The calorie restriction (CR) and insulin/IGF-I-like signalling (IIS) are two pathways regulating the lifespan of C. elegans. Recent studies showed that glucose restriction extends the lifespan of C. elegans while excessive glucose shortens the lifespan of the worms. The first step of the glucose metabolism is the transport of glucose across the plasma membrane by the glucose transporters. The work described in this thesis aims to identify glucose transporters in C. elegans and to provide a primary investigation of the in vitro and in vivo function of the identified glucose transporter. Nine putative transporters have been cloned and expressed. Out of the nice cloned putative transporters in the C. elegans genome, H17B01.1 (H17) only is identified as a fully functional glucose transporter using an oocyte expression system in which glucose transport activity is directly measured. The two transcripts of H17 are both capable of transporting glucose with high affinity, as well as transporting trehalose. Heterologous expression of H17 in mammalian CHO-T cells suggests that the protein is localised both on the plasma membrane and in the cytosol. In vitro studies of H17 show that the protein does not respond to insulin stimulation when expressed in mammalian CHO-T cell and rat primary adipocyte systems. In vivo functional studies using H17 RNAi indicate that the worm’s lifespan is not affected by the H17 knockdown. However, glucose metabolism of C. elegans (as measured by glucose oxidation to CO2 and incorporation into fat reserves) is influenced by the decreased expression of H17, especially in the daf-2 mutant strain, e1370. However, the increase of glucose metabolism caused by H17 knockdown observed in daf-2 mutant is inhibited in the age-1 and akt-1 mutant strains. The findings reported in this thesis suggest that the H17 glucose transporter may play an important role glucose metabolism in C. elegans and that this transport and metabolism is influenced by insulin receptor activity and serine kinase cascades.

Understanding how the human brain functions on a molecular and cellular level is nearly impossible with current technology and ethical considerations. Utilizing the small nematode, Caenorhabditis elegans, and its innate behavioral responses to olfactory social cues, we can begin to unravel the mechanisms underlying social behavior. This is made possible given that innate behaviors are crucial for survival, and therefore hardwired into the genome of organisms. This allows for genetic-level analysis of neural circuitries driving behavior. Studying the neuronal mechanisms underlying C. elegans’ behavioral responses to social cues will not only assist in our overall understanding of how the brain perceives stimuli to enact a behavioral response at the cellular and molecular level, but also our understanding as to how the nervous system properly integrates information to enact social behavioral responses: mis-integration and social abnormalities are commonalities seen in many neuropsychiatric disorders, and these studies will provide fruitful insights into the defects observed in these disorders. Lastly, by comparing the perception of several different types of social chemicals, we can further our understanding of neural coding strategies for the various behaviors crucial for survival. Chapter One of this thesis orients the reader to social, innate behavior, and the usefulness of C. elegans as a tool for understanding behavioral coding. Chapter Two explores and establishes the required components of a socially aversive pheromone, providing insight into signaling evolution and co-option of biological machineries. Chapter Three examines how multiple, competing stimuli are integrated to modulate behavioral output, furthering our understanding of molecular and cellular integration and decision making within the nervous system. Chapter Four highlights the importance of predator pressure, and provides insights into circuit strategies of redundant and promiscuous networks of threat detection. Lastly, Chapter Five considers the implications of these findings as a whole, in the perspective of evolutionary strategies leading to neuronal coding of different behavioral outputs. Taken together, this dissertation aimed to fill the void in our understanding of social behavior neural circuitries, and how integration governed at the molecular and cellular level of the nervous system affects those behaviors.

C. elegans is as an extremely powerful model for the study of innate immunity. MAP kinase signaling pathways in C. elegans are involved in the response of C. elegans to infection by pathogenic bacteria. The yeast S. cerevisiae can infect C. elegans, producing pathogenic effects. In this project, we tested whether several MAP kinase pathways are important for C. elegans¡¯ resistance to yeast infection. We tested members of several MAP kinase pathways including tir-1, nsy-1, sek-1 and pmk-1 in the p38 pathway, mek-1, jnk-1 and kgb-1 in JNK pathway and mek-2 and mpk-1 in the ERK pathway. We used survival assays to compare the responses of mutants of components of these pathways to the control responses of wild-type C. elegans. In the survival assay, we found that mutants in all three MAP kinase pathways showed a decreased survival relative to wild type; therefore all three pathways are important for innate immunity against the yeast pathogen. With respect to the p38 pathway, mutations affected survival but not the deformed anal region (Dar) phenotype, a putative defensive response induced by yeast in wild-type C. elegans. This indicates that for the p38 pathway, survival depends on some other immune response besides Dar. Finally, we hypothesize that cross talk occurs between p38 and JNK MAPK pathways in the C. elegans immune responses.

