The field of neuroscience has recently seen optogenetics emerge as a highly utilized and powerful method of non-invasive neural activation and inhibition. This thesis seeks to enhance the optogenetic toolbox through the design, construction, and evaluation of a number of hardware and software modules for research in Caenorhabditis elegans neuroscience. In the first aim, we combine optogenetics, microfluidics, and automated image processing, to create a system capable of high-throughput analysis of synaptic function. In the second aim, we develop a multi-modal illumination system for the manipulation of optogenetic reagents. The system is capable of multi-spectral illumination in definable patterns, with the ability to dynamically alter the intensity, color, and shape of the illumination. The illumination system is controlled by a set of software programs introduced in aim three, and is demonstrated through a set of experiments in aim four where we selectively activate and inhibit specific neural nodes expressing optogenetic reagents in freely moving C. elegans. With the ability to target specific nodes in a freely moving animal, we can correlate specific neural states to behaviors allowing for the dissection of neural circuits. Taken together, the developed technologies for optogenetic researchers will allow for experimentation with previously unattainable speed, precision and flexibility.

<p>Mitochondria are the primary energy producers of the cell and play key roles in cellular signaling, apoptosis and reactive oxygen species (ROS) production. Mitochondria are the only organelles that contain their own genome which encodes for a small subset of electron transport chain (ETC) proteins as well as the necessary tRNAs and ribosomal subunits to translate these proteins. Over 300 pathogenic mitochondrial DNA (mtDNA) mutations have been shown to cause a number of mitochondrial diseases emphasizing the importance of mtDNA maintenance and integrity to human health. Additionally, mitochondrial dysfunction and mtDNA instability are linked to many wide-spread diseases associated with aging including cancer and neurodegeneration. Mitochondria lack the ability to repair certain helix-distorting lesions that are induced at high levels in mtDNA by important environmental genotoxins including polycyclic aromatic hydrocarbons, ultraviolet C radiation (UVC) and mycotoxins. These lesions are irreparable and persistent in the short term, but their long-term fate is unknown. Degradation of mitochondria and mtDNA is carried out by autophagy. Autophagy is protective against cell stress and apoptosis resulting from exposure to mitochondrial toxicants suggesting that it plays an important role in removal of unstable mitochondria that can serve as a source of ROS or initiate apoptotic cell death. Furthermore, dysfunctional mitochondria can be specifically targeted for degradation by the more specific process of mitophagy influenced in part by the processes of mitochondrial dynamics (i.e., fusion and fission). </p><p>The goals of this dissertation were to investigate the long-term fate of helix-distorting mtDNA damage and determine the significance of autophagy and mitochondrial dynamics in removal of and recovery from persistent mtDNA damage. Removal of irreparable mtDNA damage and the necessity of autophagy, mitophagy, fusion and fission genes in removal of this damage were examined using genetic approaches in adult <italic>Caenorhabditis elegans</italic>. In order to investigate the significance of autophagy, fusion and fission genes in recovery from mtDNA damage-induced mitochondrial dysfunction <italic>in vivo</italic>, an experimental method was developed to specifically induce persistent mtDNA damage and mitochondrial dysfunction without persistent nDNA damage in developing <italic>C. elegans</italic>. Additionally, the effect of persistent helix-distorting DNA damage on mitochondrial morphology, mitochondrial function and autophagy was investigated in <italic>C. elegans</italic> and in mammalian cell culture. The rate and specificity of mitochondrial degradation was further examined in cell culture using live-cell fluorescence microscopy and transmission electron microscopy. </p><p>Removal of UVC-induced mtDNA damage was detectable by 72 hours in <italic>C. elegans</italic> and mammalian cell culture, and required mitochondrial fusion, fission and autophagy, providing genetic evidence for a novel mtDNA damage removal pathway. UVC exposure induced autophagy with no detectable effect on mitochondrial morphology in both systems; mitochondrial function was inhibited in the <italic>C. elegans</italic> system but not in the cell culture system in which the degree of mtDNA damage induced was less. Furthermore, mutations in genes involved in these processes as well as pharmacological inhibition of autophagy exacerbated mtDNA damage-mediated larval arrest, illustrating the <italic>in vivo</italic> relevance of removal of persistent mtDNA damage. Mutations in genes in these pathways exist in the human population, demonstrating the potential for important gene-environment interactions affecting mitochondrial health after genotoxin exposure.</p> / Dissertation

In my thesis I aimed to contribute to the understanding of the mechanism of cytokinesis in C. elegans embryos. I wanted to analyze the relative contributions of different spindle parts – microtubule asters and the midzone - to cytokinesis furrow positioning. I developed a UV laser-based severing assay that allows the spatial separation of the region midway between the asters and the spindle midzone. The spindle is severed asymmetrically between one aster and the midzone. I found that the spindle provides two consecutive signals that can each position a cytokinesis furrow: microtubule asters provide a first signal, and the spindle midzone provides a second signal. The use of mutants that do not form a midzone suggested that the aster-positioned furrow is able to divide the cell alone without a spindle midzone. Analysis of cytokinesis in hypercontracile mutants suggests that the aster-positioned cytokinesis furrow and the midzone positioned furrow inhibit each other by competing for cortical contractile elements. I then wanted to identify the molecular pathway responsible for cytokinesis furrow positioning in response to the microtubule asters. To this end, I performed an RNAi screen, which identified a role for LET-99 in cytokinesis: LET-99 appeared to be required for aster-positioned cytokinesis but not midzone-positioned cytokinesis. LET-99 localizes as a cortical band that overlaps with the cytokinesis furrow. Mechanical displacement of the spindle demonstrated that the spindle positions cortical LET-99 at the site of furrow formation. The furrow localization of LET-99 depended on G proteins, and consistent with this finding, G proteins are also required for aster-positioned cytokinesis. (Anlage: Quick time movies, 466, 67 MB)

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "August 2000." Typescript. Vita. Includes bibliographical references (leaves 107-112). Also available on the Internet.

