The Gram-negative bacterium Legionella pneumophila is an intracellular parasite of aquatic protozoa. It exhibits a distinct dimorphic lifecycle that alternates between vegetative replicative form (RF) and infectious cyst-like form (CLF). Inadvertent inhalation of aerosolized CLFs by immunocompromised individuals leads to an infection in alveolar macrophages causing Legionnaires' disease. To further study a Legionella infection the use of the multicellular organism Caenorhabditis elegans was done. Differential Interference Contrast (DIC) microscopy of live L. pneumophila infected nematodes shows Legionella-containing vacuoles (LCVs) with motile forms. Transmission Electron Microscopy (TEM) defined the ultrastructure of L. pneumophila forms found in the primary infection site of the intestinal lumen and the secondary infection site in the gonadal tissues. These findings suggest the possible intracellular replication cycle of Legionella occurring in the gonadal tissues of the nematode. Providing insight and a plausible evolutionary origin of the ability of L. pneumophila to manipulate the macrophage innate immune system. / October 2014

Down syndrome is one of the most common genetic disorders, resulting in a range of neurological and neuromuscular disabilities. Although the presence of specific disabilities varies among individuals with Down syndrome, all individuals with Down syndrome are born with hypotonia (low muscle tone) and over half with congenital heart defects. Later in life, all individuals demonstrate intellectual disabilities to varying degrees, while many also develop early-onset Alzheimer’s disease. While the cause of Down syndrome is known to be a triplication of the 21st chromosome, it is unknown how this extraneous genetic material causes the development of these phenotypes. We have begun research into the biological basis of these disabilities using the tiny nematode, Caenorhabditis elegans as a genetic model. We used the technique RNA interference (RNAi), which allows us to study the in vivo function of genes by knocking down their expression one at a time in a living, behaving animal. We have used this technique to systematically study the in vivo function for genes involved in Down syndrome. To this end, we identified and knocked down C. elegans genes with sequence similarity to 67% of genes on the human 21st chromosome genes. We used these RNAi-treated worms to investigate the neuromuscular function of human chromosome 21 gene equivalents by assaying locomotion and pharyngeal pumping in a blinded screen. We used locomotion as a measure of neurological and neuromuscular function, while we used pharyngeal pumping as a model for cardiac function. We also performed an aldicarb screen to examine the role of some of these genes in the function of the synapse. Our experiments have provided valuable insight into the in vivo function of the vast majority of genes on the human 21st chromosome. This will be vital to identify genes that are potentially involved in eliciting Down syndrome-related phenotypes, laying the groundwork for further studies into the neurobiology of Down Syndrome. / text

Transposons are a diverse collection of mobile genetic elements and are important components of nearly every genome. Because of their mobile and repetitive nature, transposons can have considerable effects on host gene expression, genome organization and evolution. The recent availability of genomic sequence information has expedited the discovery and study of transposons, as exemplified in this thesis by the complete genome analysis of the model plant system Arabidopsis thaliana. Data mining in Arabidopsis has revealed a rich diversity of transposons, of which Basho and Terminal-repeat Retrotransposons In Miniature (TRIM) elements were previously unknown types. The identification of Related to Empty Sites (RESites) provide evidence for past transposition events. Examples of elements contributing to coding regions, acquiring cellular sequences, along with in-depth analysis of the insertions, their target sites and their distribution illustrate the impact of transposons on gene and genome structures. Computer-based searches of genomic sequences has also improved our understanding of previously identified transposon families, such as the origin, classification and mobilization of Tourist elements. In addition, information on transposons gathered from in silico analysis of genomic sequences has served to design in vivo experiments. In a whole genome strategy, Transposon Display was used to investigate transposition and regulation of mobility of Tourist-like elements in A. thaliana and in the nematode Caenorhabditis elegans.

In this thesis I used Caenorhabitis elegans as a model of Fetal Alcohol Spectrum Disorder (FASD) to study the physical and behavioral effects of ethanol exposure during embryonic development. Davis et al. (2008) found that ethanol exposure during larval development in C. elegans produced physical/developmental and behavioral effects; however, whether exposure during embryonic development might produce similar outcomes remained to be elucidated. Because the type and degree of effects caused by developmental ethanol exposure was dependent on the pattern of ethanol treatment, in the first part of the thesis I investigated the physical/developmental effects of embryonic exposure to various ethanol doses, exposure durations, onsets and frequencies. I found that exposure to >30% ethanol for an hour during embryonic development was necessary to lower hatch rate, delay reproductive onset, and reduce body size in C. elegans. Furthermore, exposure during early embryonic development caused a larger effect than exposure during later stages, and multiple exposures produced a worse outcome than a single exposure for a comparable duration. In the second part of the thesis, I investigated locomotory activities and habituation of adult C. elegans exposed to various patterns of embryonic ethanol treatment. I found that the rate of locomotion was altered differently by chronic and acute embryonic ethanol exposure, but I did not find any effect in short- or long-term habituation. In summary, I have characterized the pattern of embryonic ethanol exposure necessary to produce physical/developmental effects in C. elegans, and identified the types of exposure conditions that would cause worse outcomes than others; in addition, I have found that embryonic ethanol exposure affects the rate of locomotion in C. elegans. In this thesis, I have established a foundation for the future investigation into the physical and motor defects caused by embryonic ethanol exposure in C. elegans.

