RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes are required downstream for interference. These findings provide evidence for germline transmission of an extragenic sequence-specific silencing factor and implicate rde-l and rde-4in the formation of the inherited agent. Other forms of homology-dependent silencing in C. elegansinclude co-suppression and transcriptional silencing of transgenes in the germline. We demonstrate that silencing of a germline transgene can be initiated by injected dsRNA, via the RNAi pathway, and then maintained on a different level. This observation indicates that post-transcriptional and transcriptional silencing of homologous genes could be connected. This dissertation also describes the connection between RNAi and developmental pathways of gene regulation in C. elegans. We show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-l), and two homologs of rde-1 (alg-l and alg-2) cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-l, alg-l, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7small temporal RNAs that regulate stage-specific development. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. Finally, this study illustrates the detection of small interfering RNAs (siRNAs), intermediates in the RNAi process, and describes requirements for their accumulation. We show that, in the course of RNAi induced by feeding dsRNA, C. elegans accumulate only siRNAs complementary to the target gene. This accumulation depends on the presence of the target sequence and requires activities of several RNAi-pathway genes. We show that selective retention or amplification of RNAi-active molecules can create a reservoir of memory antisense siRNAs that prevent future expression of the genes with complementary sequence. This suggests a parallel at the molecular level with the clonal selection of antibody forming cells and in the vertebrate immune system.

145 p. Advisers: John Conery (Computer and Information Science)and Shawn Lockery (Biology) / A print copy of this title is available through the UO Libraries under the call number: SCIENCE QA76.87 .D96 2006 / This thesis makes two major contributions: it introduces a novel method for analysis of artificial neural networks and provides new models of the nematode Caenorhabditis elegans nervous system. The analysis method extracts neural network motifs,or subnetworks of recurring neuronal function, from optimized neural networks. The method first creates models for each neuron relating network stimulus to neuronal response, then clusters the model parameters, and finally combines the neurons into multi-neuron motifs based on their cluster category. To infer biological function, this analysis method was applied to neural networks optimized to reproduce C. elegans behavior, which converged upon a small number of motifs. This allowed both a quantitative exploration of network function as well as discovery of larger motifs. Neural network models of C. elegans anatomical connectivity were optimized to reproduce two C. elegans behaviors: chemotaxis (orientation towards a maximum chemical attractant concentration) and thermotaxis (orientation towards a set temperature). Three chemotaxis motifs were identified. Experimental evidence suggests that chemotaxis is driven by a differentiator motif with two important features. The first feature was a fast, excitatory pathway in parallel with one or more slow, inhibitory pathways. The second feature was inhibitory feedback on all self-connections and recurrent loops, which regulates neuronal response. Six thermotaxis motifs were identified. Every motif consisted of two circuits, each a previously discovered chemotaxis motif with most having a dedicated sensory neuron. One circuit was thermophilic (heat-seeking) and the other was cryophilic (cold-seeking). Experimental evidence suggests that the cryophilic circuit is a differentiator motif and the thermophilic circuit functions by klinokinesis. / NSF: IBN-0080068

During embryonic development of the nematode Caenorhabditis elegans, cell fates are specified by asymmetric segregation of cell fate determinants and via cell-cell signaling events. Specification of the eight-cell stage blastomere E, the endoderm progenitor cell, requires both cell signaling and asymmetric cell division. At the four-cell stage, a polarity-inducing signal from the P2 cell is required for the EMS cell to divide asymmetrically to produce an anterior daughter MS, and posterior daughter E. In the absence of signal, the EMS cell divides symmetrically to produce two daughters that adopt the MS fate. This thesis describes the identification and analyses of seven genes required to tranduce this polarity-inducing signal and specify endoderm formation. The mom-1, mom-2, mom-5, apr-1, and wrm-1 genes are homologous to components of the Wnt/Wingless signal transduction pathway, and the mom-4, and lit-1 genes are related to components of the mitogen-activated protein kinase pathway. Biochemical analysis of these signaling molecules reveal a novel convergence of these pathways at the level of the LIT-1 and WRM-1 proteins, which appear to function as a kinase complex and are required for the downregulation of POP-1. Together these genes constitute components of a complex genetic pathway required for specification of the E cell fate.

