The ubiquitin ligase Highwire is responsible for cell-autonomously promoting synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects in synaptic function and extra branching at the axon terminal, corresponding to transient branching that occur in the course of giant synapse formation during metamorphosis. The MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner, suggesting different molecular mechanisms downstream of the MAP kinase pathway govern function and pruning. A novel role for Highwire is revealed, regulating the giant fiber axon’s ability to respond to external cues regulated by Fos. When expression of the transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Oxidant stress and injury is inherent in many human diseases such as ischemic vascular and respiratory diseases, heart failure, myocardial infarction, stroke, perinatal and placental insufficiencies, diabetes, cancer, and numerous psychiatric and neurodegenerative disorders. Finding novel therapeutics to combat the deleterious effects of oxidative stress is critical to create better therapeutic strategies for many conditions that have few treatment options. This study used the anoxia-tolerant fruit fly, Drosophila melanogaster, to investigate endogenous cellular protection mechanisms and potential interactions to determine their ability to regulate synaptic functional tolerance and cell survival during acute oxidative stress. The Drosophila larval neuromuscular junction (NMJ) was used to analyze synaptic transmission and specific motor axon contributions. Drosophila Schneider 2 (S2) cells were used to assess viability. Acute oxidative stress was induced using p harmacological paradigms that generate physiologically relevant oxidant species: mitochondrial superoxide production induced by sodium azide (NaN3) and hydroxyl radical formation via hydrogen peroxide (H2O2). A combination of genetic and pharmacological approaches were used to explore the hypothesis that endogenous protection mechanisms control cellular responses to stress by manipulating ion channel conductance and neurotransmission. Furthermore, this study analyzed a group of marine natural products, pseudopterosins, to identify compounds capable of modulating synaptic transmission during acute oxidative stress and potential novel neuromodulatory agents. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Sox proteins all contain a single ~70 amino acid High Mobility Group (HMG) DNA-binding domain with strong homology to that of Sry, the mammalian testisdetermining factor. In Drosophila melanogaster, there are four closely related members of the B group, Dichaete (D), Sox Neuro (Sox N), Sox 21a, and Sox 21b that each exhibit ~90% sequence identity within the HMG domain.The previous study has shown that Dichaete plays a major role in embryonic nervous system development and is expressed in several clusters of neurons in the brain, including intermingled olfactory LNs and central-complex neurons strongly expressed in local interneuron of the olfactory system. However, little is known about the possible expression and functions of the related group B Sox genes in the larval and adult brain. In particular, it is unclear if Sox N may function along with Dichaete in controlling the development or physiology of the adult olfactory system. Our data suggests Sox N potential role in the elaboration of the olfactory circuit formation. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

The classic guidance molecules, Netrin and its receptor Frazzled (Fra), dictate the strength of synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating gap junction localization in the pre-synaptic terminal. In Netrin mutant animals the synaptic coupling between a giant interneuron and the jump motor neuron was weakened. Dye-coupling between these two neurons was severely compromised or absent. These mutants exhibited anatomically and physiologically defective synapses between the giant fiber (GF) and tergotrochanteral motor neuron (TTMn). In cases where Netrin mutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses. Dye-coupling between the giant fiber and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in the Netrin mutants. When we examined the gap junctions with antibodies to Shaking-B Innexin (ShakB), they were significantly decreased or absent in the pre-synaptic terminal of the mutant GF. This data is the first to show that Netrin and Frazzled regulate placement of gap junctions pre-synaptically at a central synapse. In the Drosophila Giant Fiber System, we demonstrate a mechanism that ensures the monoinnervation of two homologous motor neurons by two homologous interneurons. In a scenario where both interneurons could synapse with both motor neuron targets, each interneuron exclusively synapsed with only one target and the circuit functions at normal physiological levels. This innervation pattern depended on the ratio of netrin-to-frazzled expression. When Netrin was over expressed in the system, shifting the ratio in favor of Netrin, both interneurons synapsed with both target motor neurons and physiological function was reduced. This resulted in the polyinnervationof a single target. In contrast, when Frazzled was over expressed in the system, one interneuron innervated both targets and excluded the remaining interneuron from making any synaptic contact. This resulted in a single interneuron mono-innervating both motor neurons and physiological function was mutant. The orphaned interneuron made no synaptic contact with either motor neuron target. Physiological function was only normal when the Netrin-Frazzled ratio was at endogenous levels and each GF monoinnervated one motor neuron. When we examined the gap junctions at this synapse in experimental animals, there was a significant reduction of gap junction hemichannels in the presynaptic terminal of axons that deviated from normal innervation patterns. While the synapse dyecoupled, the reduction in gap junction hemichannels reduced function in the circuit. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2013.

