The prototype uncoupling protein 1 (UCP1) mediates proton leak-dependent thermogenesis in mammals, but the physiological functions of the novel UCP2-5 are unclear. Nematodes only express one uncoupling protein that is most similar to UCP4 in the human brain, which is believed to be the most evolutionarily conserved of the uncoupling proteins. Consistent with reported UCP functions in mammals, we observed that ceUCP4-null nematodes had decreased metabolic rates and increased adiposity compared to wild type. Surprisingly, these phenotypes corresponded to decreased succinate-mediated mitochondrial respiration without apparent changes in mitochondrial uncoupling. ceUCP4-null mitochondria exhibited normal electron transport chain functions, but had a decreased capacity for succinate import. Supporting the functional importance of ceUCP4-dependent complex II regulation in vivo, ceUCP4 deficiency was demonstrated to result in a selectively lethal response to genetic and pharmacological inhibition of Complex I. Similarly, ceUCP4-deficiency significantly prolonged lifespan in the short-lived mev-1 mutant that generates deleterious complex II-derived reactive oxidants. These results define a new physiological function for the ancestral ceUCP4 in the regulation of complex II-mediated oxidative phosphorylation through an unexpected effect on mitochondrial succinate transport. The data described in this dissertation also describe a novel mechanism by which uncoupling proteins mediate mitochondrial bioenergetics. / text

<p>Early life exposure to mitochondrial toxicants, including paraquat, rotenone, and manganese, has been hypothesized to promote early onset of genetic mitochondrial disorders as well as common degenerative diseases such as Parkinson's Disease and Alzheimer's Disease. This dissertation aimed to investigate the biochemical and physiological effects of early life exposure to mitochondrial genotoxicants during development in the whole organism model<i>C. elegans</i>. In the first experiment, a panel of model mammalian neurotoxicants and heavy metal ions was screened for mitochondrial genotoxicity by measuring mitochondrial DNA (mtDNA) copy number and damage in <i>C. elegans</i>. Exposures to paraquat, cumene hydroperoxide, rotenone, maneb, cadmium (II) chloride, and manganese (II) chloride have no significant effect on the mtDNA : nuclear DNA (nuDNA) ratio; only exposure to paraquat resulted in higher mtDNA than nuDNA damage level.In the second experiment, a laboratory method was developed to generate persistent mtDNA damage in larval <i>C. elegans</i> using serial ultraviolent C (UVC) exposures. While the mitochondrial DNA damage persisted from L1 to L4 stage, there was no difference between mitochondrial copy number of the control and UVC treated worms. The UVC treatment significantly inhibited both ATP level and oxygen consumption 24 and 48 hr after the exposure, while the mitochondrial mRNA expression was inhibited 3 hr after the exposure. The <i>pink-1</i> mutation, a mitochondrial serine/threonine-protein kinase involved in the mitophagy process, appeared to limit the growth inhibitory effect of UVC treatment and increase the mitochondrial DNA content of the organism. In the third experiment,larval <i>C. elegans</i> was exposed to UVC and paraquat and examined using differential interference contrast and fluorescence confocal microscopy. Both resulted in detectable, dose-dependent lesions in dopaminergic CEP neurons in adult <i>C. elegans</i>. Neither significant lesions in the GABAergic dorsal nerve cord nor any sign of pharyngeal necrosis were detected. This work demonstrated a mechanism in which early life exposure to mitochondrial genotoxicants could result in both biochemical and physiological changes in later stages of life, thereby highlighting the potential health hazard of time-delayed effects of these chemicals in the environment.</p> / Dissertation

In humans, the functional regulation of facilitative glucose transporters (GLUTs) by insulin plays a central role in the maintenance of glucose homeostasis. The insensitivity of tissues to this regulation results in diabetes mellitus, however, the underlying mechanisms remain largely unknown. To establish Caenorhabditis elegans (C. elegans) as a model system to study the mechanisms of insulin regulation of GLUTs because of the well-conserved insulin/IGF-like signaling (IIS) and many unique advantages of this organism, we functionally characterized 9 candidate genes of human GLUT homologues in C. elegans based on their sequence homologies to GLUTs. We found that FGT-1 is the only functional GLUT homologue with the ability to transport 2-deoxy-D-glucose (2DG) in Xenopus oocytes. FGT-1 mediated 2DG transport could be inhibited by the GLUT inhibitor phloretin and exhibited a Michaelis constant (Km) of 2.8 mM, which is smaller than the Km values of human GLUT1 and GLUT4. In addition to glucose, FGT-1 could also transport mannose, galactose, and fructose. Using a FGT-1::GFP fusion construct under the control of the 5 kb promoter sequence of the fgt-1a gene, FGT-1 was shown to be ubiquitously expressed in C. elegans tissues and cells, including the digestive tract, neurons, and body wall muscle. Two FGT-1 alternative splicing isoforms, FGT-1A and FGT-1B, showed similar transport activity and tissue localization. To study the function of FGT-1 and its regulation by IIS, the changes in several phenotypes that are known to be regulated by IIS were observed in FGT-1-knockdown worms or null strains in the presence or absence of IIS activity. FGT-1 knockdown resulted in fat accumulation but had no effects on dauer formation or brood size in both wild-type and daf-2 (insulin receptor) gene mutant strains. However, the function of FGT-1 in animal growth and aging was dependent on the IIS background, suggesting IIS regulation of FGT-1 function. Consistently, FGT-1 mediated glucose uptake was almost completely defective in the daf-2 and age-1 (PI3 kinase) mutants, and phloretin could only marginally inhibit 2DG uptake in these strains. This defect was only partially related to the approximately 60% decrease in FGT-1 protein levels in these mutants, suggesting the involvements of both post-transcriptional and post-translational regulatory mechanisms. We also found that OGA-1, an O-GlcNAcase, is essential for the function of FGT-1, implying possible regulation of FGT-1 function by glycosylation. In summary, our study has established C. elegans as a powerful model to study the mechanism by which insulin regulates glucose transporters and has provided insights into the mechanism of defective glucose uptake by tissues in patients with diabetes.