Phosphorescent transition metal complexes make up an important group of compounds that continues to attract intense research owing to their intrinsic bioimaging applications that arise from bright emissions, relatively long excited state lifetimes, and large stokes shifts. Now for biomaging assay a model organism is required which must meet certain criteria for practical applications. The organism needs to be small, with a high turn-over of progeny (high fecundity), a short lifecycle, and low maintenance and assay costs. Our model organism C. elegans met all the criteria. The ideal phosphor has low toxicity in the model organism. In this work the strongly phosphorescent platinum (II) pyrophosphito-complex was tested for biological applications as a potential in vivo hypoxia sensor. The suitability of the phosphor was derived from its water solubility, bright phosphorescence at room temperature, and long excited state lifetime (~ 10 µs). The applications branched off to include testing of C. elegans survival when treated with the phosphor, which included lifespan and fecundity assays, toxicity assays including the determination of the LC50, and recovery after paraquat poisoning. Quenching experiments were performed using some well knows oxygen derivatives, and the quenching mechanisms were derived from Stern-Volmer plots. Reaction stoichiometries were derived from Job plots, while percent scavenging (or antioxidant) activities were determined graphically. The high photochemical reactivity of the complex was clearly manifested in these reactions.

Alzheimer’s disease (AD) is symptomized by amyloid-beta plaques in the brain and accounts for more than 65 percent of dementia cases. Vitamin B12 (cobalamin) deficiency can result in similar cognitive impairment and roughly 15% of the elderly are vitamin B12 deficient. Vitamin B12 deficiency results in the accumulation of toxic methylmalonic acid and homocysteine. Hyperhomocysteinemia is a strong risk factor for AD. To test if vitamin B12 deficiency stimulates amyloid-beta toxicity, Caenorhabditis elegans expressing amyloid-beta in muscle were fed either vitamin B12-deficient OP50-1 or vitamin B12-rich HT115(DE3) E. coli bacteria. Increased amyloid-beta toxicity was found in worms fed the 0P50-1 diet. Supplementation of the OP50-1 diet with vitamin B12 did not rescue the increased C. elegans toxicity. Knockdown of either of the only two C. elegans vitamin B12-dependent enzymes metr-1 or mmmc-1 protected against toxicity. Therefore, vitamin B12 deficiency does not stimulate Alzheimer’s amyloid-beta-mediated toxicity in C. elegans.

6 Abstract Lipopolysaccharides are composed of covalently bound saccharides. They are a characteristic component of the cell wall of gram-negative bacteria. They are the cause of severe sepsis in humans and complications in human medicine. Lipopolysaccharides are a constant part of the infections of gram-negative bacteria. We expect an evolutionarily conserved non-specific immune response and protection. The question is whether there is an immune response in the model organism Caenorhabditis elegans. If so, what mechanism is controlled and regulated. We submitted lipopolysaccharides from the bacteria Pseudomona aeruginosa with the bacteria Escherichia coli OP50 and observed the influence of lipopolysaccharides on the expression of selected genes. We examined metabolism and development. We have shown the influence of lipopolysaccharides on gene expression of C-type lectine clec-60 a clec-71, nextna lys-5, hsp-60 a F44G.3.2.1 genes. We incubated Caenorhabditis elegans on some components of lipopolysaccharide. We found regulation of these selected genes with hydrophobic components of lipopolysacharide, lipid A. We did not observe regulation with saccharide components of lipopolysaccharide, glucose and galatose. The metabolism of lipids had changed. We demonstrated a reduction of neutral lipids and changes in...

Aging can be defined as the accumulation of changes affecting the maintenance of homeostatic processes over time, leading to functional decline and increased risk for disease and death. In its simplicity, aging is the systemwide deterioration of an organism. Genetic studies have identified many potential molecular mechanisms of aging including DNA damage, telomere shortening, mitochondrial dysfunction, increased oxidative stress, uncontrolled inflammation, and hormone dysregulation (reviewed in [1]). However, in reality, aging is likely to be a combination of some (or potentially all) of these mechanisms. Interestingly, aging and metabolism are tightly coordinated. Aging is a major contributor to metabolic decline and related diseases, including type 2 diabetes, metabolic syndrome, and cancer. One of the best characterized metabolic pathways implicated in aging is the insulin/IGF-1 signaling (IIS) pathway. Downstream signaling components of the IIS pathway receptor have been well studied and include an interconnected network of signaling events that regulate many physiological outputs. However, less is known about the role of upstream signaling components and how intracellular pathways and physiology are regulated accordingly. In Part I, I present my work towards understanding upstream IIS pathway components using a systems biology approach. The goal of this study is to gain insight into the redundancy and specificity of the insulin gene family responsible for initiating IIS pathway activity in Caenorhabditis elegans. The information gained will serve as a foundation for future studies dissecting the molecular mechanisms of this pathway in efforts to uncouple the downstream signaling and physiological outputs. The clear impact of metabolism on aging and disease stimulated questions regarding the potential of promoting health and longevity through diet and dietary mimetics. Recent findings indicate reduced food intake, meal timing and nutritional modulation of the gut microbiome can ameliorate signs of aging and age-associated diseases. Aging, therefore, is also the result of dynamic and complex interplay between genes of an organism and its environment. In Part II, I will discuss my efforts to gain insight into how diet influences aging. This preliminary study has demonstrated that diet can affect lifespan in the model organism, C. elegans. Additionally, we observe diet-specific effects on drug efficacy that, in turn, modulates C. elegans lifespan and reproduction. The implications of these experiments, while limited, illustrate a potentially greater role in diet- and drug-mediated effects on lifespan.