In the developing CNS, presynaptic neurons often have exuberant overgrowth and form excess (and overlapping) postsynaptic connections. Importantly, these excess connections are refined during circuit maturation so that only the appropriate connections remain. This synaptic rearrangement phenomenon has been studied extensively in vertebrates but many of those models involve complex neuronal circuits with multiple presynaptic inputs and postsynaptic outputs. Using a simple escape circuit in Drosophila melanogaster (the giant fiber circuit), we developed tools that enabled us to study the molecular development of this circuit; which consists of a bilaterally symmetrical pair of presynaptic interneurons and postsynaptic motorneurons. In the adult circuit, each presynaptic interneuron (giant fiber) forms a single connection with the ipsilateral, postsynaptic motorneuron (TTMn). Using new tools that we developed we labeled both giant fibers throughout their development and saw that these neurons overgrew their targets and formed overlapping connections. As the circuit matured, giant fibers pruned their terminals and refined their connectivity such that only a single postsynaptic connection remained with the ipsilateral target. Furthermore, if we ablated one of the two giant fibers during development in wildtype animals, the remaining giant fiber often retained excess connections with the contralateral target that persisted into adulthood. After demonstrating that the giant fiber circuit was suitable to study synaptic rearrangement, we investigated two proteins that might mediate this process. First, we were able to prevent giant fibers from refining their connectivity by knocking out highwire, a ubiquitin ligase that prevented pruning. Second, we investigated whether overexpressing Netrin (or Frazzled), part of a canonical axon guidance system, would affect the refinement of giant fiber connectivity. We found that overexpressing Netrin (or Frazzled) pre- & postsynaptically resulted in some giant fibers forming or retaining excess connections, while exclusively presynaptic (or postsynaptic) expression of either protein had no effect. We further showed that by simultaneously reducing (Slit-Robo) midline repulsion and elevating Netrin (or Frazzled) pre- & postsynaptically, we significantly enhanced the proportion of giant fibers that formed excess connections. Our findings suggest that Netrin-Frazzled and Slit-Robo signaling play a significant role in refining synaptic circuits and shaping giant fiber circuit connectivity. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

O fungo leveduriforme Malassezia pachydermatis está presente na microbiota da pele dos animais. Alterações na imunidade da pele do hospedeiro fazem com que a levedura sofra adaptações modificando a expressão de fatores de virulência, causando otite, dermatite e até mesmo infecção sistêmica em animais e humanos. O tratamento de M. pachydermatis é relativamente simples, à base de antifúngicos azólicos. No entanto, o aparecimento de cepas resistentes e de infecções recorrentes tem gerado preocupação recentemente. Drosophila melanogaster tem sido um modelo promissor no estudo de microrganismos, apresentando duas vias de sinalização da resposta imune, a via Imd contra bactérias gram-negativas e a via Toll contra bactérias gram-positivas e fungos, as quais desencadeiam a produção dos peptídeos antimicrobianos locais e sistêmicos. A virulência de M. pachydermatis em moscas D. melanogaster wild-type e Toll-deficientes foi testada com inóculos nas concentrações entre 103 e 107 unidades formadoras de colônia (UFC)/ml e a infecção ocorreu pela punção com agulha no tórax das moscas, que foram colocadas em frascos com alimento e incubados à 29 °C por sete dias, com contagem diária da sobrevivência. Ainda foram realizados a contagem de UFC/ml/mosca e a histopatologia após os sete dias de observação. As moscas wild-type se mostraram resistentes à infecção. Curvas de mortalidade de moscas Toll-deficientes, em concentrações a partir de 104 leveduras/ml, diferiram estatisticamente do grupo controle. Após o sétimo dia de infecção, moscas wild-type inoculadas com 1 x 107 UFC/ml tinham carga fúngica variando entre 0,33 a 1 x 102 UFC/mosca, enquanto em moscas Toll-deficientes a carga fúngica variou de 2,3 x 103 a 1,3 x 104 UFC/mosca. Portanto, D. melanogaster Toll-deficientes são suscetíveis à infecção por M. pachydermatis devido à falta do sistema imune para o reconhecimento