Serial Analysis of Gene Expression (SAGE) on Caenorhabditis elegans RNA from FACS sorted embryonic body wall muscle cells has identified nearly 8000 genes expressed in nematode body wall muscle. Approximately 60% of these are genes are expressed at low levels (<5 tags/~50,000-100,000 tag library). Low-abundance transcripts have typically been overlooked since most are considered experimental or contamination errors. Consequently, research has been focused on transcripts that are most enriched in the particular tissue of interest. Here I focus on the analysis of low-expressed transcripts in the muscle SAGE libraries in order to investigate what percentage of these are in fact expressed in muscle and are not false positives. Most well characterized C. elegans body wall muscle genes are not expressed at low levels, therefore I anticipate that focusing on these rarely expressed genes will allow for the identification of muscle components that have been previously unrecognized. RT-PCR was performed on RNA isolated from purified body wall muscle cells to initially estimate what fraction of these low abundance transcripts present in the SAGE data are indeed expressed in muscle. I examined 128 genes, of which 84 were represented by a single SAGE tag. From this initial list, 38% of the low-expressed transcripts were verified for their presence in body wall muscle. Subsequently, reporter GFP fusions were used to deduce if these low-expressed transcripts are indeed expressed in vivo within muscle. Of the low-expressed genes that tested positive via RT-PCR, 42% showed in vivo expression in body wall muscle. When the results from the RT-PCR and in vivo expression experiments are combined, I can extrapolate that at least 16% of low-expressed genes identified by the SAGE libraries are in fact expressed in muscle and are not false positives. RNAi and knockout analysis were performed in order to investigate the role of low-expressed muscle genes in myofilament structure. RNAi results show that 14/34 (41%) of the genes screened had mild defects in myofilament organization. The SAGE libraries identified 6388 low-expressed transcripts, this work suggests that at least 16% (1022 genes) of these are in fact expressed in muscle and may reveal new components previously overlooked by other approaches.

Understanding gene function and genetic relationships is elemental in our efforts to better understand biological systems. Here, I describe a reliable high-throughput approach, Systematic Genetic Interaction analysis (SGI), capable of revealing both weak and strong genetic interactions in the nematode Caenorhabditis elegans. I also present evidence that UNC-73 functions cell-autonomously in an UNC-40 pathway to direct muscle arm extension in C. elegans. Previous efforts to systematically describe genetic interactions between redundant genes on a global scale either have focused on core biological processes in protozoans or have surveyed catastrophic interactions in metazoans. I investigated synthetic genetic interactions between eleven ‘query’ mutants in conserved signal transduction pathways and hundreds of ‘target’ genes compromised by RNAi. A network of 1246 genetic interactions was uncovered through an unbiased global analysis of the interaction matrix, establishing the largest metazoan genetic interaction network to date. To investigate how genetic interactions connect genes on a systems-wide level, the SGI network was superimposed with existing networks of physical, genetic, phenotypic and co-expression interactions. Fifty-six putative functional modules were identified within the superimposed network, one of which regulates fat accumulation and is coordinated by bar-1(ga80)/β-catenin interactions. This led to the discovery that SGI interactions link distinct functional modules on a global scale, which is a previously unappreciated level of organization within metazoan systems. In addition, I present evidence that the properties of genetic networks are conserved between C. elegans and S. cerevisiae, but that the connectivity of the interactions within the current networks is not. Although the buffering between functional modules may differ between species, studying these differences may provide insight into the evolution of divergent form and function. In C. elegans the postsynaptic membrane of the neuromuscular junction reaches its destination through an active process of guided cell extension. The worm has 95 body wall muscles (BWMs) that extend projections called 'muscle arms' to motor axons. The muscle arms harbour the postsynaptic elements of neuromuscular junctions. The stereotypical pattern of muscle arm extension was exploited in a forward genetic screen for new genes required for guided cell migration by looking for mutations that caused a reduction in the number of arms that extend to the motor axons. One of the resulting mutants was tr117, which extended half the number of arms compared to wild type animals. Genetic mapping, complementation tests, and sequencing revealed that tr117 was a mutation in unc-73/Trio, which encodes a guanine nucleotide exchange factor. Expression of UNC-73 specifically in the BWMs rescued the muscle arm development defects of unc-73(e936) mutants, indicating that UNC-73 functions cell-autonomously to regulate muscle arm extension. UNC-73::CFP was localized to muscle arm termini in a pattern similar to that of UNC-40/Dcc, which directs muscle arm extension. UNC-73 over-expression suppressed the Madd phenotype of unc-40 null worms and unc-73(e936) suppressed ectopic myopodia induced by UNC-40 over-expression. These results indicate that UNC-73 functions downstream of UNC-40 in a pathway that regulates muscle arm extension.

