Despite being integral to the practice of surgery, the mechanisms by which general anesthetics mediate their effects remain unknown. For this reason, it is difficult to predict adverse side-effects and to determine how treatment should be modified for specific patient populations. Recent clinical studies have reported post-operative neuropsychological and behavioral abnormalities in children and protracted periods of post-operative cognitive decline in elderly patients. Definitively linking these post-operative consequences to the agents used to induce anesthesia has been difficult, due to a lack of proper clinical controls and an abundance of confounding health factors. Animal studies, have repeatedly shown that general anesthetics can be neurotoxic and lead to lasting impairments in learning and memory acquisition in both the very young and old. However, the scope and causes of these post-exposure impairments and the reasons why age seems to measurably affect outcomes remain unclear. Here we employ multi-neuronal fluorescence imaging in the nematode Caenorhabditis elegans to measure changes in neuronal activity and connectivity across the animal’s nervous system, following exposure to the volatile anesthetic isoflurane during neurodevelopment and senescence. Employing transgenic expression of the fluorescent calcium indicator GCaMP6s, we measure neuronal activity of specific command interneurons as well as across the majority of the nervous system with single cell resolution. Isoflurane exposure during developing, results changes in the transition rate between neuronal activity states and an overall increase in excitatory connectivity. Importantly these effects are dependent on cellular stress pathways involved mTOR and daf-16 but not on apoptotic cell death (medatied by ced-3). Measuring neuronal activity across the animals lifespan, we identify substantial age-related alterations to neural activity, connectivity and functional organization of the system. These include a progressive loss of system-wide organization and a corresponding shift in individual neuron activity toward higher frequencies. We also observe a specific loss of anti-correlative (i.e. inhibitory) signaling between neurons, resulting in an overall shift in the excitatory/inhibitory balance of the system. In support of this, we find that application of the GABAA agonist muscimol diminishes certain aspects of nervous system decline in aged animals. We further identify genes that either hasten or delay the progression toward senescent neural activity patterns, including the presynaptic voltage-gated Ca2+ channel UNC-2/CaV2, and also CED-4, a key mediator of the conserved cell-death pathway. Finally, imaging post-exposure consequences of isoflurane during senescence reveals long term effects on neuronal signaling that involved a decrease in excitatory connectivity, the opposite of what is observed during development. We conclude that anesthetic exposure during development cause permanent alteration in neuronal activity and signaling which involves cellular stress pathways but that these effects are distinct from long-term effect of anesthetic exposure we observe in age animals. Our studies also begin to define the changes in neuronal dynamics with age and demonstrate the importance of excitatory/inhibitory balance in this processes. Through comprehensive multi-neuronal imaging in C. elegans, we are able to measure the progressive breakdown of neuronal activity and system dynamics with age and isoflurane exposure and begin to identify the cellular processes and changes in synaptic signaling that contribute to these declines. Moreover, we leverage this platform to gain insight into the age-dependency of isoflurane-induced insult to neural systems.

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Lhx genes are a sub-family of Hox genes that play important roles in animal development. In Caenorhabditis elegans there are seven Lhx genes, including the founding family member lin-11. The lin-11 gene is necessary for the specification of neuronal and reproductive tissues. My thesis work has involved understanding the mechanism of lin-11 regulation and its function in these tissues. To this end, I addressed two distinct but complementary questions, one of which focused on how transcriptional regulation of lin-11 occurs and the second on the role of LIN-11 protein domains/regions. My work on the transcriptional regulation has uncovered important roles of two of the largest lin-11 introns, intron 3 and intron 7. These introns promote lin-11 expression in non-overlapping sets of amphid neurons. Based on gene expression patterns and behavioural assays, intron 3 is capable of restoring lin-11 function in lin-11(n389 ) null mutant allele. Comparison of intron 3-driven reporter expression in the neuronal cell types between C. elegans and C. briggsae has revealed cis and trans evolutionary changes in lin-11 regulation between the two species. Functional dissection of the introns in C. elegans has led to the identification of three distinct non-overlapping enhancers, each specific for a single amphid neuron, i.e., RIC, AIZ, and AVG. I have also identified four transcription factors, SKN-1, CEH-6, CRH-1, and CES-1, that act through these enhancers to regulate neuronal expression of lin-11. Furthermore, I have characterized the function of the LIM domains and a proline-rich (PRR) C-terminus region of LIN-11 in the specification of neuronal and reproductive tissues. My work shows that while the LIM domains are required for LIN-11 function in these tissues, the PRR region is dispensable. I have also examined the functional conservation of lin-11 domains using two other Lhx genes, Drosophila melanogaster (dLim1) and Mus musculus (Lhx1 ), and found that both of these genes were able to rescue lin-11 defects. Together, my work has significantly advanced our understanding of transcriptional regulation of lin-11, the importance of LIM domains in tissue formation, and functional conservation of Lhx genes across phyla. / Thesis / Doctor of Philosophy (PhD)