e montagem da resposta imune frente ao fungo, semelhante ao que ocorre em pacientes imunocomprometidos, dado o sistema imune conservado entre os mamíferos e a mosca-das-frutas. Deste modo, D. melanogaster é um modelo promissor e inovador para estudos de patogenicidade e de prospecção de fármacos frente a M. pachydermatis. / The yeast Malassezia pachydermatis is present in the normal microbiota of the skin of animals. Changes in the host's immunity cause the yeast to undergo adaptations modifying the expression of virulence factors, causing otitis, dermatitis and even systemic infection in animals and humans. The treatment of M. pachydermatis is relatively simple, based on the use of azole antifungals. However, the emergence of resistant strains and recurrent infections has generated concern recently. Drosophila melanogaster has been a promising model in the study of microorganisms, presenting two pathways of immune response signaling, the Imd pathway against gram-negative bacteria and the Toll pathway against gram-positive bacteria and fungi, which trigger the production of local and systemic antimicrobial peptides. The virulence of M. pachydermatis in wild-type and Toll-deficient D. melanogaster flies was tested with inoculum concentrations ranging between 103 and 107 colony forming units (CFU)/ml and the infection occurred by needle puncture in the thorax of the flies, which were placed in vials with food and incubated at 29 °C for seven days, with daily survival counts. Fungal burden counts and the histopathology were also performed after seven days of observation. Wild-type flies were resistant to infection. Mortality curves of Toll-deficient flies, at concentrations superior of 104 yeasts/ml, differed statistically from the control group. After the seventh day of infection, wild-type flies inoculated with 1 x 107 CFU/ml had fungal load ranging from 0.33 to 1 x 102 CFU/fly, while in Toll-deficient flies the fungal load ranged from 2.3 x 103 to 1.3 x 104 CFU/fly. We concluded that Toll-deficient D. melanogaster flies are susceptible to infection by M. pachydermatis due to lack of immune system for the recognition and assembly of immune response to the fungus, similar to that occurring in immunocompromised patients, given the conserved immune system among mammals and fruit flies. Therefore, D. melanogaster is a promising and innovative model for pathogenicity studies and prospection of drugs against M. pachydermatis.

Understanding how organisms function is central to Biology. Assessing how animals respond to fluctuations in their environment and determining inter-individual variation in phenotypic plasticity is paramount to identifying the physiology of traits, the selective pressures which have shaped them, and how we can manipulate them to benefit human life. The over-arching goal of my thesis is to understand the effects of sex, diet and pathogen exposure on the physiology of the fruit fly to assess the versatility of their individual traits in response to these natural factors. Chapter 2 investigates how the sexes utilise nutrition towards their lifespan and reproduction, providing evidence that the reproduction of males and females requires different dietary components while lifespan does not. Chapter 3 reveals that the sexes also differ in how they utilise nutrients for pathogen resistance identifying that females are highly protein-limited and more susceptible to infection than males. Chapter 4 provides the first comprehensive study of how organisms alter their dietary intake in response to infection, finding that flies behaviourally ingest less and consume higher protein:carbohydrate ratio diets when exposed to live fungal spores. Chapter 5 explores the phenomenon of trait-enhnacing external stresses, a response often termed hormesis. This study reveals that the beneficial physiological response from inactive fungal spore exposure, a potential form of hormesis, incurs immune costs. The implications of my results to the field of physiology are discussed in Chapter 6 where I also highlight the limitations of my work and potential consequences for life history research. Overall it is determined that studies investigating the natural physiological response of organisms or potentially beneficial treatments for our own species, must consider sex-specific effects, physiological consequences in a variety of traits, and how organisms may utilise variation within their environment to alter their phenotypic condition.

