Many obese individuals and type 2 diabetes mellitus (T2DM) patients have elevated levels of insulin. Hyperinsulinemia is a major cancer risk factor in T2DM individuals and activated insulin receptor (IR) has been linked to many types of cancer and poor survival. However, the mechanisms that account for the link between the hyper-active insulin signaling and cancer risk is not well understood. PTEN plays an antagonistic role in the canonical insulin signaling pathway, and is the second most commonly mutated tumour suppressor (after p53) found in human cancers. In many cancers the PTEN gene is not deleted, but instead the protein is lost. Therefore the regulation of PTEN protein in humans is of great importance. Here we hypothesized that the activated insulin signaling down-regulates PTEN. Considering that insulin signaling is highly conserved from C. elegans to human, I used C. elegans as a model and showed that DAF-2, the worm homolog of IR, is a negative regulator of DAF-18, the worm homolog of PTEN. In addition, I showed that DAF-28, the worm homolog of insulin, also negatively regulates DAF-18/PTEN. I used western blot and immunostaining to show that the protein level of DAF-18/PTEN is increased in the daf-2/IR and daf-28/insulin mutants. I further showed that daf-18/Pten is genetically epistatic to daf-2/IR in regulating neuronal development. I then employed human cell culture experiments and reported that this negative regulation is conserved in human cancer cell lines. I showed that knocking-down IR through siRNA up-regulates PTEN, and over-expressing a gain-of-function IR down-regulates PTEN. I also showed that insulin stimulation dramatically decreased PTEN and this decrease is dependent on IR. I further confirmed a physical association between IR and PTEN in both human and C. elegans, and reported that IR could phosphorylate PTEN. To provide mechanistic insight to DAF-18/PTEN regulation, I identified another protein, which is a ubiquitin ligase, that functions in insulin signaling to down-regulate DAF-18/PTEN. Additionally, I also provided evidence that insulin signaling cross talks with Eph receptor signaling. In summary, my findings will be informative for cancer biologists to study the roles of these genes in carcinogenesis. / Thesis (Ph.D, Biology) -- Queen's University, 2013-02-04 14:37:29.376

Helicases perform vital roles in the cell by unwinding D N A to make it accessible for the essential processes of replication, transcription and repair. In Caenorhabditis elegans, the DOG- 1 helicase-like protein is required for polyG/polyC-tract (G/C-tract) maintenance, as dog-l animals have a mutator phenotype characterized by deletions that initiate in G/C-tracts. DOG-1 may unwind secondary structures that form in polyguanine D N A during lagging strand replication. In order to more completely understand the role of dog-1, genetic interactors were identified, dog-1 functionally interacts with the him-6/BLM helicase. Absence of recombinational repair-implicated proteins in the dog-1 background, including HIM-6/BLM, RAD-51, BRD-1/BARD1 and HIM-9/XPF, as well as the trans-lesion synthesis polymerases polKMD po/7 increased the frequency of animals with G/C-tract deletions, indicating that these pathways are important mechanisms for repair at G/C-tracts in the absence of DOG-1. These data support the hypothesis that persisting D N A secondary structures can cause replication fork stalling, which can be resolved by deletion-free or deletion-prone mechanisms. DOG-1 has highest sequence identity to human BR1P1/FANCJ, which is mutated in patients from the Fanconi Anemia (FA) subgroup J. D N A damage sensitivity experiments indicated that, like chicken F A N C J cells, dog-1 mutants were not significantly sensitive to DNA damage from X-ray or UV-irradiation, but were extremely hypersensitive to the D N A interstrand cross-linking agent UVA-activated trimethylpsoralen. Thus, DOG-1 appears to have a conserved role in cross-link repair and is the C. elegans F A N C J homolog. Characterization of the dog-1/FANCJ-relatsd helicase, Jrh-1, revealed that mutants for this putative helicase are moderately sensitive to cross-linking agents, dog-1 jrh-1 double mutants displayed a synthetic lethal phenotype characterized by excessive recombination intermediates and mitotic catastrophe in the germline. However, absence of JRH-1 did not have any effect on G/C-tract deletions, indicating that JRH-1 does not have a redundant function with DOG-1 at G/C-tracts. Absence of JRH-1 reduced the fitness of eTl and nTl translocation hétérozygotes, but not translocation homozygotes, jrh-1 was synthetically lethal with him-6/BLM and with the endonuclease mus-81, suggesting a possible role for JRH-1 in regulating the balance between different types of repair.