The worm Caenorhabditis elegans is a well-studied model organism in numerous aspects of its biology. This small free living nematode has less than 1,000 cells, but shows clear conservation in both signaling and behavior to mammals in aspects of appetite control. This is of importance to humans, where failure of appetite control is a major factor in the unprecedented obesity epidemic that we see today. In general, worm behavior reflects its internal nutritional state and the availability and quality of food. Specifically, worms show a behavioral state that mimics aspects of the mammalian behavioral satiety sequence, which has been termed satiety quiescence. We have used locomotion tracking and Hidden Markov Model analysis to identify worm behavioral state over time, finding quiescence along with the established worm locomotive behaviors roaming and dwelling. Using this analysis as well as more conventional cell biology and genetic approaches we have further investigated satiety signaling pathways. We have found that the neuron ASI is a major center of integration of signals regarding the internal nutritional state of the worms as well as the nutritional content of its environment. Our results show that cGMP causes levels of the TGFβ ligand to be increased in fasted worms, which is then released and binds to its receptor on the RIM and RIC neurons. This signaling connects nutritional state to behavioral response, promoting the sleep-like behavioral state satiety quiescence. Additionally, we have begun a candidate approach examining several other groups of signaling molecules for potential roles in satiety quiescence signaling including cannabinoids, multidrug resistance proteins, and neuropeptides. The result of this investigation is a better understanding of mechanisms of satiety quiescence signaling as well as a new tool that provides highly quantitative, unbiased, and automated data to aid in our ongoing work.

Marfan Syndrome (MFS) is one of the most common monogenic diseases and affects approximately 1 in 5,000 individuals worldwide. The syndrome is characterized by elongated extremities, tall stature, slender frame, and cardiac, and vision abnormalities due to severe connective tissue defects. It is caused by mutations in the fbn1 gene, which encodes an extracellular matrix glycoprotein, and is required for proper cardiac and skeletal development and for sequestration of TGFβ (transforming growth factor beta) and BMP (bone morphogenetic protein) within the extracellular matrix (ECM). The primary objective of this study was to establish a C.elegans MFS model and use this model to determine which genes interact with a C.elegans fbn1 homolog, MUA-3 and ascertain the role of metabolic rate in the development of MFS pathology. We isolated a temperature sensitive mutant of mua-3, a fbn1 homolog. We found that at the fourth larval molt, when animals shed the exoskeleton and rebuild a new one, the mutants die due to extensive mechanical stress in connective tissue shown as fragmented internal structures. Using this mutant, an unbiased forward genetic screen to isolate the genetic interactors of the fibrillin gene homolog, was completed. A collagen gene, that has been implicated to genetically interact with a bone morphogenetic protein (BMP), was isolated. This suggests that mua-3(uy19) may interact with genes involved in TGFβ regulation during the L4 molt and that fibrillin-1, TGF-β, and metalloproteases may act in-concert to modulate TGFβ availability and connective tissue integrity in C. elegans. In addition, we found that two independent mutations of mua-3 show temperature-sensitive phenotypes. Based on this result, we propose that increase of temperature aggravates the phenotype potentially due to increased metabolism. This hypothesis, if correct, will suggest a potential connection between metabolic rate and severity of MFS pathology.

Alcohol abuse is a problem in our society. There are few treatments available, in part due to the unclear molecular mechanisms of ethanol’s effects. Human studies indicate that there is a genetic component influencing disease susceptibility, and that an individual’s initial response to alcohol can predict their development of addiction. We have taken a forward genetics approach to study one component of initial response, acute functional tolerance (AFT), in Caenorhabditis elegans. We identified bet11, a mutation that causes animals to be defective in the development of AFT. Genetic analysis suggested that the gene that bet11 disrupts participates in a synthetic genetic interaction with an unlinked natural allelic variant in another gene that alters ethanol response. We also examined the role of lipid membrane composition in the response to ethanol. Identification of modulators that are responsible for alcohol-induced responses will provide a greater understanding of the mechanisms that cause alcohol-related diseases.

