Master of Science / Department of Biology / S. K. Chapes / Ehrlichia chaffeensis is a Gram-negative, obligatorily intracytoplasmic bacterium and the causative agent of a tick-borne disease, human monocytic ehrlichiosis. In vertebrates, E. chaffeensis exhibits tropism for monocytes /macrophages. However, no clear requirements for cell tropism have been defined in ticks. Previously, our group identified two host genes that control E. chaffeensis replication in vivo in Drosophila. We used these two genes, CG6364 and separation anxiety (san) to test the hypothesis that E. chaffeensis replicates in arthropod hemocytes. Using the UAS/GAL4 RNAi system, we generated F1 flies (RNAi flies) and confirmed ubiquitous-or tissue-specific reduction in the transcript levels of the targeted genes. When RNAi flies were screened for Ehrlichia infections, we found that when either CG6364 or san were specifically suppressed in the hemocytes or in the fat body E. chaffeensis failed to replicate or cause infection. Deletion of these genes in the eyes, wings or the salivary glands did not impact fly susceptibility or bacterial replication within these organs. Our data demonstrate that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals. This study provides insights about replication sites of E. chaffeensis in arthropods.

The oxidation of methionine (Met) into methionine sulfoxide (met-(o)) leads to deleterious modifications to a variety of cellular constituents. These deleterious alterations can be reversed by enzymes known as methionine sulfoxide reductases (Msr). The Msr (MsrA and MsrB) family of enzymes have been studied extensively for their biological roles in reducing oxidized Met residues back into functional Met. A wide range of studies have focused on Msr both in vivo and in vitro using a variety of model organisms. More specifically, studies have noted numerous processes affected by the overexpression, under expression, and silencing of MsrA and MsrB. Collectively, the results of these studies have shown that Msr is involved in lifespan and the management of oxidative stress. More recent evidence is emerging that supports existing biological functions of Msr and theorizes the involvement of Msr in numerous biological pathways. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

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

Cone snails are venomous marine predators whose venom is a complex mixture of modified peptides (conopeptides). Conopeptides have direct specificity towards voltage- and ligand-gated ion channels and G-protein coupled receptors. More specifically, alpha conotoxins target nicotinic acetylcholine receptors (nAChR) and are of great interest as probes for different nAChR subtypes involved in a broad range of neurological function. Typically, the amount of peptide provided directly from the cone snails (from either dissected or “milked” venom) is minimal, thus hindering the wide use of bioassay-guided approaches for compound discovery. Biochemical-based approaches for discovery by means of identification and characterization of venom components can be used due to their compatibility with the small quantities of cone snail venom available; however, no direct assessment of the bioactivity can be gleaned from these approaches. Therefore, newly discovered conotoxins must be acquired synthetically, which can be difficult due to their complicated folding motifs. The ability to test small quantities of peptide for bioactivity during the purification process can lead to the discovery of novel components using more direct approaches. Presented here is the description of use of an effective method of bioassay-guided fractionation for the discovery of novel alpha conotoxins as well as further biological characterization of other known alpha conotoxins. This method requires minimal amounts of sample and evaluates, via in vivo electrophysiological measurements, the effect of conotoxins on the functional outputs of a well-characterized neuronal circuit in Drosophila melanogaster known as the giant fiber system. Our approach uses reversed-phase HPLC fractions from venom dissected from the ducts of Conus brunneus in addition to synthetic alpha conotoxins. Fractions were individually tested for activity, re-fractionated, and re-tested to narrow down the compound responsible for activity. A novel alpha conotoxin, bru1b, was discovered via the aforementioned approach. It has been fully characterized in the giant fiber system through the use of mutant flies, as well as tested in Xenopus oocytes expressing nicotinic acetylcholine channels and against the acetylcholine binding protein. Other well-known alpha conotoxins have also been characterized in the giant fiber system. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Organisms face a constant risk of attack from parasites. While classic immunology has revealed numerous physiological and molecular mechanisms that underpin host immunity, the recently developed field of ecological immunology has attempted to understand the ecological and evolutionary causes that explain the diversity of such immune mechanisms. However, progress in the field has been hampered by the complex relationship between immunity and fitness as well as the methodological limitations of our experiments. There is an urgent need for eco-immunological studies that combine life history theory with experimentally tractable but ecologically realistic host and pathogen models. In this thesis, I tackle three novel aspects of host defence against parasites in an established model for insect immunity, the fruit fly Drosophila melanogaster, with the entomopathogenic fungus Metarhizium robertsii, one of the most successful natural insect pathogens. In particular, I show in Chapter 2 that an immune and stress response gene, Turandot M, provides specific immunity against sexually transmitted fungal infections; but, this protective effect comes at a cost to life history in the absence of infection. In Chapter 3, I show that when exposed to the fungal pathogen, the fruit fly alters its temperature preference by seeking out cooler temperatures, which results in a dramatic shift in its life history strategy while simultaneously enhancing antifungal resistance, though not tolerance. Finally, I demonstrate in Chapter 4 that exposure to fungal parasites induces fitness-associated maternal effects on offspring meiotic recombination and life history, both of which have the potential to accelerate adaptive evolution. Taken together, these results demonstrate the benefits of integrating life history theory in eco-immunological research. They show that life history responses are an integral component of host defence against parasites, and that Drosophila-Metarhizium is a promising model system for ecological immunology.