Like most organisms, Drosophila melanogaster can synchronize its physiological and behavioural processes by possessing internal circadian clock that regulates. Naturally fluctuating timing cues, like light and temperature (also known as Zeitgebers), synchronize these endogenous and self‐sustained clocks with external time. In Drosophila, synchronization of the circadian clock by light has been studied in detail, but much less is known about the molecular mechanisms underlying temperature entrainment. Previous data from our lab shows that Nocte, a Chordotonal organ (Ch organ) located protein, is required for normal temperature entrainment in Drosophila. However, neither the function of Nocte in temperature entrainment nor the molecular underlying mechanisms are clear. To address these issues, a proteomics strategy of combing co‐immunoprecipitation and MS/MS sequencing was applied to isolate potential interactors of Nocte. IR25a was one of the most promising candidates, which was later confirmed by behavioural tests using RNA interference: Reducing IR25a expression in Chorgan resulted in abnormal behaviour during temperature cycles, similar to what had been described for Nocte mutant. To further confirm the interaction between Nocte and IR25a, I showed that IR25a physically interacts with Nocte in vivo. Moreover, using an IR25a‐gal4 line, I was able to show that IR25a is expressed in subsets of chordotonal organs (Ch organ) including Johnston's Organs (JO), where Nocte is also highly expressed. These results, along with the behavioural data mentioned above are consistent with the proteomics results and suggest that Nocte and IR25a physically and functionally interact. IR25a mutants were employed to further investigate the function of IR25a in temperature entrainment. First of all, I found that both central and peripheral clocks in wild type flies can be synchronized to temperature cycles with only two degree differences (12h: 12h, 27 °C: 25 °C). In contrast, synchronization of locomotor activity rhythms in the IR25a null mutants to the same temperature cycles and other TC's with 2°C amplitude was eliminated. Under the same conditions, the oscillations of the core clock proteins TIMLESS (TIM) and PERIOD (PER) that normally occur in fly heads were completely abolished inIR25a null mutants, suggesting that IR25a is required for temperature entrainment of peripheral clocks. In the central brain pacemaker neurons, the oscillations of TIM in dorsal and lateral neurons were also affected by the IR25a mutants. On the contrary, IR25a is not required for light entrainment and temperature compensation, suggesting that IR25a is specifically involved in temperature synchronization. Moreover, temperature entrainment of the IR25a null mutants can be partially restored by applying larger temperature intervals (29°C: 25°C) indicating that IR25amay function as amplitude detector independent of absolute temperature values. Finally, neuronal activity in IR25a+ neurons is crucial for the synchronization of circadian clocks to low amplitude temperature cycles. Re‐constitution of functional olfactory receptors required the assembly of IR25a with IR76a and IR76b. Interestingly, IR76a and IR76b are neither required for temperature entrainment at the behavioural level nor expressed in the Ch organs. To check if other potential IRs interacting with IR25a exist, I screened the expression pattern of most divergent IRs using IR‐gal4/UAS‐GFP flies. IR56a was isolated as a potential partner of IR25a because it is also expressed in the femur chordotonal organs. To investigate the function of IR56a in temperature entrainment, I generated a null mutant of IR56a. Surprisingly, this gene is not required for synchronizing clocks to a temperature cycle (27°C: 25°C) at the behavioural level. However, the behavioural and molecular phenotypes of IR56a mutant under different temperature cycles need to be further characterized.

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