How behaviors are generated by neural circuits is one of the central questions in neurobiology. Under standard culture conditions, Caenorhabditis elegans travel by propagating sinusoidal waves, moving primarily forward, punctuated by brief runs of backing. How these behaviors are generated and altered is not well understood. Using a combination of behavioral analyses and neuronal imaging, I reveal that an activity imbalance between cholinergic A- and B-motoneurons is the key determinant of directional locomotion. Furthermore, heterotypic gap junctions that couple command interneurons and motoneurons of the backward motor circuit, mediated by innexins UNC-7 in AVA and UNC-9 in A-motoneurons, respectively, establish the B>A activity pattern required for forward movement. Loss of this coupling results in both the hyperactivation of AVA backward interneurons revealing the unregulated, endogenous activity of A-motoneurons. With equal A-motoneuron activity levels as B-motoneurons, innexin mutant animals exhibit irregular body bending (kinking) instead of executing forward motion, as well as increased backing. Through a genetic screen, I identified two stomatin-like proteins as regulators of innexin UNC-9 activity that affect C. elegans’ directional movement. The loss of function of stomatin-like unc-1 leads to the same kinker phenotype as unc-7 or unc-9 mutants. Like UNC-9, UNC-1 functions primarily in the A-motoneurons to allow forward motion, suggesting that UNC-1 is required for effective UNC-7-UNC-9 coupling between AVA and A-motoneurons. Dominant mutations in UNC-1, and another stomatin-like protein STO-6, exhibit genetic interactions with these innexin mutants. These mutations partially restore the forward movement of unc-7 mutants, in an UNC-9-dependent manner, indicating that they regulate UNC-9 channel activity in motoneurons to re-establish the B>A-motoneuron activity pattern in the absence of heterotypic gap junctions between interneurons and motoneurons. These studies describe a role of gap junctions as regulators of circuit dynamics by establishing an imbalanced motoneuron activity pattern that favors forward motion, which can be modulated by upper layer inputs. This study also identifies stomatin-like regulators of innexin hemichannel and gap junction function. Future work will focus on understanding mechanisms through which these stomatins regulate the activity of specific innexin channels in C. elegans motoneurons, as well as their contribution to the dynamic output of the C. elegans motor circuit.

NCA-1 (putative nematode calcium channel) and NCA-2 are two cation channel-like proteins in Caenorhabditis elegans that function redundantly to regulate locomotion through unknown mechanisms. A recent study from our lab showed that in vivo Ca2+ imaging analyses of egg-laying neurons in nca-1 loss- and gain-of-function mutants implicate that NCA channels regulate Ca2+ flux at synapses, without affecting Ca2+ dynamics in neuron somas. Furthermore, we observed that NCA-1 localizes to non-synaptic region along axons, strongly suggesting that NCA channels propagate electrical signals from cell bodies to synapses. To identify molecular components that function in the nca-1 genetic pathway, I performed a genetic suppressor screen that led to the identification of behavioral suppressors of nca-1 gain-of-function mutant. Possible NCA auxiliary subunits, UNC-79 (uncoordinated) and UNC-80, were identified from this screen. Molecular characterization of other suppressors will help to identify other regulators and downstream signaling components through which NCA channels transmit electrical signals.

No description available.

The anti-nematodal drug ivermectin hyperactivates invertebrate-specific glutamate-gated chloride channels (GluCls) causing pharyngeal paralysis and a cessation of feeding and growth. I find that for C. elegans even brief exposure to ivermectin can lead to irreversible pharyngeal paralysis. Ivermectin induces heterogeneous vacuolation in the pharynx that appears slowly and accumulates over several days. This vacuolation is almost completely rescued by a mutation in avr-15, which codes for the alpha-subunit of pharyngeal GluCls. The vacuoles stain strongly with Lysotracker Red and are therefore likely to be acidic compartments of the endosomal-lysosomal system. Examination of mutants defective for endocytosis (rme-1, rme-8, and cup-5) uncovers the presence of acidic vacuoles identical in appearance to ivermectin-induced vacuoles. Further, RME-1, a marker for recycling endosomes, is shown to redistribute soon after ivermectin exposure. Examination of the effects of ivermectin on extrapharyngeal neurons expressing ectopic avr-15 reveals an apoptotic phenotype that is shown to be ced-independent.