I present a novel method for the unsupervised discovery of behavioural motifs in larval Drosophila melanogaster and Caenorhabditis elegans. Most current approaches to behavioural annotation suffer from the requirement of training data. As a result, automated programs carry the same observational biases as the humans who have annotated the data. The key novel element of my work is that it does not require training data; rather, behavioural motifs are discovered from the data itself. The method is based on an eigenshape representation of posture. Hence, my approach is called the eigenshape annotator (ESA). First, I examine the annotation consistency for a specific behaviour, the Omega turn of C. elegans, and find significant inconsistency in both expert annotation and the various Omega turn detection algorithms. This finding highlights the need for unbiased tools to study behaviour. A behavioural motif is defined as a particular sequence of postures that recurs frequently. In ESA, posture is represented by an eigenshape time series, and motifs are discovered in this representation. To find motifs, the time series is segmented, and the resulting segments are then clustered. The result is a set of self-similar time series segments, i.e. motifs. The advantage of this novel framework over the popular sliding windows approaches is twofold. First, it does not rely on the ‘closest neighbours’ definition of motifs, by which every motif has exactly two instances. Second, it does not require the assumption of exactly equal length for motifs of the same class. Behavioural motifs discovered using the segmentation-clustering framework are used as the basis of the ESA annotator. ESA is fully probabilistic, therefore avoiding rigid threshold values and allowing classification uncertainty to be quantified. I apply eigenshape annotation to both larval Drosophila and C. elegans, and produce a close match to hand annotation of behavioural states. However, many behavioural events cannot be unambiguously classified. By comparing the results to eigenshape annotation of an artificial agent’s behaviour, I argue that the ambiguity is due to greater continuity between behavioural states than is generally assumed for these organisms.

How neurons choose appropriate synaptic partners to form functional neural circuits is not well understood. Two subfamilies of Drosophila immunoglobulin superfamily (IgSF) cell surface proteins, Dprs (defective proboscis response) and DIPs (Dpr interacting proteins) are broadly expressed in the nervous system and involved in the development of neural circuits. A qualitative interactome developed from high-throughput experiments has shown that each DIP interacts with a unique set of Dpr proteins. Neurons with distinct synaptic specificities express distinct combinations of Dprs, while a subset of their synaptic partners express the complementary DIPs. These findings are consistent with the idea that the specificity of interactions between Dprs and DIPs help to define the synaptic connectivity of the neurons in which they are expressed. Thus, it is essential to fully understand interactions between members of these two protein families. Using surface plasmon resonance (SPR), we have generated a quantitative Dpr and DIP interactome, which contained several novel features. We determined the binding affinities of the majority of Dpr-DIP interactions, revealing binding groups that span a range of affinities and reflect DIP and Dpr phylogeny. Crystal structures of Dpr-DIP heterocomplexes were determined and used to design site-specific mutants that, along with SPR experiments, reveal the major determinants of Dpr-DIP binding specificity. Using analytical ultracentrifugation (AUC), we show that some Dpr and DIP family members form homophilic dimers as well. Multiple crystal structures of DIP homodimers reveal the molecular determinants of homophilic binding and structure-guided mutants along with AUC experiments further validated their mechanism of interaction. The existence of DIP and Dpr homodimers suggests the possibility of still-unknown mechanisms of Dprs and DIPs in neural circuit formation. Based on information derived from our crystal structures and biophysical experiments, we designed, produced, and tested Dpr and DIP proteins with altered binding properties. Many of the structural and biophysical studies described in this thesis were undertaken to produce tools to probe Dpr and DIP function in an in vivo setting. Parallel studies utilizing many of the mutant proteins described here (and other reagents that are not described here) are underway in the Zipursky lab, and are not described herein.

