In this study, we investigated the mechanism of nitrogen excretion in the soil nematode Caenorhabditis elegans. Utilizing the scanning ion electrode technique (SIET), it was shown for the first time in nematodes that the excretory cell promotes a secretion of ions, including Na+, K+, H+ and Ca2+. In addition, observations from experiments exposing the animal to various environmental pH regimes suggested that the mode of ammonia excretion is dependent on acidification of the unstirred boundary layer, supported also by a detected H+-net-excretion over the hypodermis employing SIET. Pharmacological experiments, SIET and enzyme activity measurements implicated the participation of a functional microtubule network, V-type H+-ATPase, carbonic anhydrase, Na+/K+-ATPase, and apical Na+-channels in the ammonia excretion mechanism of this roundworm. Most importantly, employing ammonia transporter deficient Saccharomyces cerevisiae we were able to show for the first time that an invertebrate Rh-like protein (Rhr-1) does indeed function as an ammonia transporter. Further, a second Rh-protein, Rhr-2, was found to be predominantly expressed in the hypodermis. Knock-out experiments on this transporter further suggested participation of Rhr-2 in the apical ammonia trapping mechanism. Overall, the results of this study provided evidence for a novel ammonia excretion mechanism over the hypodermis, which exhibits features commonly seen in both freshwater (ammonia trapping) and seawater inhabiting species (vesicular transport and exocytosis). / October 2015

The nervous system is a complex organ that functions in most metazoans to sense and respond to a constantly changing world. How the nervous system does this is a major focus of systems-level neuroscience. This dissertation investigates the neural basis of the sensorimotor transformation underlying a spatial orientation strategy in the nematode Caenorhabditis elegans. Motile organisms rely on spatial orientation strategies to navigate to environments that are conducive to organismal fitness and comfort, e.g. environments with the correct temperature, light level, or access to food and mates. As such, spatial orientation strategies as a class represent a key behavior common to most forms of life on earth. To explore the behavioral mechanism used by C. elegans for spatial orientation, we designed and manufactured a microfluidic device that breaks the feedback loop between self-motion and environmental change by partially restraining the animal. The device takes advantage of laminar flow at small scale to provide distinct environments across the dorsoventral undulation that constitutes locomotion in this animal without using a physical barrier. This device allowed us to conclude that worms use the change in chemical concentration sensed between lateral extremes of the locomotion cycle to direct forward locomotion toward a favorable stimulus, an orientation strategy termed klinotaxis. We then investigated the neuronal basis of this behavior using laser ablation, calcium imaging, and optogenetic stimulation. We found a minimal neuronal network for klinotaxis to sodium chloride including the ASE, AIY, AIZ, and SMB neuron classes that displays left/right asymmetry across the sensory neuron, interneuron, and motor neuron levels. We extended these results by ablating other neurons that have been implicated in klinotaxis in other studies. Finally, we imaged the ASE neurons during klinotaxis in microfluidic device and found that these neurons are active on the timescale of individual head swings. Additionally, we found anecdotal evidence that photostimulation of ASE neurons expressing the light sensitive ion channel Channel Rhodopsin (CHR2) is sufficient to stimulate klinotaxis behavior. This dissertation includes previously published co-authored material.

