Organisms must coordinate behavioral and physiological responses to changingenvironmental conditions. In the nematode C. elegans, the presence or absence of foodin the environment affects many metabolic and behavioral responses, including fathomeostasis, lifespan, and male mating. Specifically, male mating behavior normallyoccurs when a well-nourished male encounters a hermaphrodite, and is repressed if themale is under-nourished. To understand how environmental changes influence the driveto carry out specific behavioral tasks, I used C. elegans male mating as a model.Previously, mutants were isolated that display male mating behavior at inappropriatetimes, i.e. in the absence of mating cues. Loss of function mutations in the ERG K+channel, UNC-103, results in spontaneous seizures of the male sex muscles.Interestingly, I found that food deprivation can suppress unc-103(lf)-induced seizures,suggesting that pathways activated under this environmental condition can suppress theexcitability of the mating circuit.Using molecular, genetic, and behavioral assays, I identified sensory andmolecular mechanisms that reduce sex-muscle excitability under food-deprived conditions. I found that mutations that affect the muscular feeding organ, the pharynx,phenocopy the effects of food deprivation, and reduce sex-muscle excitability. Idemonstrated that mutations in the pharyngeal muscle protein, tropomyosin, cause thepharyngeal neurosecretory motor neurons (NSMs) to increase pharyngeal excitabilityand reduce sex-muscle excitability. Additionally, I found that olfactory neurons (AWCs)with sensory cilia exposed to the environment are up-regulated in the absence of foodstimuli, and also send inhibitory signals to the sex muscles. To determine howchemosensory and pharyngeal neurons in the head can signal to the genitalia, Ihypothesized that one mechanism could be via secretion of metabolic hormones. To testthis, I examined loss-of-function mutations in the insulin-like receptor, DAF-2, which isknown to regulate many behavioral and physiological responses to food. I demonstratedthat DAF-2 activity in the sex muscles is required for food-deprivation suppression ofunc-103(0)-induced seizures. I then identified components of a novel-insulin-like/DAF-2signaling pathway that reduces excitability. Specifically, I propose that ligand binding toDAF-2 activates PLC- and leads to increased cystolic Ca2+. This Ca2+ influx activatesCaMKII, which can phosphorylate/activate EAG-like K+ channels, thereby reducing cellexcitability.

The circuits that allow organisms to control behavioral timing need to be tightly regulated to ensure execution of appropriate environmental responses. Disrupting such regulation results in individuals unable to perform tasks necessary for survival and propagation. Identifying the molecular components regulating behaviors will enable compensation where behavioral impediments to survival exist. To identify circuits of behavioral regulation, I studied male mating behavior in the nematode Caenorhabditis elegans. Specifically, I focused on the step wherein the male inserts his copulatory spicules into the hermaphrodite vulva, as vulva penetration is required for successful sperm transfer. This step must be tightly regulated; if the spicules protract too soon or not at all, vulva penetration and thus successful mating will not occur. In this dissertation, I elucidate the circuits regulating sex-muscle excitability under standard conditions and describe how these pathways are augmented to further reduce excitability under food deprivation conditions. I employ a variety of assays to identify and analyze these circuits, including genetic manipulation, biochemical techniques, and behavioral assays. Under standard conditions the calcium/calmodulin-dependent protein kinase II (CaMKII) encoded by unc-43 is required to inhibit C. elegans male sex-muscle seizures; under conditions where food is scarce, I propose that CaMKII is further up-regulated to activate the EAG K+ channel EGL-2 through a direct interaction. The CaMKII/EGL-2 interaction functions to attenuate calcium influx from L-type voltage-sensitive calcium channels (L-VGCCs), while CaMKII also downregulates calcium influx from ryanodine receptors. Additionally, another K+ channel, the voltage- and calcium-sensitive big current channel SLO-1, attenuates sex-muscle excitability by inhibiting L-VGCCs under food deprivation conditions. In conclusion, CaMKII and EGL-2?s paralog, UNC-103/ERG-like K+ channel, are required when food is plentiful to prevent premature sex-muscle contractions, while food deprivation reduces cell excitability and thereby inhibits inappropriate seizures through CaMKII, EGL-2, and SLO-1.