Despite their small brains, insects are capable of incredible navigational feats. Even Drosophila melanogaster (the common fruit fly) uses visual cues to remember locations in the environment. Investigating sophisticated navigation behaviors, like visual place learning, in a genetic model organism enables targeted studies of the neural circuits that give rise to these behaviors. Recent work has shown that the ellipsoid body, a midline structure deep within the fly brain, is critical for certain navigation behaviors. However, nearly all aspects of visual place learning remain mysterious. What visual features are used to encode place? What is the site of learning? How do the learned actions integrate with the core navigation circuits? To begin to address these questions I have established an experimental platform where I can measure neural activity using a genetically encoded calcium indicator in head-fixed behaving Drosophila. I further developed a virtual reality paradigm where flies are conditioned to prefer certain orientations within a virtual environment. In dendrites of ellipsoid body neurons, I observe a range of specific visual responses that are modified by this training. Remarkably, I find that distinct calcium responses are observed during presentation of preferred visual features. These studies reveal learning-associated neural activity changes in the inputs to a navigation center of the insect brain.

A key feature of sleep is reduced responsiveness to the environment, which puts animals in a particularly vulnerable state; yet, sleep has been conserved throughout evolution, indicating that it fulfills a vital purpose. A core function of sleep across species has not been identified, but substantial advances in sleep research have been made in recent years using the genetically tractable model organism, Drosophila melanogaster. While a standard approach in sleep research is to study the effects of short-term sleep deprivation on an animal, tools are now available to genetically manipulate sleep amount in the fruit fly. In particular, a number of short-sleeping Drosophila mutants have been identified that model the long-term sleep restriction that is widespread in modern society. This thesis describes a body of work in which short-sleeping Drosophila mutants, as well as other genetic and pharmacological tools, were used to shed light on the function and regulation of sleep.

Walking is an essential behavior across the animal kingdom. To navigate complex environments, animals must have highly robust, yet flexible locomotor behaviors. One crucial aspect of this process is the selection of an appropriate walking speed. Speed shifts entail not only the scaling of behavioral parameters (such as faster steps) but also changes in coordination to produce different gaits, and the details of how this switch occurs are currently unknown. Modulatory substances, particularly small biogenic amine neurotransmitters, can alter the output and even the connectivity of motor circuits. This work addresses the hypothesis that one such neuromodulator – serotonin (5HT) – is a key regulator of walking speed at the level of motor circuitry. To explore this question, I use the model organism Drosophila melanogaster which, like vertebrates, displays complex coordinated locomotion at a wide range of speeds. In Chapter 2, I will describe our efforts to characterize the anatomy of the serotonergic cell populations that provide direct input to motor circuitry. I find that innervation of the neuropil of the ventral nerve cord - a structure roughly analagous to the mammalian spinal cord - is provided primarily by local modulatory interneurons. Using stochastic single cell labeling techniques, I will detail the specific anatomy of individual neuromodulatory cells, and also the distribution of synapses across their processes. In Chapter 3, I will show that optogenetic activation or tonic inhibition of VNC serotonergic neurons produces opposing shifts in walking speed. To analyze behavior, I will use two complementary approaches. On the one hand, I will use an arena assay to holistically assess walking velocity and frequency. On the other, I will use a behavioral assay developed in the lab - the Flywalker - to assess walking kinematics at high resolution. The combination of these technique will give us a broad and specific picture of how the VNC serotonergic system modulates walking. In Chapter 4, I will identify natural behavioral contexts under which serotonin is used to shift walking behavior. I will use a variety of paradigms that induce animals to shift their speed, from changes in orientation and nutrition state, to pulses of light, odor, and a vibration. I will assess the requirement for the VNC serotonergic system under all of these conditions, to build a clearer picture of its role in modulating behavioral adaptation. In Chapter 5, I will describe our efforts, in collaboration with Pavan Ramdya's lab at EPFL, to functionally image VNC serotonergic cells while the animal is walking, to understand how activity is endogenously regulated in this population. Finally, in Chapter 6 I will characterize the circuit elements which might be responsible for serotonin's effect on walking. I will use recently developed mutant lines to identify the particular serotonergic receptors responsible for enacting shifts in walking behavior. Using genetic labeling tools, I will identify potential targets of serotonergic signaling in the VNC, and formulate a model by which action on these targets could adjust locomotor output. Altogether, this work seeks to characterize the anatomy and behavioral role of the VNC serotonergic system in Drosophila. I hope that through this work, I will gain a deeper understanding of not only this particular modulatory system in this particular behavioral context, but also of how static circuits are conferred with essential flexibility in behaving animals.