o presente trabalho teve como objetivo realizar o estudo químico das sementes de Trichilia elegans ssp. Elegans A. Juss. (coletadas no município de Corumbá, MS), visando o isolamento e identificação ou elucidação estrutural dos seus metabólitos secundários, particularmente limonóides. Da fase diclorometânica, obtida de uma partição efetuada com o extrato etanólico das sementes, foram isoladas, através de técnicas cromatográficas de separação (cromatografia em colunas de sílica gel, de Sephadex LH 20 e CLAE em fase reversa), dezoito substâncias, compreendendo: dois protolimonóides, onze limonóides com esqueleto do tipo obacunol (abertura dos anéis A e D), quatro com esqueleto do tipo ivorensato de metila (abertura dos anéis A, B e D) e 3β-O-β-D-glicopiranosilsitosterol. Todas as substâncias são inéditas, com exceção do esteróide glicosilado e de dois limonóides com esqueleto do tipo obacunol (kihadaninas A e B). As determinações estruturais foram efetuadas com base em dados espectroscópicos de RMN 1H e 13C, incluindo experimentos bidimensionais (COSY 1H-1H, COSY 1H-13C, HMQC, NOESY e HMBC); a partir de informações obtidas dos espectros de massas e na região do IV e através de dados de difração de raios-X. Quatro dos limonóides obtidos foram submetidos a um ensaio biológico de atividade antitumoral, utilizando-se linhagens mutantes de Saccharomyces cerevisiae, porém, mostraram-se inativos. / The present work describes the isolation and identification or structural elucidation of the chemical constituents of the seeds of Trichilia elegans ssp. Elegans A. Juss., collected in Corumbá, MS. From the dichloromethane solubles, obtained from partition of the ethanolic extract from the seeds, eighteen substances have been isolated, after a combination of column and flash chromatography on silica gel, gel filtration and reversed phase HPLC separations. The isolated substances have been characterized as two new protolimonoids, nine new obacunol- and four new methyl ivorensate-type limonoids, in addition to two known limonoids belonging to the obacunol group (kihadanins A and B) and 3-O-&#946-D-glucopyranosyl-sitosterol. The structures of these compounds have been established on the basis of 1D (1H, 13C) and 2D (1H-1H and 1H-13C COSY, HMQC, HMBC and NOESY) NMR spectroscopic techniques, IR and mass spectral data and X-ray crystallographic analyses. Four of the isolated limonoids have been tested against DNA reparr deficient mutants of Saccharomyces cerevisiae but, nevertheless, shown to be inactive.

Seizures are a symptom of epilepsy, characterized by spontaneous firing due to an imbalance of excitatory and inhibitory features. While mammalian seizure models receive the most attention, the simplicity and tractability of invertebrate model systems, specifically C. elegans and D. melanogaster, have many advantages in understanding the molecular and cellular mechanisms of seizure behavior. This research explores C. elegans and D. melanogaster as electroconvulsive seizure models to investigate methods to both modulate and better understand seizure susceptibility. A common underlying feature of seizures in mammals, worms, and flies involves regulating excitation and inhibition. The C. elegans locomotor circuit is regulated via well characterized GABAergic and cholingeric motoneurons that innervate two rows of dorsal and ventral body wall muscles. In this research, we developed an electroconvulsive seizure assay which utilizes the locomotor circuit as a behavioral read out of neuronal function. When inhibition is decreased in the circuit, for example by decreasing GABAergic input, we find a general increase in the time to recovery from a seizure. After establishing the contribution of excitation and inhibition to seizure recovery, we explored a ubiquitin ligase, associated with comorbidity of an X-linked Intellectual Disorder and epilepsy in humans, and established that the worm homolog, eel-1, contributes to seizure susceptibility similarly to the human gene. Next, we investigated a cGMP-dependent protein kinase (PKG) that functions in the nervous system of both worms and flies and determined that increasing PKG activity, decreases the time to recovery from an electroconvulsive seizure. These experiments suggest a potential novel role for a major protein, PKG, in seizure susceptibility and that the C. elegans and D. melanogaster electroconvulsive seizure assays can be used to investigate possible genes involved in seizure susceptibility and future therapeutic to treat epilepsy. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

The calorie restriction (CR) and insulin/IGF-I-like signalling (IIS) are two pathways regulating the lifespan of C. elegans. Recent studies showed that glucose restriction extends the lifespan of C. elegans while excessive glucose shortens the lifespan of the worms. The first step of the glucose metabolism is the transport of glucose across the plasma membrane by the glucose transporters. The work described in this thesis aims to identify glucose transporters in C. elegans and to provide a primary investigation of the in vitro and in vivo function of the identified glucose transporter. Nine putative transporters have been cloned and expressed. Out of the nice cloned putative transporters in the C. elegans genome, H17B01.1 (H17) only is identified as a fully functional glucose transporter using an oocyte expression system in which glucose transport activity is directly measured. The two transcripts of H17 are both capable of transporting glucose with high affinity, as well as transporting trehalose. Heterologous expression of H17 in mammalian CHO-T cells suggests that the protein is localised both on the plasma membrane and in the cytosol. In vitro studies of H17 show that the protein does not respond to insulin stimulation when expressed in mammalian CHO-T cell and rat primary adipocyte systems. In vivo functional studies using H17 RNAi indicate that the worm’s lifespan is not affected by the H17 knockdown. However, glucose metabolism of C. elegans (as measured by glucose oxidation to CO2 and incorporation into fat reserves) is influenced by the decreased expression of H17, especially in the daf-2 mutant strain, e1370. However, the increase of glucose metabolism caused by H17 knockdown observed in daf-2 mutant is inhibited in the age-1 and akt-1 mutant strains. The findings reported in this thesis suggest that the H17 glucose transporter may play an important role glucose metabolism in C. elegans and that this transport and metabolism is influenced by insulin receptor activity and serine kinase cascades.