<p>Recently, the study of invertebrate innate immunity has garnered considerable attention after the discovery that mammalian homologues of the <italic>Drosophila melanogaster</italic> </p><p>Toll pathway play a role in mammalian innate immunity. One invertebrate model system that has begun to be intensely studied is the nematode <italic>Caenorhabditis elegans</italic>. Immunity in <italic>C. elegans</italic> has been shown to be inducible in that it responds uniquely to different pathogens. These changes in gene expression require transcription factors in order for certain genes to be transcribed. We utilized an RNA interference screen of potential transcription factors to identify the GATA transcription factor ELT-2 as a possible transcription factor involved in immunity. We then demonstrated that ELT-2 was required for resistance to a wide range of pathogens and was responsible for regulating expression of the C-type lectin <italic>clec-67</italic>, a marker of immunity. </p><p>We also studied another transcription factor known to play a role in C. elegans immune function, the FOXO transcription factor DAF-16. We specifically focused in on the role of DAF-16 in germline-deficient mutants, and we demonstrated that such mutants are resistant to many different pathogens. This led to further investigation of the germline-deficient mutant glp-4, which should also show broad range resistance to pathogens but fails to do so. Through whole genome sequencing, we identified mutations that may be responsible for the glp-4 phenotype. We also demonstrated that DAF-16 was active in glp-4 mutants, leading to us proposing a model where glp-4 plays a role in influencing <italic>C. elegans</italic> immunity besides its involvement in germline development.</p> / Dissertation

The ability to switch between different forms of locomotion is critical to many aspects of survival, whether it is switching from walking to running to evade predators, or switching to a slower gait to obtain food. Uncovering the mechanisms behind gait transitions has implications for many fields, from treating Parkinson Disease to understanding the impact of drugs of abuse on movement. However, the mechanisms of gait transitions are not well understood. The experiments outlined in this thesis sought to understand the neuronal basis for gait switching. This work employed the nematode Caenorhabditis elegans, a unique model organism chosen for its genetic tractability and fully characterized nervous system. C. elegans displays different forms of motion: crawling on land and swimming in liquid. First, I sought to determine the mechanisms for switching between these forms of motion in collaboration with Dr. Andres Vidal-Gadea. In the process, we discovered that crawling and swimming actually represent distinct gaits in contrast to recent reports that suggested they were merely a single gait. We further elucidated mechanisms for gait transition in C. elegans. For instance, we found that the transition to crawling required viii the D1-like dopamine receptors DOP-1 and DOP-4; and activation of dopamine neurons via the light-activated cation channel Channelrhodopsin2 was sufficient to induce crawling behavior in worms immersed in liquid. Conversely, photoactivation of serotonergic neurons expressing Channelrhodopsin2 induced swim-like behavior on land. Finally, laser microablation of dopaminergic or serotonergic neurons was sufficient to impair the transition to crawl or swim, respectively. Together these results show that transitions to crawling and swimming are controlled by dopamine and serotonin respectively. Next I wanted to better understand how gait transitions are impaired by a drug of abuse, alcohol. I found that, as in other organisms, ethanol disrupts gait transitions, causing worms in water to inappropriately transition from swim to crawl and to display other land-specific behaviors. Animals lacking the D1-like dopamine receptor DOP-1 were resistant to the ethanol-induced transition to crawl. Finally, I found that several interneurons required for the transition to crawl. Specifically, laser microablation of the DOP-4 receptor-expressing neuron RID or the DOP-1-expressing neurons PQR or RIS resulted in a significant impairment in the time to crawl onset. Overall, the findings presented in this thesis represent the first evidence that C. elegans uses an evolutionarily conserved mechanism to transition between gaits and provides the beginning of a molecular description of gait transitions. / text

