branched DNA structures, and arrest in G₂/M after a transient exposure to MMS. These cells are proficient in DNA damage checkpoint activation. Deletion of SLX1, SLX4, MUS81 or MUS81 in the rmi1-1 strain causes synthetic lethality, which is associated with cell cycle defects. Following a transient exposure to MMS, rmi1-1 mutants accumulate homologous recombination intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity in the absence of functional Rmi1. This resolution depends upon Mus81-Mms4, but not on Slx1-Slx4 or Yen1. I propose that while the Sgs1-Top3-Rmi1 complex constitutes the main pathway for removal of homologous recombination intermediates following a perturbed S-phase, Mus81-Mms4 can act as a back up for resolution of these intermediates, which most likely represent double Holliday junctions. In this study, I also present screens for high copy suppressors of rmi1-1 phenotypes, and for novel Rmi1 interaction partners.