PNPase, polynucleotide phosphorylase, is a multifunctional exoribonuclease protein with 3` terminal oligonucleotide polymerase activity. Coded by the PNPT1 gene, the protein is associated with mitochondrial homeostasis and functions as a possible target for cancer therapy. In this study, C. elegans was used to investigate the effect of mutation and overexpression of pnpt-1, the gene that encodes PNPase. It was determined that two specific mutations in pnpt-1 did not affect PNPase expression nor did they produce deleterious phenotypes that affected polycistronic transcript accumulation or ROS production. Creation of a stable overexpression model was achieved through Fusion PCR. However, different transgenic strains overexpressing PNPase produced opposite results for polycistronic transcript accumulation while ROS production saw no significant change, suggesting a mosaic overexpression model. In a cancer model, exogenous PNPase was present in the pachytene region of the germline and where expressed the cells were in non-germline cells suggesting differentiation mechanisms associated with overexpression of PNPase. However, further analysis of different mutations in pnpt-1 or optimizations to the overexpression model are necessary to provide a better understanding of PNPase function with mitochondria homeostasis and in a cancer model setting.

No description available.

Large numbers of people suffer from nervous system injuries and neurodegenerative diseases each year, with little success in regaining lost neural functions. Attempts to successfully regenerate nervous tissue in the mammalian Central Nervous System have meet with limited success. Simpler models have thus been useful in determining conserved mechanisms in the enhancement of neural regeneration. One such mechanism is intracellular calcium signaling. We used <italic>Caenorhabditis elegans</italic> as a model system to study the effects of optogenetic stimulation on regeneration. Using a femtosecond laser we cut individual <italic>C. elegans</italic> axons <italic>in vivo</italic> and then periodically stimulated the neurons by activating the genetically encoded light activated channel, Channelrodopsin-2. We found that periodic photo-activation could increase regeneration over 24h by at least 31%. We repeated these experiments with dantrolene treatment and in <italic>unc-68(e540)</italic> mutants to assess the effects from a lack of internal calcium ion signaling in these worms. In both cases, we found a complete suppression of stimulated regeneration when calcium signaling was blocked. This indicates that intracellular calcium ion signaling is crucial in the initiation of neural regeneration in the first 24 hours and mediates the enhanced outgrowth we observe with periodic photo-activation. The importance of intracellular calcium ion signaling can lead to further studies to enhance the stimulation of neural regeneration, and improve therapies for patients with neural damage and loss of neural functions.

This thesis describes the use of a micropipette deflection technique to measure the viscous forces experienced by the millimeter sized undulatory swimmer and model organism C. elegans. Using a specialized pipette, we are able to simultaneously measure both the lateral and propulsive forces acting on the worm. We find that the measured force curves are well described by Resistive Force Theory, which is a low Reynolds number hydrodynamic model. This work constitutes the first justification of its applicability at Reynolds numbers of this magnitude (roughly 0.1). Through our comparison with Resistive Force Theory, we extract the worm's drag coefficients, which are in agreement with an existing theoretical prediction. Through a simple scaling argument, we obtain a relationship between the size of the worm and the typical viscous forces, which is in good agreement with our data. We also present a study aimed at measuring how the hydrodynamic forces on the worm change in proximity to solid boundaries. Using micropipette deflection, forces are measured at controlled distances from a single planar boundary and midway in between two parallel boundaries. We find the viscous forces and drag coefficients to increase significantly as the worm approaches a boundary. We find a constant value for the ratio of normal to tangential drag coefficients at all distances from a single boundary, but measure it to increase significantly as the worm is confined between two boundaries. In addition, the worm is seen to undergo a continuous gait modulation, primarily characterized by a decreased swimming amplitude, as it is subject to larger drag forces in confinement. Finally, the interactions between two worms swimming nearby one another are probed. Worms are held adjacent to one another using micropipettes, and are found to tangle with each other, rather than interact hydrodynamically. We develop simple models that well capture the onset and probability of tangles as a function of the separation distance between the worms. / Thesis / Master of Science (MSc)