Insulin signaling is highly conserved across animals, and is known for its ubiquitous function in all aspects of animal physiology. Despite its relatively well-studied role in metabolism and energy expenditure, how it is involved in learning and memory remains a mystery, due to the complex nature of the nervous system. In this thesis, I have used C. elegans, a tractable model organism with a sophisticated behavioral repertoire, to investigate molecular and cellular mechanisms of insulin signaling in learning.

Radiation induced bystander effects have changed our understanding of the biological effects of ionizing radiations. The original assumption was that biological effects require direct damage to DNA. The bystander effect eliminated that requirement and has become one main stream in radiation research ever since first reported over 20 years ago. Most bystander studies to date have been carried out by using conventional single cell in vitro systems , 2D cell array and 3D tissue samples, which are useful tools to characterize basic cellular and molecular responses. But to reveal the complexity of radiation responses and cellular communication, live animal models have many advantages. In recent years, models such as C. elegans and Zebrafish have been utilized in bystander effects research. In the Loma Linda/TAMU experiment, a L1 larva C. elegans model was devloped to study the radiation bystander effects by irradiating single intestine cell nuclei with a microbeam of protons. Due to the stochastic nature of particle interactions with matter and changing stopping power when protons slow down, precise dosimetry in the target nucleus is a difficult problem. This research was undertaken to provide a detailed description of the energy deposition in the targeted and surrounding non-targeted cell nuclei, and to evaluate the probabilities of the non-targeted cell nucleus being irradiated. A low probability is required to exclude the possibility of radiation biological an effect in non-targeted cells is caused by scattered particles. Mathematical models of the microbeam system and the worm body were constructed in this research. Performing Monte Carlo simulations with computer code, Geant 4, this research provided dosimetry data in cell nuclei in different positions and Geant 4, this research provided dosimetry data in cell nuclei in different positions and probabilities of scattering to non-targeted cell nuclei in various microbeam collima- tor configurations. The data provided will be useful for future collimated microbeam design.

Small molecules are powerful reagents for biological investigation. They provide an alternative to genetic perturbation and may offer more control over a target’s activity. C. elegans has recently gained prominence as a platform to discover new chemical tools. Through large-scale screens for compounds that induce phenotypes consistent with the disruption of conserved pathways, we identified two previously uncharacterized molecules of interest that we named nemadipine and migrazole. Here, I describe my efforts to understand their mechanism of action. Nemadipine is structurally analogous to 1,4-dihydropyridines (DHPs), which target the Cav1 calcium channel and are used clinically to lower blood pressure. Phenotypic and genetic evidence suggest that nemadipine targets the worm Cav1 channel, EGL-19. To identify the target of nemadipine in an unbiased manner, I performed a forward genetic screen for mutants resistant to its effects. The majority of the mutants from my screen had polymorphisms in EGL-19, providing additional evidence that it is the target of nemadipine. I also found that nemadipine is the only DHP that robustly elicits phenotypes in the worm. Therefore, I used this unique chemical to investigate the in vivo interactions between DHPs and the Cav1 channel. I identified residues in EGL-19 important for DHP-sensitivity in worms and showed that some of these residues are also important for mammalian DHP-interaction. Other labs have since exploited nemadipine’s in vivo properties to demonstrate new biological insights for EGL-19. Chemical genetic analyses indicated that migrazole disrupts multiple signal transduction pathways. This, together with experiments that I performed in yeast, suggests that migrazole may affect multiple pathways by perturbation of protein transport. To identify migrazole’s target, I performed a forward genetic screen for mutants resistant to migrazole’s effects. However, I was unable to identify the target of migrazole through analysis of the mutants I isolated. This result illustrates that while forward genetic screens can be very successful for target identification, their effectiveness is likely dependent on the nature of the compound-target interaction. My work shows that all aspects of developing a small molecule into a tool for biological analysis, from its discovery to its characterization, can be accomplished using C. elegans.