Mobile genetic elements represent a large portion of the genome in many species. Posing a danger to the integrity of genetic information, silencing and structural machinery has evolved to suppress the mobility of foreign and transposable elements within the genome. Condensin proteins – which regulate chromosome structure to promote chromosome segregation – have been demonstrated to function in repetitive gene regulation and transposon silencing in several species. In model system Caenorhabditis elegans, microarray analysis studies have implicated Condensin II subunit HCP-6 in the silencing of multiple loci, including DNA transposon MIRAGE. To address the hypothesis that HCP-6 has a direct function in transcriptional gene silencing of the MIRAGE transposon, we queried MIRAGE expression and chromatin profiles in wild-type and hcp-6 mutant animals. Our evidence confirms that HCP-6 does indeed function during silencing of MIRAGE. However, we found no significant indication that HCP-6 binds to MIRAGE, nor that HCP-6 mediates MIRAGE enrichment of H3K9me3, the repressive heterochromatin mark observed at regions undergoing transcriptional silencing. We suggest that the silencing of MIRAGE, a newly evolved transposon and the only tested mobile element considerably derepressed upon loss of HCP-6, is managed by HCP-6 indirectly.

It has become increasingly clear that transcription factors (TFs) play crucial roles in the development and day-to-day homeostasis that all biological systems experience. TFs target particular genes in a genome, at the appropriate place and time, to regulate their expression so as to elicit the most appropriate biological response from a cell or multicellular organism. TFs can often be grouped into families based on the presence of similar DNA binding domains, and these families are believed to have expanded and diverged throughout evolution by several rounds of gene duplication and mutation. The extent to which TFs within a family have functionally diverged, however, has remained unclear. We propose that systematic analysis of multiple aspects, or parameters, of TF functionality for entire families of TFs could provide clues as to how divergent paralogous TFs really are. We present here a multiparameter integrated network of the activity of the basic helix-loop-helix (bHLH) TFs from the nematode Caenorhabditis elegans. Our data, and the resulting network, indicate that several parameters of bHLH function contribute to their divergence and that many bHLH TFs and their associated parameters exhibit a wide range of connectivity in the network, some being uniquely associated to one another, whereas others are highly connected to multiple parameter associations. We find that 34 bHLH proteins dimerize to form 30 bHLH dimers, which are expressed in a wide range of tissues and cell types, particularly during the development of the nematode. These dimers bind to E-Box DNA sequences and E-Box-like sequences with specificity for nucleotides central to and flanking those E-Boxes and related sequences. Our integrated network is the first such network for a multicellular organism, describing the dimerization specificity, spatiotemporal expression patterns, and DNA binding specificities of an entire family of TFs. The network elucidates the state of bHLH TF divergence in C. elegans with respect to multiple functional parameters and suggests that each bHLH TF, despite many molecular similarities, is distinct from its family members. This functional distinction may indeed explain how TFs from a single family can acquire different biological functions despite descending from common genetic ancestry.

How does a nervous system orchestrate compound behaviors? Finding the neural basis of behavior requires knowing which neurons control the behavior and how they are connected. To accomplish this we measured and manipulated neural activity in a live, behaving animal with a completely defined connectome. The C. elegans escape response is a compound behavior consisting of a sequence of behavioral motifs. Gentle touch induces a reversal and suppression of head movements, followed by a deep turn allowing the animal to navigate away from the stimulus. The connectome provides a framework for the neural circuit that controls this behavior. We used optical physiology to determine the activity patterns of individual neurons during the behavior. Calcium imaging of locomotion interneurons and motor neurons reveal unique activity profiles during different motifs of the escape response. Furthermore, we used optogenetics and laser ablations to determine the contribution of individual neurons to each motif. We show these that the suppression of head movements and turning motifs are distinct motor programs and can be uncoupled from the reversal. The molecular mechanisms that regulate these motifs involve from signaling with the neurotransmitter tyramine. Tyramine signaling and gap junctions between locomotion interneurons and motor neurons regulate the temporal orchestration of the turning motif with the reversal. Additionally, tyramine signaling through a GPCR in GABAergic neurons facilitates the asymmetric turning during forward viii locomotion. The combination of optical tools and genetics allows us to dissect a how a neural circuit converts sensory information into a compound behavior.