Organisms must coordinate behavioral and physiological responses to changingenvironmental conditions. In the nematode C. elegans, the presence or absence of foodin the environment affects many metabolic and behavioral responses, including fathomeostasis, lifespan, and male mating. Specifically, male mating behavior normallyoccurs when a well-nourished male encounters a hermaphrodite, and is repressed if themale is under-nourished. To understand how environmental changes influence the driveto carry out specific behavioral tasks, I used C. elegans male mating as a model.Previously, mutants were isolated that display male mating behavior at inappropriatetimes, i.e. in the absence of mating cues. Loss of function mutations in the ERG K+channel, UNC-103, results in spontaneous seizures of the male sex muscles.Interestingly, I found that food deprivation can suppress unc-103(lf)-induced seizures,suggesting that pathways activated under this environmental condition can suppress theexcitability of the mating circuit.Using molecular, genetic, and behavioral assays, I identified sensory andmolecular mechanisms that reduce sex-muscle excitability under food-deprived conditions. I found that mutations that affect the muscular feeding organ, the pharynx,phenocopy the effects of food deprivation, and reduce sex-muscle excitability. Idemonstrated that mutations in the pharyngeal muscle protein, tropomyosin, cause thepharyngeal neurosecretory motor neurons (NSMs) to increase pharyngeal excitabilityand reduce sex-muscle excitability. Additionally, I found that olfactory neurons (AWCs)with sensory cilia exposed to the environment are up-regulated in the absence of foodstimuli, and also send inhibitory signals to the sex muscles. To determine howchemosensory and pharyngeal neurons in the head can signal to the genitalia, Ihypothesized that one mechanism could be via secretion of metabolic hormones. To testthis, I examined loss-of-function mutations in the insulin-like receptor, DAF-2, which isknown to regulate many behavioral and physiological responses to food. I demonstratedthat DAF-2 activity in the sex muscles is required for food-deprivation suppression ofunc-103(0)-induced seizures. I then identified components of a novel-insulin-like/DAF-2signaling pathway that reduces excitability. Specifically, I propose that ligand binding toDAF-2 activates PLC- and leads to increased cystolic Ca2+. This Ca2+ influx activatesCaMKII, which can phosphorylate/activate EAG-like K+ channels, thereby reducing cellexcitability.

Insulin signaling plays a prominent role in regulation of dauer formation and longevity in Caenorhabditis elegans. Here, I show that insulin signaling also is required in benzaldehyde-starvation associative plasticity, where worms pre-exposed to the odor attractant benzaldehyde in the absence of food subsequently demonstrate a conditioned aversion response towards the odorant. Animals with mutations in ins-1, daf-2, and age-1 which encode the homolog of human insulin, insulin/IGF-1 receptor, and PI-3 kinase, respectively, have significant deficits in benzaldehyde-starvation associative plasticity. Using a conditional allele I show that the behavioral roles of DAF-2 signaling in associative plasticity can be dissociated, with DAF-2 signaling playing a more significant role in the memory retrieval than in memory acquisition. I propose DAF-2 signaling acts as a learning specific starvation signal in the memory acquisition phase of benzaldehyde-starvation associative plasticity but functions to switch benzaldehyde-sensing AWC neurons into an avoidance signaling mode during memory retrieval.

E3 ubiquitin ligases are important regulators of several cellular processes, including apoptosis. To determine the extent to which E3 ligases regulate DNA damage-induced apoptotic signalling in C. elegans, a high-throughput RNAi screen was performed in our laboratory. We identified the E3 ubiquitin ligase EEL-1 as a positive regulator of DNA damage-induced germ cell apoptosis. ARF-BP1, the mammalian EEL-1 ortholog, negatively regulates both the tumour suppressor protein p53 and the anti-apoptotic protein Mcl-1. In C. elegans, we found that eel-1 regulates DNA damage-induced germ cell apoptosis by a mechanism downstream of cep-1/p53 and upstream of ced-9/mcl-1. My results show that unlike ARF-BP1, EEL-1 does not regulate CED-9/Mcl-1 protein levels, suggesting a novel mechanism of apoptosis regulation in C. elegans for this E3 ligase. Unexpectedly, eel-1 causes synthetic sterility in ced-9 loss-of-function mutants that is suppressed by ablation of the Apaf-1 orthologue ced-4, suggesting an additional role for these genes in oogenesis.

E3 ubiquitin ligases are important regulators of several cellular processes, including apoptosis. To determine the extent to which E3 ligases regulate DNA damage-induced apoptotic signalling in C. elegans, a high-throughput RNAi screen was performed in our laboratory. We identified the E3 ubiquitin ligase EEL-1 as a positive regulator of DNA damage-induced germ cell apoptosis. ARF-BP1, the mammalian EEL-1 ortholog, negatively regulates both the tumour suppressor protein p53 and the anti-apoptotic protein Mcl-1. In C. elegans, we found that eel-1 regulates DNA damage-induced germ cell apoptosis by a mechanism downstream of cep-1/p53 and upstream of ced-9/mcl-1. My results show that unlike ARF-BP1, EEL-1 does not regulate CED-9/Mcl-1 protein levels, suggesting a novel mechanism of apoptosis regulation in C. elegans for this E3 ligase. Unexpectedly, eel-1 causes synthetic sterility in ced-9 loss-of-function mutants that is suppressed by ablation of the Apaf-1 orthologue ced-4, suggesting an additional role for these genes in oogenesis.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

Insulin signaling plays a prominent role in regulation of dauer formation and longevity in Caenorhabditis elegans. Here, I show that insulin signaling also is required in benzaldehyde-starvation associative plasticity, where worms pre-exposed to the odor attractant benzaldehyde in the absence of food subsequently demonstrate a conditioned aversion response towards the odorant. Animals with mutations in ins-1, daf-2, and age-1 which encode the homolog of human insulin, insulin/IGF-1 receptor, and PI-3 kinase, respectively, have significant deficits in benzaldehyde-starvation associative plasticity. Using a conditional allele I show that the behavioral roles of DAF-2 signaling in associative plasticity can be dissociated, with DAF-2 signaling playing a more significant role in the memory retrieval than in memory acquisition. I propose DAF-2 signaling acts as a learning specific starvation signal in the memory acquisition phase of benzaldehyde-starvation associative plasticity but functions to switch benzaldehyde-sensing AWC neurons into an avoidance signaling mode during memory retrieval.

p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.