Understanding how the human brain functions on a molecular and cellular level is nearly impossible with current technology and ethical considerations. Utilizing the small nematode, Caenorhabditis elegans, and its innate behavioral responses to olfactory social cues, we can begin to unravel the mechanisms underlying social behavior. This is made possible given that innate behaviors are crucial for survival, and therefore hardwired into the genome of organisms. This allows for genetic-level analysis of neural circuitries driving behavior. Studying the neuronal mechanisms underlying C. elegans’ behavioral responses to social cues will not only assist in our overall understanding of how the brain perceives stimuli to enact a behavioral response at the cellular and molecular level, but also our understanding as to how the nervous system properly integrates information to enact social behavioral responses: mis-integration and social abnormalities are commonalities seen in many neuropsychiatric disorders, and these studies will provide fruitful insights into the defects observed in these disorders. Lastly, by comparing the perception of several different types of social chemicals, we can further our understanding of neural coding strategies for the various behaviors crucial for survival. Chapter One of this thesis orients the reader to social, innate behavior, and the usefulness of C. elegans as a tool for understanding behavioral coding. Chapter Two explores and establishes the required components of a socially aversive pheromone, providing insight into signaling evolution and co-option of biological machineries. Chapter Three examines how multiple, competing stimuli are integrated to modulate behavioral output, furthering our understanding of molecular and cellular integration and decision making within the nervous system. Chapter Four highlights the importance of predator pressure, and provides insights into circuit strategies of redundant and promiscuous networks of threat detection. Lastly, Chapter Five considers the implications of these findings as a whole, in the perspective of evolutionary strategies leading to neuronal coding of different behavioral outputs. Taken together, this dissertation aimed to fill the void in our understanding of social behavior neural circuitries, and how integration governed at the molecular and cellular level of the nervous system affects those behaviors.

C. elegans is as an extremely powerful model for the study of innate immunity. MAP kinase signaling pathways in C. elegans are involved in the response of C. elegans to infection by pathogenic bacteria. The yeast S. cerevisiae can infect C. elegans, producing pathogenic effects. In this project, we tested whether several MAP kinase pathways are important for C. elegans¡¯ resistance to yeast infection. We tested members of several MAP kinase pathways including tir-1, nsy-1, sek-1 and pmk-1 in the p38 pathway, mek-1, jnk-1 and kgb-1 in JNK pathway and mek-2 and mpk-1 in the ERK pathway. We used survival assays to compare the responses of mutants of components of these pathways to the control responses of wild-type C. elegans. In the survival assay, we found that mutants in all three MAP kinase pathways showed a decreased survival relative to wild type; therefore all three pathways are important for innate immunity against the yeast pathogen. With respect to the p38 pathway, mutations affected survival but not the deformed anal region (Dar) phenotype, a putative defensive response induced by yeast in wild-type C. elegans. This indicates that for the p38 pathway, survival depends on some other immune response besides Dar. Finally, we hypothesize that cross talk occurs between p38 and JNK MAPK pathways in the C. elegans immune responses.

The division of one cell to form two cells, or cytokinesis, is fundamental to the development of all known multi-cellular organisms, as well as the propagation of life between generations. The intracellular mechanisms that mediate the physical deformation of the cell membrane during division have proven to be remarkably robust, with multiple processes functioning together to achieve bisection. Here, I present my doctoral work, which seeks to illuminate the dynamic molecular interplay that coordinates and drives cytokinesis in the Caenorhabditis elegans single-cell zygote. In Chapter 1, I begin with an introduction on cytokinesis and the many proteins known to regulate cell division. Chapter 2 presents a detailed review of three intracellular signaling molecules that mediate the spatial control of cytokinesis, known as Rho family small GTPases. In Chapter 3, I present work in which we inactivated specific cytokinesis protein functions at precise stages of the division process, in order to map out the first “temporal atlas” of essential cytokinetic functions. In Chapter 4, I present evidence that the GTPase CDC-42 and the cortical polarity machinery sequester cytokinesis-inhibiting proteins away from the division plane and protect the fidelity of cytokinesis. Chapter 5 lays out preliminary evidence that another GTPase, RAC-1, is a suppresser of cytokinesis and must be inactivated in the division plane specifically by a spindle-associated regulatory protein. Through this body of work, I have attempted to elucidate the underpinnings of the complex intracellular orchestra that drives cytokinesis. This work provides valuable insight, not only into how this vital process occurs, but also how the disruption of its components could lead to the development of complex diseases like cancer.