<p> Caenorhabditis elegans (C. elegans) is a model organism which is known for its transparent body, small body size, high reproductivity and short lifecycle. Several important genes and signal transduction pathways are well conserved in C. elegans. lin//, a LIM homeobox family member, plays a crucial role in the development of the vulva in C. elegans. LIM homeobox genes are a subgroup of Homeobox family that play fundemental role in animal development. In C. elegans lin-If mutant animals fail to form a functional vulva and vulval-uterine connection and consequently exhibit egg-laying defective phenotype. The cell lineage and marker gene expression studies have shown that lin-// is required for the patterning of all primary and secondary lineage vulval cells. lin- II also functions in the nervous system. </p> <p> lin-// expression is mainly observed in the developing vulval cells and in the pi cells which are involved in the formation of vulval-uterine connection. lin-If expression is also seen in VCs and in some of the head and tail neurons. The completed genome sequences of closely related species in Caenorhabditis genus serve as a power tool to do systematic comparative studies. The lin-If regulatory sequences from these species have been compared along with the expression patterns. </p> <p> We looked at the regulation of lin-// in closely related nematode species like C. elegans, C. briggsae, C. remanei and Caenorhabditis n species. </p> <p> Consistent with this. expression of lin-11 is observed in the developing vulval cells. We are interested in understanding evolutionary changes in the regulation and function of lin-II in reproductive system </p> <p> /in-11 is a LIM homeodomain family member which IS involved in several developmental events. lin-11 role is documented in the thermoregulatory circuit specifying AIY interneuron, in chemosensory neurons like A W A and olfactory neurons A WS. During vulval development lin-II expression is dynamically expressed in subset of secondary lineage cells and is broadly expressed in all the cells indicating its role in cell identity and cell fusion of the vulval cells. lin-II is also required for the formation of vulval uterine connection which is the passage to lay eggs in the hermaphrodite. linllloss of function hermaphrodites have change in the axis of the secondary lineage cells during vulval development, uterine Jt cell migration defect, defects in the AIY, A W A and A WS interneurons resulting in egg-laying defect and protruding vulva and neuronal defects and reduced mating efficiency. </p> <p> The expression pattern of lin-If in closely related species is highly similar but not identical. From the sequence comparison of lin-If regulatory sequences a 1 kb conserved block of sequences have been identified which includes the regulatory sequences responsible for the expression of lin-If in vulva and Jt cells. We propose that cisregulatory elements controlling lin-If gene expression are slowly evolving though there is no change in the function which indicates that lin-If plays critical role during the development of the vulva and other tissues. </p> / Thesis / Master of Science (MSc)

Environmental factors, such as diet, can have a significant impact on the health of animals, influencing lifespan, development, and disease progression. The model organism Caenorhabditis elegans is a bacterivore whose development is characterized by an invariant pattern of cell division. This study investigated how C. elegans developmental rate is altered in response to 48 different bacterial diets. The bacterial species studied had a wide range of effects on developmental progression, with some bacteria dramatically decreasing developmental rate, while others caused developmental arrest in early larval stages. From these analyses, Staphylococcus species that caused very slow development of animals in the L1 stage were selected for further characterization. The slow developmental rate observed in these animals was rescued by supplementation with essential amino acids. Genetic analysis revealed that these effects were mediated through TOR signaling and were independent of insulin signaling. Loss of daf-15(raptor), a central component of TOR complex 1, inhibited the rescuing effects of the amino acid supplementation on developmental rate, suggesting that the slow development induced by Staphylococcus could be rescued by activating the TORC1 pathway. Autophagy, negatively regulated by TOR, is increased in worms fed Staphylococcus species, suggesting TOR activity is reduced, and supplementation through amino acids reduced autophagy. These data suggest that TOR signaling is reduced in C. elegans in response to certain bacterial diets, resulting in decreased developmental rate, and that this effect may be due to amino acid deficiency. / Thesis / Master of Science (MSc)

Cell migration is critical in generating complex animal forms during development; misregulation of migration contributes to pathological conditions such as cancer metastasis. Thanks to its easily traceable cell lineages in a transparent body and a compact genome accessible to a wealth of genetic manipulations, the use of the nematode C. elegans as a model system has greatly advanced our understanding of mechanisms governing cell migration conserved through higher organisms. Among several migration processes in C. elegans, sex myoblast (SM) migration is an attractive system that has a simple and well-defined migratory route along the ventral side from the posterior to the precise center of the gonad. A multitude of guidance mechanisms control SM migration, many of which are likely to be conserved in other migratory processes. Similar to vertebrate systems, C. elegans uses Rho family small GTPases to regulate the engine of cell motility, the actin cytoskeleton, in response to guidance cues. The differential utilizations of Rho GTPases in distinct processes in vivo remain a central question in the study of Rho GTPases. I investigated how Rho GTPases regulate different aspects of SM migration, and found that Cdc-42/CDC42 functions in the anteroposterior migration, whereas MIG-2/RhoG and CED-10/Rac1 control ventral restriction independently of FGF and SLIT/Robo signaling. The relative difficulty in perturbing SM migration using constitutively active Rho GTPases compared to other migration processes illustrates the robustness of the mechanisms that control SM migration. On a technical aspect, I established a nematode larval cell culture system that allows access to postembryonic cells. Compared to the flourishing genetic researches in C. elegans, there are few studies of molecules that also extend to the subcellular level in postembryonic development, mainly due to the lack of a larval cell culture system. I developed a novel method combining SDS-DTT presensitization of larval cuticles and subsequent pronase E digestion. My method efficiently isolates both low- and high-abundance cell types from all larval stages. This technical advance will not only facilitate studies such as regulation of actin dynamics with high-resolution microscopy, but is beginning to be used by researchers to tackle cell-type specific questions through profiling methods as gene expression analysis. / Ph. D.