Small molecules are powerful reagents for biological investigation. They provide an alternative to genetic perturbation and may offer more control over a target’s activity. C. elegans has recently gained prominence as a platform to discover new chemical tools. Through large-scale screens for compounds that induce phenotypes consistent with the disruption of conserved pathways, we identified two previously uncharacterized molecules of interest that we named nemadipine and migrazole. Here, I describe my efforts to understand their mechanism of action. Nemadipine is structurally analogous to 1,4-dihydropyridines (DHPs), which target the Cav1 calcium channel and are used clinically to lower blood pressure. Phenotypic and genetic evidence suggest that nemadipine targets the worm Cav1 channel, EGL-19. To identify the target of nemadipine in an unbiased manner, I performed a forward genetic screen for mutants resistant to its effects. The majority of the mutants from my screen had polymorphisms in EGL-19, providing additional evidence that it is the target of nemadipine. I also found that nemadipine is the only DHP that robustly elicits phenotypes in the worm. Therefore, I used this unique chemical to investigate the in vivo interactions between DHPs and the Cav1 channel. I identified residues in EGL-19 important for DHP-sensitivity in worms and showed that some of these residues are also important for mammalian DHP-interaction. Other labs have since exploited nemadipine’s in vivo properties to demonstrate new biological insights for EGL-19. Chemical genetic analyses indicated that migrazole disrupts multiple signal transduction pathways. This, together with experiments that I performed in yeast, suggests that migrazole may affect multiple pathways by perturbation of protein transport. To identify migrazole’s target, I performed a forward genetic screen for mutants resistant to migrazole’s effects. However, I was unable to identify the target of migrazole through analysis of the mutants I isolated. This result illustrates that while forward genetic screens can be very successful for target identification, their effectiveness is likely dependent on the nature of the compound-target interaction. My work shows that all aspects of developing a small molecule into a tool for biological analysis, from its discovery to its characterization, can be accomplished using C. elegans.

In vertebrate and invertebrate systems, the role of glia-neuron interactions during development and behavior is becoming apparent. Recent studies have been aimed at characterizing glial-expressed proteins that affect the modulation of activities traditionally thought to be regulated by the neuron itself. The soil nematode Caenorhabditis elegans has recently emerged as an important invertebrate model to study glial roles in nervous system function and development. My dissertation work focuses on the characterization of HLH-17, a C. elegans basic helix-loop-helix transcription factor that is strongly and constitutively expressed in the glial cells that associate with four of the cephalic (CEP) neurons in the head of the animal. The CEP neurons are four of eight dopaminergic neurons with well characterized roles in the modulation of a number of behavioral activities in the worm. Although HLH-17 is required for neither the specification nor the development of the CEPsh glia or the CEP neurons, it does have a defined role during dopamine responses. We show that HLH-17 functions upstream of the dopamine receptors DOP-1, DOP-3 and the dopamine transporter DAT-1 to affect DA-dependent behaviors. Also, our microarray analyses provide preliminary evidence that HLH-17 targets factors responsible for receiving and transducing signaling molecules that are involved in the modulation of synaptic events in the worm nervous system. Together these results point to a role for HLH-17 in glia-neuron interactions in C. elegans. My dissertation studies therefore provide further support for the role of glial-expressed proteins in the regulation of activities mediated by the nervous system.

Abstract Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline residues in collagens. The vertebrate enzymes are α2β2 tetramers in which the β subunit is identical to protein disulphide isomerase (PDI). Two isoforms of the catalytic α subunit have been identified in vertebrates, forming type I [α(I)]2β2 and type II [α(II)]2β2 collagen prolyl 4-hydroxylase tetramers. This thesis reports on the cloning and characterization of a third vertebrate α subunit isoform, α(III). The recombinant human α(III) isoform associates with PDI to form an active type III collagen prolyl 4-hydroxylase tetramer, and its Km values for the cosubstrates are very similar to those of the type I and II enzymes, those for a peptide substrate and an inhibitor being found to lie between the two. The α(III) mRNA is expressed in all tissues studied but at much lower levels than the α(I) mRNA. A novel mixed tetramer PHY-1/PHY-2/(PDI-2)2 was found to be the main collagen prolyl 4-hydroxylase form produced in the nematode Caenorhabditis elegans in vivo and in vitro. However, mutant nematodes can compensate for the lack of the mixed tetramer by increasing the assembly of PHY-1/PDI-2 and PHY-2/PDI-2 dimers, these forms also being unique. The catalytic properties of the recombinant mixed tetramer were characterized, and it was shown by the analysis of mutant worms that PHY-1 and PHY-2 represent the only catalytic subunits needed for the hydroxylation of cuticular collagens. The roles of the two catalytic sites in a collagen prolyl 4-hydroxylase tetramer were studied by using the C. elegans mixed tetramer and a hybrid C. elegans PHY-1/human PDI dimer. An increase in the chain length of the peptide substrate led to an identical decrease in the Km values in both enzyme forms. It is thus clear that two catalytic sites are not required for efficient hydroxylation of long peptides, and their low Km values most probably result from more effective binding to the peptide-substrate-binding domain. Inactivation of one catalytic site in the mixed tetramer reduced the activity by more than 50%, indicating that the remaining wild-type subunit cannot function fully independently.