Helicases perform vital roles in the cell by unwinding D N A to make it accessible for the essential processes of replication, transcription and repair. In Caenorhabditis elegans, the DOG- 1 helicase-like protein is required for polyG/polyC-tract (G/C-tract) maintenance, as dog-l animals have a mutator phenotype characterized by deletions that initiate in G/C-tracts. DOG-1 may unwind secondary structures that form in polyguanine D N A during lagging strand replication. In order to more completely understand the role of dog-1, genetic interactors were identified, dog-1 functionally interacts with the him-6/BLM helicase. Absence of recombinational repair-implicated proteins in the dog-1 background, including HIM-6/BLM, RAD-51, BRD-1/BARD1 and HIM-9/XPF, as well as the trans-lesion synthesis polymerases polKMD po/7 increased the frequency of animals with G/C-tract deletions, indicating that these pathways are important mechanisms for repair at G/C-tracts in the absence of DOG-1. These data support the hypothesis that persisting D N A secondary structures can cause replication fork stalling, which can be resolved by deletion-free or deletion-prone mechanisms. DOG-1 has highest sequence identity to human BR1P1/FANCJ, which is mutated in patients from the Fanconi Anemia (FA) subgroup J. D N A damage sensitivity experiments indicated that, like chicken F A N C J cells, dog-1 mutants were not significantly sensitive to DNA damage from X-ray or UV-irradiation, but were extremely hypersensitive to the D N A interstrand cross-linking agent UVA-activated trimethylpsoralen. Thus, DOG-1 appears to have a conserved role in cross-link repair and is the C. elegans F A N C J homolog. Characterization of the dog-1/FANCJ-relatsd helicase, Jrh-1, revealed that mutants for this putative helicase are moderately sensitive to cross-linking agents, dog-1 jrh-1 double mutants displayed a synthetic lethal phenotype characterized by excessive recombination intermediates and mitotic catastrophe in the germline. However, absence of JRH-1 did not have any effect on G/C-tract deletions, indicating that JRH-1 does not have a redundant function with DOG-1 at G/C-tracts. Absence of JRH-1 reduced the fitness of eTl and nTl translocation hétérozygotes, but not translocation homozygotes, jrh-1 was synthetically lethal with him-6/BLM and with the endonuclease mus-81, suggesting a possible role for JRH-1 in regulating the balance between different types of repair.

Helicases perform vital roles in the cell by unwinding D N A to make it accessible for the essential processes of replication, transcription and repair. In Caenorhabditis elegans, the DOG- 1 helicase-like protein is required for polyG/polyC-tract (G/C-tract) maintenance, as dog-l animals have a mutator phenotype characterized by deletions that initiate in G/C-tracts. DOG-1 may unwind secondary structures that form in polyguanine D N A during lagging strand replication. In order to more completely understand the role of dog-1, genetic interactors were identified, dog-1 functionally interacts with the him-6/BLM helicase. Absence of recombinational repair-implicated proteins in the dog-1 background, including HIM-6/BLM, RAD-51, BRD-1/BARD1 and HIM-9/XPF, as well as the trans-lesion synthesis polymerases polKMD po/7 increased the frequency of animals with G/C-tract deletions, indicating that these pathways are important mechanisms for repair at G/C-tracts in the absence of DOG-1. These data support the hypothesis that persisting D N A secondary structures can cause replication fork stalling, which can be resolved by deletion-free or deletion-prone mechanisms. DOG-1 has highest sequence identity to human BR1P1/FANCJ, which is mutated in patients from the Fanconi Anemia (FA) subgroup J. D N A damage sensitivity experiments indicated that, like chicken F A N C J cells, dog-1 mutants were not significantly sensitive to DNA damage from X-ray or UV-irradiation, but were extremely hypersensitive to the D N A interstrand cross-linking agent UVA-activated trimethylpsoralen. Thus, DOG-1 appears to have a conserved role in cross-link repair and is the C. elegans F A N C J homolog. Characterization of the dog-1/FANCJ-relatsd helicase, Jrh-1, revealed that mutants for this putative helicase are moderately sensitive to cross-linking agents, dog-1 jrh-1 double mutants displayed a synthetic lethal phenotype characterized by excessive recombination intermediates and mitotic catastrophe in the germline. However, absence of JRH-1 did not have any effect on G/C-tract deletions, indicating that JRH-1 does not have a redundant function with DOG-1 at G/C-tracts. Absence of JRH-1 reduced the fitness of eTl and nTl translocation hétérozygotes, but not translocation homozygotes, jrh-1 was synthetically lethal with him-6/BLM and with the endonuclease mus-81, suggesting a possible role for JRH-1 in regulating the balance between different types of repair. