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Ubiquitin is a multifunctional 76 amino acid protein which plays critical roles in many aspects of cellular metabolism. Ubiquitin protein structure and gene structure are highly conserved among eukaryotes. In C. elegans the major source of ubiquitin RNA was shown to be the polyubiquitin locus, UbiA. UbiA was shown to be transcribed as a polycistronic mRNA which contained eleven tandem repeats of ubiquitin sequence and possessed a two amino acid carboxy terminal extension on the final repeat. Mature UbiA mRNA was demonstrated to acquire a 22 nucleotide leader sequence via a trans splicing reaction involving a 100 nucleotide splice leader RNA derived from a different chromosome. UbiA was also shown to be unique among known polyubiquitin genes in containing four cis spliced introns within its coding sequence. Thus UbiA was shown to be one of a small class of genes found in higher eukaryotes whose hnRNA undergoes both cis and trans splicing. The expression of the UbiA gene was studied under various heat shock conditions, and was monitored during larval molting and throughout the major stages of development. These studies indicated that the expression of the UbiA gene was not inducible by acute or chronic heat shock, and did not appear to be under nutritional or developmental regulation. A second ubiquitin gene, UbiB, was cloned from C. elegans and a related nematode species C. briggsae. This gene was comprised of (at least) one ubiquitin unit followed by a basic 52 amino acid tail sequence. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

In this thesis, chromosome I rearrangements were used to study the organization of essential genes and regions important for chromosome behaviour in the nematode Caenorhabditis elegans. To facilitate the genetic mapping of mutations in essential genes, rearrangements were isolated using a procedure designed to recover derivative chromosome I duplications shortened by gamma radiation from existing duplications. Sixty-two duplications were isolated in this way. These duplications, along with three deletions isolated in this study and 9 existing deletions of the region, divided the left half of chromosome I into at least 24 regions. Protocols were developed and used to rapidly map mutations into the regions defined by the breakpoints. The techniques and results described demonstrate the feasibility of carrying out a similar analysis on the whole genome. The majority of duplications behaved as if they were free; that is they segregated independently of the euploid chromosome set. While size was an important determinant of mitotic stability, clear exceptions to a size - stability correlation were observed. For example, despite its larger size, hDp72 was lost during cell division more frequently than hDpl8, suggesting features of chromosome structure were important. Shortening of duplications in the unc-11 dpy-5 region caused greater reductions in mitotic stability than similar sized shortenings in the dpy-5 unc-13 region. Therefore, specific sequences appear to influence duplication stability. Some free duplications were also observed to break spontaneously. Breakage occurred at different frequencies for different duplications and correlated with mitotic instability. The meiotic properties of four translocations involving chromosome I were examined. No recombination was observed in any of the translocation heterozygotes along the left (let-362 - unc-13) portion of chromosome I. By isolating a half-translocation chromosome as a free duplication, I mapped the breakpoints of three of the translocations. The boundaries of cross-over suppression coincided with the physical breakpoints. These results agree with the proposal that DNA sequences at the right end of chromosome I are essential for homologue recognition followed by meiotic synapsis and recombination. The published data of other translocations and duplications indicates that each of the other five C. elegans chromosomes has DNA sequences localized to one end that are required for homologue recognition and recombination. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

This thesis describes the genetic analysis of a small region of the Caenorhabditis elegans genome. The region analyzed was defined by the 0.5 map unit interval between the breakpoints of the duplications hDp16 and hDp19, which lies within the dpy-5 unc-13 region on chromosome I. The analysis consisted of the identification and characterization of essential genes in this region. All the lethal mutations analyzed came from a set of 495 EMS-induced, sDp2-rescued lethals described in Howell (1989). The lethal mutations have been maintained as homozygotes, made viable by the presence of a wild-type allele on the sDp2 duplication. I used a combination of mapping techniques to analyze over 200 EMS-induced lethal mutations. A total of 189 new lethal mutations were positioned within the dpy-5 unc-13 interval. Three methods were used for mapping: recombination analysis, lethal rescue using duplications, and deficiency mapping. Duplication mapping was found to be the fastest and most precise method used. 178 of the new lethal mutations