The nematode C. elegans is a multicellular model organism to study the neuronal-basis of behaviour. C. elegans demonstrates an innate response to swim towards the cathode in the presence of a DC electric field(EF), a behaviour known as “electrotaxis”. We examined mutants affecting sensory and dopaminergic neurons and found that these mutants moved with reduced speed with intermittent pauses, abnormal turning, and slower body bend. A similar phenotype was observed in worms treated with neurotoxins 6-OHDA, MPTP and rotenone. Pre-exposing worms to a known neuroprotective compound acetaminophen could suppress the effects of neurotoxin on movement. Further, this study demonstrates that dopamine and the D2-type dopamine receptor are necessary to modulate electrotactic movements in worms. A reduction in extracellular dopamine leads to a significant increase in the swimming speed as judged by the analysis of bas-1(dopa decarboxylase) and cat-1(VMAT) mutants. The dopamine transporter dat-1 acts genetically downstream of bas-1 and cat-1 since dat-1 mutants efficiently suppress bas-1 and cat-1 phenotypes. We also found that DOP-3(D2-type receptor) acts as the sole receptor for dopamine-mediated regulation of electrotaxis. Interestingly, we found that prolonged exposure to EF resulted in a gradual decline in the swimming speed such that animals were 40% slower at the end of ten minutes exercise period. This change is mediated by DOP-3 since dop-3 mutants continue to swim at the initial speed and don’t slow down. This conclusion is supported by the analysis of animals treated with Heloperidol(D2 antagonist) and SKF38393(D1 agonist). Overall, our work demonstrates that D2 receptor-mediated neuronal signalling is required to restrict muscle activity not only during the initial phase of electrotaxis swimming but also for the entire duration of the assay. We suggest that such a role of dopamine signalling might serve as an important and conserved mechanism to limit muscle overuse during prolonged physical exercise. / Thesis / Doctor of Science (PhD)

No description available.

Neurotrophic factors are proteins involved in the maturation, differentiation and survival of neurons. Due to their neuroprotective properties, they have been regarded as potent candidates for the treatment of neurodegenerative diseases. Recently, a novel family of neurotrophic factors was discovered comprising mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF). These factors have been shown to protect against the degeneration of nigrostriatal dopaminergic neurons in mammalian models of Parkinson's disease, however their neuroprotective mechanisms of action are not yet understood. Although distinct in vertebrates, MANF and CDNF constitute a single homolog in invertebrates. In the present study, we have characterized the in vivo expression and function of the C. elegans homolog manf-1. We have shown that manf-1 is not essential for neuronal development, however when knocked down, mutants exhibit enhanced age-related dopaminergic neuronal degeneration accompanied by an increase in the endogenous ER stress response. Loss of manf-1 function also results in enhanced alpha-synuclein expression and aggregation, a pathological hallmark of Parkinson’s disease. / Thesis / Master of Science (MSc)

The nematode, Caenorhabditis elegans is an ideal animal model to study conserved mechanisms of developmental and postdevelopmental processes. Here, I describe the role of an Axin family member, pry-1, in aging, lipid metabolism, and seam cell development. Our analysis of pry-1 animals showed a catastrophic collapse of adult lifespan, which was accompanied with hallmarks of accelerated aging. Transcriptome profiling of pry-1 mutants revealed altered expression of genes associated with aging and lipid metabolism such as vitellogenins, fatty acid desaturases, lipases, fatty acid transporters and genes involved in cuticle synthesis. Consistent with this, pry-1 animals display significantly reduced levels of somatic lipids. Knockdowns of vitellogenins in the pry-1 background restored lifespan and lipid levels, suggesting that vitellogenins are necessary to mediate pry-1 function in aging and lipid metabolic processes. Additionally, lowered expression of desaturases and lipidomics analysis provided evidence of reduced fatty acid synthesis in pry-1 animals. In agreement with this, an exogenous supply of oleic acid restored depleted lipids in somatic tissues in addition to suppressing the short-lived phenotype of worms. In addition, transcriptome profiling for differentially expressed miRNAs in pry-1(mu38) identified heterochronic miRNAs (lin-4 and let-7 -family members) to act downstream of pry-1 /Axin. In C. elegans, these miRNAs are known to robustly regulate the stem-like, seam cell division. Loss of pry-1 function caused heterochronic defects such that the seam cells divide precociously to produce additional cells. The pry-1-miRNAs are involved in mediating silencing of the heterochronic gene, hbl-1, a C. elegans hunchback homolog, to regulate seam cell division. Furthermore, I report identification of novel miRNAs from C. elegans and C. briggsae. Overall, our findings demonstrate a novel role of the Wnt signaling regulator, pry-1/Axin, in the maintenance of adult lifespan that involves lipid homeostasis and regulation of heterochronic miRNA to control the developmental timing of seam cell division in C. elegans. / Thesis / Doctor of Philosophy (PhD)