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The Candida genus is full of fungi that are subtle parts of the human microbiome, but they can cause complications if they overgrow within the body—specifically the mouth and throat, the genitalia, and the entire body through infection of the bloodstream. Candida species are a rising problem for many across the world, and this can be seen in the recent threat of Candida auris hospitalizing patients and being regularly resistant to anti-fungal medications. Beyond C. auris, Candida albicans is the most common Candida species that humans must combat because it causes the most infections in humans—mostly vaginal yeast infections. C. albicans does have natural competitors that can either inhibit its growth or kill it in general, and the competition that we took advantage of was with the Alcaligenes species. Alcaligenes faecalis and Alcaligenes viscolactis have been shown to at least inhibit C. albicans growth and maybe even kill the fungus. This rate of infection from C. albicans places it at the forefront of Candida research, and we attempted to further this research by utilizing both A. faecalis and A. viscolactis to create a co-infection model for Caenorhabditis elegans—a simple nematode lifeform. It is known that A. faecalis and A. viscolactis do not commonly adversely affect humans, so little research has been done concerning their clinical effects. We were looking to find a possible answer to C. albicans infections beyond antifungal drugs because we know that antibiotic resistance is on the rise. We performed liquid assays to test the survivability of C. elegans nematodes in various bacterial/fungal circumstances. We subjected batches of C. elegans to E. coli OP50 as a control, A. faecalis, A. viscolactis, C. albicans, A. faecalis and C. albicans, and A. viscolactis and C. albicans. This procedure was followed in order to determine the viability of using the Alcaligenes species to either help the C. elegans survive the infection or prevent them from getting infected at all. After following through with the project, we found that there was a noticeable increase in the survivability of C. elegans when subjected to both one of the Alcaligenes species and C. albicans as opposed to the C. albicans alone. The data, although early, shows the possibility of Alcaligenes species being used to combat C. albicans infections in lifeforms.

This study investigated spatial learning in Caenorabditis elegans; the ability to associate reinforcing cues with a location. Naive, wildtype C. elegans were trained in a microfluidic t-maze in the presence of diacetyl (a volatile attractant associated with food) and subsequently tested to see if they could associate diacetyl with one arm of the t-maze. 70-80% of the subjects chemotaxed towards diacetyl during training phase, but they randomly chose left or right when diacetyl was absent (number of subjects that chose diacetyl being under 65%). From our experiments, it is unlikely that the worms are associating diacetyl with one arm of the t-maze, but appears to be using some component of the atmosphere as a cue.

Aging is a complex process by which a combination of environmental, genetic and stochastic factors generate whole-system changes that modify organ and tissue function and alter physiological processes. Over the last few decades, many genetic and environmental modulators of aging have been found to be highly conserved between humans and a diverse group of model organisms. Yet, an integrative understanding of how these environmental and genetic variables interact over time in a whole organism to modulate the systemic changes involved in aging is lacking. The goal of this thesis project is to advance a systems perspective of aging by providing the experimental tools and conceptual framework for dissecting the regulatory connection between environmental inputs, molecular outputs and long term aging phenotypes in Caenorhabditis elegans, an experimentally tractable multi-cellular model for aging. Specifically, this work advances the quantitative imaging toolsets available to biologists by developing and refining microfluidic, hardware, computer vision, and software integration tools for high-throughput, high-content imaging of C. elegans. As a result of these technological advances, new roles for the TGF-beta and serotonin signaling pathways in encoding environmental food signals to influence longevity were uncovered and quantitatively characterized. Moreover, this work develops and integrates new microfluidic technologies with off-chip support systems to establish a platform for long-term tracking of the health and longevity trajectories of large numbers of individual C. elegans. The capabilities of this platform have the potential to address many important questions in aging including addressing environmental determinants of aging, the sources of inter-individual variability, the time course of aging-related declines and the effects of interventional strategies to improve health outcomes. Together, the toolsets for quantitative imaging and the long-term culture platform permit the large-scale investigation of both the internal state and long-term behavioral and health outputs of an important multicellular model organism for aging.