Wnt signalling belongs to conserved pathways and mediates cell fate decision, development, regeneration and adult tissue homeostasis. Disruption or misregulation of Wnt signalling pathway often leads to disease. Wnt proteins are hydrophobic glycoproteins which need a special receptor for transport from Golgi Apparatus to cell surface, which is called MIG-14 in Caenorhabditis elegans and Wntless (Wls) in mammals. In this study, I focus on understanding mechanisms that regulate MTM-6 protein activity. MTM-6, a lipid phosphatase associated with endosomal membrane, has been recently identified as a regulator of MIG-14/Wls trafficking in Caenorhabditis elegans. Silencing of mtm-6 leads to misregulation of some Wnt-directed processes, such as migration of Q neuroblasts progeny. This study reports identification of novel mtm-6 genetic interactors that have been found to influence migration of Q neuroblasts progeny through Wnt signalling. New knowledge about mtm-6 genetic interactions bring us near to understanding of Wnt signalling regulation. Keywords: Caenorhabditis elegans, MTM-6, SEL-5, Wntless, Wnt, endosomes, phosphoinositides, retromer

Master of Science / Division of Biology / Michael A. Herman / Caenorhabditis elegans are free-living bacterivorous nematodes that naturally consume bacteria as food source. As an excellent genetic model, C. elegans has proven to be a successful system to study innate immune responses to human pathogens, which resulted in identification of many evolutionarily conserved defense pathways. Most of these studies examined innate immune pathway mutants in a single genetic background in response to monoculture of human pathogens that worms might not necessarily encounter in the wild. While this has led to the successful genetic dissection of these defense pathways, in order to fully understand their biological functions, the relevant ecological and evolutionary context needs to be taken into account. The bacterial environment C. elegans naturally encounter is likely to be highly heterogeneous. While many bacteria are mainly considered as dietary resource for worms, some could be potential pathogens. Worms thus constantly face the challenge to defend against the pathogens mixed in the food. Stenotrophomonas maltophilia is one such bacterium. S. maltophilia is a ubiquitous bacterium that has been found associated with native nematodes. But it can also cause nosocomial infections in human, especially in immune-compromised individuals. Due to its natural resistance to multiple antibiotics, it has been emerging as an opportunistic human pathogen. Our lab isolated a S. maltophilia strain, JCMS, which was found being pathogenic to C. elegans. Both C. elegans strains, N2 (Bristol, England) and CB4856 (Hawaii), showed decreased survivorship when fed on S. maltophilia JCMS compared to E. coli OP50. However, more interestingly, the specific responses towards bacteria are different between strains. This indicates that survivorship of C. elegans is determined by not only genetic and environmental factors, but also genotype by environment (G×E) interactions (GEI). In order to identify the underlying genetic basis, we mapped quantitative trait loci (QTL) in a N2×CB4856 recombinant inbred panel for the survivorship in response to E. coli OP50 and S. maltophilia JCMS.

No description available.

Thesis advisor: John Wing / Thesis advisor: Stephen Wicks / Molecular biology and genetics, single nucleotide polymorphism genetic mapping, phenotypic assays including behavioral assessment, and fluorescent microscopy of GFP-tagged proteins were used to study ciliary defects in the nematode Caenorhabditis elegans. Mammalian cilia are multifunctional. Some of the physiological roles in which they are involved include sensing developmental signaling molecules and ligands as well as creating flows of mucus and cerebrospinal fluid that function as flow meters and mechanosensors. Due to the multifunctional nature of cilia, it is not surprising that many human diseases can be caused by ciliary defects. Bardet-Biedl Syndrome is a rare genetic ciliopathy characterized by retinal degeneration, polydactyly, obesity, cystic kidneys, mental retardation, and many other ailments. We have identified osm- 12/bbs-7 to be a C. elegans homologue of human BBS7, a gene known to cause Bardet-Biedl Syndrome when mutated. With the help of Michel Leroux’s group, I showed the BBS-7 protein to be localized to the base of cilia and to undergo intraflagellar transport along the ciliary axoneme. Our findings suggest that BBS- 7 plays a role in the assembly and/or functioning of the IFT complex. I also performed a mutagenesis and phenotypic screen for animals defective in the uptake of DiI into a subset of their ciliated neurons in order to identify new components involved in ciliogenesis and IFT. I describe an extended bulked segregant analysis (BSA) mapping methodology, which can save time and resources by filtering out alleles of previously known genes without performing time-consuming interval mapping. In addition, I identified one of the 11 dyefilling defective alleles from the screen to be a novel allele of dyf-3, which encodes a protein required for sensory cilia formation. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.