were mapped relative to the duplication breakpoints of hDp13, hDp16, and hDp19. These three duplications divided the dpy-5 unc-13 region into three approximately equal-sized zones. This study completes the mapping of the 495 lethals in the sDp2 set. In addition, thirty-three previously identified essential genes lying between dpy-5 unc-13 were positioned with respect to the breakpoints of the duplications hDp12, hDp13, hDp15, hDp16, hDp17, and hDp19. The dpy-5 unc-13 region carries a relatively large number of loci, therefore, I decided to concentrate on the smaller hDp16/hDp19 interval within this region. Complementation analysis was used to define the number of essential genes in the hDP16/hDp19 region. A total of eight new genes were described, six lying in the hDp16/hDP19 region, two lying just outside this region. This brings the total number of essential genes in the hDP16/hDp19 region to sixteen. In addition, as a result of my mapping data, the hDp16/hDp19 region has been subdivided into six intervals with respect to duplication and deficiency breakpoints. The stage of developmental arrest was determined for both the essential genes, and the new lethal mutations (ie. not yet defined by complementation tests), in the dpy-5 unc-13 interval. Although the number of genes studied was not great, the data suggests a relationship between map position and time of developmental arrest. The average forward mutation rate for C. elegans genes was determined to be 5.8 X 10⁻⁵mutations per gene. I have made a comparison of the forward mutation rates of the essential genes in the hDp16/hDp19 region, bli-4 was found to be the most mutable target in the region with nine mutant alleles, giving a forward mutation rate six times higher than average. In the hDp16/hDp19 region, ten of the sixteen essential genes were represented by more than one allele. The minimum estimate of the number of essential genes in the region using a truncated Poisson calculation was twenty. Therefore, the sixteen genes identified represent 80% of the essential genes in this region. This data was extrapolated to give a minimum estimate of approximately 225 essential genes in the 15 m.u. sDp2 region, and 4,500 essential genes in the C. elegans genome. This research has established the hDp16/hDp19 region as a genetically well-defined system for studying the genetic organization of essential genes, as well as the developmental regulation of gene expression, and the functional relationship between adjacent genes. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The objective of these experiments was to explore long-term memory in Caenorhabditis elegans. This examination of memory in a simple organism with accessible genetics and a well understood biology may permit later work to define the cellular processes that underlie long-term memory. Habituation training with a vibrational stimulus was administered on Day 1, and the retention test of a block of stimuli was given 24 h after the end of training on Day 2. Long-term retention of habituation was evident as a lower level of responding on Day 2 relative to the level of responding on Day 2 of untrained controls or the initial level of responding of worms on Day 1. In Experiments 1 and 2, a habituation training protocol that produced long-term retention of habituation was established, and the effects of stimulus number, interstimulus interval (ISI), and distribution of training on both short-term and long-term habituation were examined. In Experiment 1 (10-s ISI), there appeared to be a floor effect which resulted in a low level of responding regardless of training on Day 1; thus no evidence for long-term habituation after training at a 10-s ISI could be found. In Experiment 2 (60-s ISI), worms that received distributed and massed habituation training with 60 stimuli showed a significantly lower level of responding relative to untrained controls. The distributed habituation training appeared to be more effective at inducing long-term habituation and was used in the subsequent experiments. To characterize the effects of heat shock treatments used in the behavioral experiments that follow, the effects of heat shock on two assays, the induction of a heat shock protein gene, hsp16, and the rate of egg-laying were measured in Experiment 3. All heat shock treatments used caused the induction of hsp16. In addition, the number of eggs laid during a fixed interval after heat shock was sensitive to the heat shock treatments given in Experiments 4 through 8. In Experiments 4 through 8, the effects of heat shock on short- and long-term habituation were examined. Heat shock, which acts as a general cellular stressor, was administered at different times before, during and after training. In Experiment 4, heat shock (45 min, 32°C) was administered, ending 2 h before training on Day 1. Heat shock before training did not affect the initial level of responding on Day 1, habituation during training, short-term retention of habituation between blocks of training or long-term retention of habituation. In Experiment 5, heat shock (45 min, 32°C) was administered during the rest periods of distributed training in the 1-h interval after each