Studying animal mechanics is crucial in order to understand how signals in the neuromuscular system contribute to an organism’s behaviour and how force-sensing organs and sensory neurons interact. In particular, the connection between the nerves and the muscles responsible for the force generation in the neuromuscular system needs to be established. Knowledge of the locomotion forces can be beneficial for the development of therapies for muscle disorders, neurodegenerative and human genetic diseases, such as muscular dystrophy. The simplicity of the nematode Caenorhabditis elegans’ (C. elegans) nervous system, which is limited to 302 neurons, has made it an excellent model organism for studying animal mechanics which include mechanosensation and locomotion at the neuronal level. The advent of miniaturized force sensing devices has led to the proposal of various approaches for measuring C. elegans locomotion forces. However, these existing devices are relatively complex, involving complicated microfabrication procedures and are incapable of measuring forces exerted by C. elegans in motion. This thesis addresses these shortcomings by introducing a force sensor capable of continuously measuring the forces generated by C. elegans in motion. The system consists of a micropillar-based device made of polydimethylsiloxane (PDMS) only and a vision-based algorithm for resolving the worm force from the deflection of the cantilever-like pillars. The measured force is horizontal and equivalent to a point force acting at half of the pillar height. The microdevice, sub-pixel resolution for visual tracking of the deflection, and experimental technique form an integrated system for measuring dynamic forces of moving C. elegans with force resolution of 3.13 uN for worm body width of 100 um. A simple device fabrication process based on soft-lithography and a basic experimental setup, which only requires a stereo microscope with off-the-shelf digital camera mean that this method is accessible to most biological science laboratories. The results demonstrate that the proposed device is capable of quantifying multipoint forces of moving C. elegans rather than single-point forces for a worm sample. This allows one to simultaneously collect force data from up to eight measurements points on different worm body parts. This is a significant step forward as it enables researchers to explicitly quantify the relative difference in forces exerted by the worm’s different body segments during the worms’ movements. The device’s capability to determine multipoint forces during nematode motion can also generate meaningful data to compare forces associated with different worm body muscles, gaining new understanding on how these muscles function. The forces measured during locomotion in the micropillars could also be used to differentiate mutant phenotypes. Apart from locomotion forces, the device is also capable of conducting concurrent measurement of other locomotion parameters such as speed, body amplitude and wavelength, as well as undulation frequency. This additional information can be useful to further quantify phenotypic behaviour of C. elegans and deepen the understanding of the theory behind worm locomotion forces. The relationship between C. elegans locomotion forces and their environment has also been analyzed by variation of the pillar arrangement and spacing. The results indicate that the microstructured environment significantly affects the worm’s contraction force, locomotion speed and the undulation frequency. In addition, an alternative measurement technique was provided to measure worm forces on other substrates, such that worm locomotion behaviour in varying environments can be investigated further. The combination of the conventional measurement technique with the findings of worm locomotion on a glass substrate reported show promise for biological measurements and other sensing application such as tactile force. Additional functions of on-chip worm selection, sorting, and imaging have also been integrated with the device, rendering its potential to accommodate for high-throughput application of C. elegans force measurement and locomotion studies in the future. The primary contributions of this thesis are centered around four topics: the development of the PDMS micropillar array and its application to study C. elegans locomotion forces, the analysis of C. elegans muscular forces and locomotion patterns in microstructured environments, the investigation of the worm locomotion forces using different substrates and finally the integration of the PDMS micropillar with PDMS microvalve for on-chip worm selection and imaging. Although the results presented in this thesis focus on wild type C. elegans, the method can be easily applied to its mutants and other organisms.