Receptors and signal transduction complexes are assembled in a precise manner at specific subdomains of the plasma membrane. Recent research has implicated scaffolding proteins in organizing these receptor and signaling complexes. One well characterized example is the C. elegans LIN-2/LIN-7 /LIN-1 0 complex. This complex is essential in the proper localization of LET -23, the EGFR ortholog, to the basolateral membrane surface of vulval epithelial cells. The mammalian orthologs of the LIN-2/LIN-7 /LIN-10 complex have been identified. CASKIVELI!Mintl/Xllalpha function as a tripartite complex in neurons, presynaptically and postsynaptically. Presynaptically, the multi protein complex aids in linking cell adhesion to ion influx, synaptic vesicle fusion with the presynaptic membrane. and subsequent neurotransmitter release. At the post-synaptic membrane, the CASKIVELI!Mintl/Xllalpha complex is hypothesized to function in the sorting and proper localization of the NMDA type glutamate receptor, reflecting the function of the C. elegans orthologs in receptor localization. We have identified the Drosophila orthologs ofLIN-2/CASK, LIN-7NELI, and LIN-10/Mintl/Xllalpha, termed CMG, dVELI and dMINT. respectively. These proteins were found to be highly conserved among species. The Drosophila YELl protein was initially identified by the McGlade laboratory, University of Toronto, where it was found to bind phosphorylated Drosophila EGFR (DER). We have mapped the chromosomal location of dveli, determined RNA transcript distribution and protein localization, and initiated a P-element mutagenesis screen to generate a dveli mutant. Furthermore, candidate genes for other proteins known to associate with LIN-7 (PALS) have been identified by sequence analysis. dVELI expression begins early in the larval stage. It is concentrated mostly in neuropil areas, sites of synaptic connections. This expression pattern continues into adult development. Within the larval CNS, dVELI protein is localized to the neuropil areas of the ventral nerve cord and brain. NMJ staining further localizes dVELI almost exclusively to the post-synaptic density. This post-synaptic localization resembles that of mammalian YELls, wherein the complex is thought to aid in glutamate receptor sorting and localization. The similarity in structure and expression patterns of dVELI to that of its mammalian orthologs suggests a model in which the Drosophila complex aids in the localization of receptors to post -synaptic specializations in neurons. / Thesis / Master of Science (MSc)

An increase in antimicrobial resistance bacteria has endangered our ability to treat infectious diseases. Lack of good in-vivo model has made it difficult to study antimicrobial resistance. In this study, we have used an inexpensive and short life span in-vivo model namely, Caenorhabditis elegans (C. elegans) to study antimicrobial treatment using pathogenic Escherichia coli O157:H7, a multidrug resistance bacterium that causes life threatening infection in humans. We have investigated the influence of live vs. heat killed non-pathogenic E. coli OP50 (OP50) as a food source on the growth and survival of infected C. elegans mutant AU37 with E. coli O157:H7 in the presence and absence of antibiotics. This is analyzed using a liquid-based C. elegans-E. coli O157:H7 infection assay. C. elegans was synchronized and grown on a lawn of live OP50 till they reached L4-young adult stage. L4-young adults were transferred to liquid medium where the C. elegans was infected with live E. coli O157:H7 or live non-pathogenic OP50 for 24 hours. After infection, C. elegans were fed live or heat killed OP50 depending on the experiment, and the life span and levels of E. coli O157:H7 were monitored, with and without ampicillin treatment in a 96 well transwell plate. Our results indicate that live OP50 is an ideal food source for C. elegans growth and survival to study antimicrobial treatment. C. elegans growth rate and survival decreased in presence of heat killed OP50, which makes heat killed OP50 as a non-ideal food source for antimicrobial assay. Moreover, using live OP50 we have discovered that the ampicillin dose 8mg/ml, 16mg/ml, and 32mg/ml are effective in increasing the survival of C. elegans infected with E. coli O157:H7. However, treatment on C. elegans infected with acid stressed E. coli O157:H7 is controversial.