Thesis (Ph. D. in Pharmacology)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

In order to develop an environmentally friendly seaweed extract treatment which will benefit both pre- and post-harvest qualities of vegetables, the effects of pre-harvest application of the brown algae Ascophyllum nodosum extracts on the nutritional quality and post-harvest storability of spinach (Spinacia oleracea L.) was investigated. Plants treated with A. nodosum extracts accumulated higher concentrations of iron, potassium, total soluble protein, and total phenolics as compared to untreated controls. 1H NMR and LC-MS analysis revealed a roughly 50% enhanced accumulation of the 9 flavonoids identified, which is partially confirmed by the elevated chalcone isomerase activity. A. nodosum extract treatment caused an increase in transcription of the genes related to plant growth, osmolyte accumulation, and antioxidative activities. Post-harvest analysis revealed that A. nodosum extract treatment caused an enhanced storability of spinach leaves in terms of visual quality, weight loss, and senescence. Lipid peroxidation and ascorbate content were correlated with visual quality during storage. Animal experiments using the Caenorhabditis elegans nematode model revealed that spinach extracts prolonged the life span of C. elegans, and A. nodosum extract-enhanced polyphenols exerted improved beneficial effects in C. elegans against oxidative and heat stresses. Taken together, the results suggest that A. nodosum extracts enhance both pre- and post-harvest quality of spinach through stimulation of flavonoid pathways, thus leading to accumulation of flavonoids and promotion of anti-radical capacity in spinach leaves, which may protect the plant tissue against reactive oxygen species and subsequent decay. Furthermore, the increased flavonoid content in spinach exerted beneficial effects in C. elegans against oxidative and heat stresses via different mechanisms.

The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex. / Thesis (Ph.D, Biology) -- Queen's University, 2011-07-28 16:20:31.957

'The rate of living' hypothesis of aging speculates that the metabolic rate of a species ultimately determines its life expectancy. Using the nematode worm Caenorhabditis elegans as model system, mutation in twp-1 (t&barbelow;ime w&barbelow;arp) gene was found to significantly delay biological timing and remarkably increase mean and maximum life span. The rate of living in twp-1 is dramatically delayed in all the biological processes we tested, including rates of rhythmic adult behaviors, development, and reproduction. Oxygen consumption, which indicates metabolic rate of an organism, is reduced to approximately two-fold in twp-1 mutant. According to my study, twp-1 and dauer genes, daf-2 and daf-16, interact to determine biological timing and adult life span. twp-1 mutation prolongs life span in a way that is at least partially different from dauer formation mutants, whose longevity might due to their high resistance to stresses, especially oxidative stress. twp-1 gene is cloned and found to encode iron-sulfur protein (ISP) in complex III, which is the major site of mitochondrial superoxide radical production, of the mitochondrial respiratory chain. This suggests that twp-1 may live long because they produce less reactive oxygen species (ROS), and thus, result in less oxidative damage. mts-1 (mitochondrial twp-1 suppressor) mutation can fully or partially rescue most of the biological timing in twp-1 mutant, including both developmental and behavioral rates, but except life span. mts-1 encodes another subunit of complex III, cytochrome b, which normally interact with ISP during function. mts-1 might somehow restore the activity of complex III, and consequently, accelerate the rate of living. Paraquat, a herbicide that induces the formation of superoxide, was used to provide an acute oxidative stress to animals. twp-1; mts-1 was found to be highly resistant to paraquat, indicating that twp-1 animals are well capable of coping with oxidative stress. According to o

Mutations in the clk-1, clk-2, clk-3 and gro-1 genes in Caenorhabditis elegans show alterations in developmental and behavioral timing and lifespan, collectively termed the Clk phenotype. While the clk-1, clk-2, and gro-1 genes have been cloned, clk-3 gene has not been identified. Gene expression changes in clk-3 mutant worms were determined using microarray expression data. I examined genes in the region to which clk-3 gene maps, for strongly reduced expression in the clk-3 mutants and identified thirteen clk-3 candidate genes. RNAi feeding vectors for all these candidate genes were picked and cultured from the RNAi library. Knock-down worm strains were generated by feeding RNAi and analyzed for Clk phenotypes. Of all the candidate genes tested, the Y48E1B.5 gene showed the most similar phenotypic profile to the clk-3 mutants. The Y48E1B.5 gene shows weak homology to a mammalian mitochondrial ribosomal protein. Primers were designed to amplify all 9 exons of the Y48E1B.5 gene. Sequence analysis was carried out on the resulting PCR products from clk-3 mutants. An amino acid change was found in exon 4.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 3 uppsatser.