Age-dependent loss of body wall muscle function and locomotion has been observed in C. elegans, however its cause has yet to be elucidated. Utilizing biochemical techniques and calcium imaging, we demonstrate that aberrant calcium (Ca2+) release via the ryanodine receptor (RyR) homologue UNC-68 contributes to age-dependent muscle weakness in C. elegans. We show that UNC-68 comprises a macromolecular complex bearing FKB-2, a C. elegans immunophilin with high homology to the stabilizing subunit calstabin (calcium channel stabilizing binding protein, or FKBP12). Furthermore, we demonstrate that as the nematode ages, UNC-68 is oxidized and depleted of FKB-2, resulting in “leaky” channels, depleted SR calcium stores, and a reduction in body wall muscle Ca2+ transients at baseline. These perturbations resulted in a motility phenotype, where fkb-2(ok3007) worms harboring a deletion mutation that abolishes FKB-2 binding to the UNC-68 macromolecular complex suffered from poor muscle performance and exercise fatigue in swimming trials. Moreover, pharmacological interventions inducing oxidization of UNC-68 and depletion FKB-2 from the channel independently cause reduced body wall muscle Ca2+ transients, strongly suggesting that UNC-68 oxidation and FKB-2 depletion contribute to muscle function loss observed in aging. UNC-68 oxidation was found to correlate with lifespan, happening earlier in short-lived mitochondrial electron transport chain strains and later in long-lived worms. Finally, preventing FKB-2 depletion from the UNC-68 macromolecular complex in aged C. elegans using the Rycal drug S107 improved muscle Ca2+ transients. Taken together, our data implicate UNC-68 dysfunction in the underlying mechanism of muscle function loss in C. elegans, analogous to observations made of RyR1 dysfunction in aged mammalian skeletal muscle, and describes for the first time a potential role for FKB-2 in C.elegans physiology.

Multicellular organisms arise from a single fertilized zygote, which must contain all information necessary to develop. As the embryo divides, the cells adopt distinct functional characteristics, and as they do so, they become committed to these fates, unable in most cases to convert from one identity to another. Though it has been well known and described for over a century now this year, this latter process, in this work referred to as cell fate restriction, is not well understood. In this thesis, I aim to contribute to the understanding of this developmental phenomenon. The tool I use is the ectopic expression of a terminal fate specifying transcription factor, CHE-1. This transcription factor normally functions to specify the fate of a pair of gustatory neurons in the nematode Caenorhabditis elegans. If ectopically expressed early in development, it is able to induce expression of its target genes, but by adulthood, most cells are refractory to its transcriptional activation, evidence of developmental cell fate restriction in most tissues of the animal. I first describe the work of Tulsi Patel to which I contributed, in which an RNAi screen revealed that PRC2 complex is responsible for preventing CHE-1 activity in the germline cells of C. elegans. I then describe a semi-clonal genetic screen in which I found many more mutants with a similar phenotype affecting germline cells, and cloned another gene that is able to induce expression specifically in the epidermis of the animals: usp-48, a highly conserved ubiquitous nuclear deubiquitinating enzyme. Next, I describe another screen where I ectopically express CHE-1 specifically in the adult epidermis, in which I found and cloned an additional six mutants: ogt-1, dot-1.1, pmk-1, sek-1, nhr-48, and C08A9.6, here named epco-1. In this screen I also isolated but was unable to clone an additional four mutants that likely represent an additional four genes. I discuss the nature of these genes and their potential roles in restricting cell fate. Lastly, I describe the optimization of a tissue-specific transcriptional profiling protocol, INTACT, for use in the characterization of the mutants. With this optimized protocol, I was able to perform detailed RNAseq on two individual neuron types from the animal, as well as wild-type epidermis. This optimized protocol will be used to characterize the mutants in the future. Together, these results tie unexpected genes to the function of cell fate restriction in the C. elegans epidermis, which will aid in our understanding of this fundamental developmental phenomenon.