training block. While heat shock during training had no significant effect on responding on Day 1, long-term habituation was blocked. In Experiment 6, the possibility that heat shock before training would prevent the disruption of long-term habituation by heat shock during training by inducing thermal tolerance was examined. This was tested by administering heat shock (45 min, 32°C) that ended 2 h before training and heat shock during training. It was found that heat shock before training did not prevent the disruption of long-term habituation by heat shock during training. In Experiment 7, the effect of heat shock that ended 2 h before the retention test on Day 2 on the retention of long-term habituation was examined. It was found that heat shock on Day 2 did not disrupt the retention of habituation. Finally, in Experiment 8, the effect of brief heat shock (15 min, 32°C) at different intervals in the rest period following the training blocks was examined in an attempt to more narrowly define a critical period for consolidation of long-term habituation. Although there was no significant effect of brief heat shock on retention of habituation, the pattern of the data suggests that there may be a period of greater vulnerability worth further investigation. In summary, heat shock given before training or before the retention test did not affect long-term habituation, while heat shock during training disrupted consolidation of long-term habituation. Taken together, these behavioral results provide the foundation for an investigation of the cellular processes underlying long-term memory in C. elegans. By exploring the dynamics of the formation of long-term habituation, intervals of time critical to the formation of long-term habituation were defined. This in turn will help to focus attention on the cellular processes whose activity during those intervals of time may be important to the consolidation of long-term memory. / Arts, Faculty of / Psychology, Department of / Graduate

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Alterations in the microbiota have been observed in many human diseases, including diseases of neurodegeneration. However, specific microbiome factors that either promote or protect against neurodegeneration are largely unknown. We examined the effects of human microbiota in tauopathies, a class of age-associated neurodegenerative diseases that are characterized by the accumulation of tau protein inclusions. Using a Caenorhabditis elegans model expressing an aggregate prone human tau protein, we examined the influence of specific bacteria present in the human respiratory tract on neurodegeneration. We identified a bacterial species, Rothia aeria, that is neuroprotective in a C. elegans model of tauopathy. We determined that the R. aeria induced neuroprotection observed in PLM neurons is fat-3 dependent. fat-3 encodes the protein Delta (6)-fatty-acid desaturase. We also showed that a lipid(s) produced by R. aeria decreases neurodegeneration in C. elegans. Further investigation is needed to identify the lipid and the underlying mechanism. / Dissertation / Doctor of Philosophy (PhD) / The human body hosts trillions of microbes, including bacteria, viruses, and fungi. These components make up the human microbiota. Gut microbiota have an essential role in human health. In correlative studies, alterations in gut microbiota have been observed in many human diseases and conditions, including Alzheimer’s disease (AD). Diet, antibiotics, and probiotics change the composition of the microbiota and could decrease or increase the risk of developing AD. Modulation of the microbiome through diet and other interventions could be beneficial for AD. AD, the most common type of dementia, is a progressive brain disorder that slowly destroys memory and mental skills. The Alzheimer’s Society of Canada estimates that over half a million Canadians live with dementia, increasing significantly by 2031. The total estimated indirect and direct costs of dementia in 2016 in Canada was $10.4 billion. Currently, no treatments slow or stop Alzheimer’s disease and available medications can only improve the symptoms in some patients. One of the main characteristics of AD is the accumulation of a protein called tau in neurons. In this research, we look at the impact of microbiota on AD using Caenorhabditis elegans as a model organism. C. elegans is a free-living nematode. These nematodes are small, transparent, feed on bacteria and have a simple nervous system. All these criteria make C. elegans a perfect model for studying the impact of microbiota on AD. We exposed the C. elegans model of AD with high tau aggregation in their neurons to different bacteria collected from human microbiota and measured their neuronal health. We have found several species of bacteria that decreased neurodegeneration in this model. Currently, we are investigating how these bacteria improve neuronal health. Our findings suggest the involvement of human microbiota in AD. This suggests future treatment and preventative measures for AD should also consider microbiota composition. The result of this research will expand our knowledge of AD development and progression.