Anesthetics have been used for nearly two centuries, and have proved to be one of the most important tools in surgical interventions, but their methods of action remain mysterious. Previous research has focused on high-level, low-resolution measurements (average activity of many neurons) or low-level, high-resolution measurements (single neurons). The nematode Caenorhabditis elegans provides an excellent model to bridge the gap between these two scales by measuring the activity of many neurons with single neuron resolution. C. elegans display analogous behaviors to humans under anesthesia. Employing confocal imaging of GCaMP, I measured neuronal activity at different isoflurane levels in C. elegans ganglia and in small behavior-controlling circuits. The activity in C. elegans ganglia is similar to that of human ganglia, as assessed using measures that are similar to EEG. Activity in the small behavior-controlling circuit is disrupted, but not suppressed, when dosed with moderate levels of isoflurane. Neural activity in the circuit is randomized resulting in a loss of coordination between neurons that define behavioral states of the system. As such, the onset of the behaviors of anesthesia appears to be the resultant of randomization rather than suppression of individual neuron activity. Employing light sheet microscopy and automated image analysis for neuronal tracking, I expanded the imaging techniques to measure activity of the majority of neurons in the animal’s head. Expansion of these measurements to the whole head region of the nematode confirms these findings, displaying significant decreases in neuron-to-neuron coordination, as well as randomization of individual neuron signals with the onset of anesthesia. These results reveal a new physiological mechanism of action for anesthetics, and provide an avenue forward for investigating the molecular mechanism including specific genetic mutations known to alter susceptibility to anesthetics. / 2021-05-26T00:00:00Z

In C. elegans, the germline is a tightly regulated tissue where silencing pathways regulate genes, allowing expression of “self” while silencing “non-self.” Doublestranded RNAs (dsRNAs), short interfering RNAs (siRNAs), and piwi-associated RNAs (piRNAs) can transmit this regulation across generations via transgenerational epigenetic inheritance (TEI) mechanisms (Bošković and Rando, 2018). Analogously, some pathways can counteract gene silencing to allow sustained expression in the germline. One such example is a non-coding DNA structure called Periodic An/Tn clusters that can prevent the silencing of transgenes in the germline (Frøkjær-Jensen et al., 2016). In this thesis, I developed a novel piRNA-based tool called piRNA interference (piRNAi), where target-specific short “guide” piRNAs (sg-piRNAs) can robustly silence endogenous genes and transgenes. I have used piRNAi to understand the rules for licensing gene expression and transgenerational epigenetic inheritance in the C. elegans germline. Initially, I describe design rules for generating transgenes with PATC-rich introns that resist germline silencing and are robustly expressed from extrachromosomal arrays. PATC-rich transgenes showed more accurate gene expression patterns and did not prevent germline regulation by 3’ untranslated regions (3’ UTRs). Next, I developed the piRNAi technique to understand the role of PATCs in licensing transgene expression and the rules for how endogenous genes can be targeted for piRNA-mediated silencing and TEI. I demonstrate that a PATC-rich gfp transgene and endogenous genes are not resistant to piRNA-mediated silencing. Finally, I used piRNAi to define rules for TEI: 1. I identified two new endogenous targets for TEI (him-5 and him-8) that can inherit silencing for four and six generations respectively, after transient exposure to sg-piRNAs. 2. I demonstrate that an endogenous gene (him-5) can be semi-permanently silenced in the absence of the piRNA/PRG-1 pathway. 3. The duration of TEI was significantly shortened in a transgene that contained PATC-rich introns. Altogether, my thesis shows that an endogenous small RNA pathway can be reprogrammed to silence endogenous genes and transgenes in the germline, which enables novel experimental paradigms for studying inherited and semipermanent silencing.