As muscle function deteriorates with age, the discovery of new ways to enhance the quality of human life by stunting inevitable aging processes, such as sarcopenia, is a subject of great interest to aging populations, to health care professionals and to nutritional companies. β-hydroxy-β-methylbutyrate (HMB) enhances muscle strength in humans and attenuates disease-induced and disuse-dependent atrophy in rodents. We investigated the feasibility of utilizing Drosophila as a model organism to study the biological effects of HMB in aging muscle. Using flight ability as an index of muscle function and monitoring the lifespan of flies, we found that normal food supplemented with 10 mg/mL HMB, supplied from eclosion through adulthood, attenuates the age-dependent decline in flight ability and enhances longevity of flies. To further discern the dietary optimization of HMB supplementation, we examined the effect of 10 mg/mL HMB supplementation from the larval stages throughout adulthood under various dietary conditions. As dietary restriction without malnutrition and HMB supplementation independently increase longevity in flies, we investigated the relationship between dietary restriction and HMB supplementation. Flies were subject to non-restricted, reduced yeast (protein), or intermittently cycled feeding regimens, with or without HMB. Both modes of dietary restriction improved flight ability, while only cycling flies on and off food increased lifespan and improved survivorship of the flies. HMB supplementation increased flight ability later in life in all groups, but had differential effects on lifespan; HMB improved the survivorship of females fed a reduced yeast diet, but decreased the survivorship of both non-restricted and intermittently fed flies. This not only suggests that HMB may act via different pathways to influence fly flight and survivorship, but that these mechanisms may differ under various dietary conditions, in different sexes. Because HMB supplemented dietary restriction had different effects on flight ability and survival than dietary restriction alone, HMB likely acts via different mechanisms than dietary restriction. Sex specific effects were found, suggesting that future HMB and dietary restriction studies should make distinctions between effects in males and females. As the flight ability and longevity of D. melanogaster was affected by HMB supplementation under various dietary conditions, future studies may use D. melanogaster as a model organism to explore the effects of HMB on age-related muscle deterioration and to help uncover the biological mechanisms of such observations, as well as potential treatments for age-associated muscle dysfunction and disease.

Doctor of Philosophy / Division of Biology / Theodore J. Morgan / Thermal stress impacts animals around the globe and understanding how organisms adapt to changes in temperature is of particular interest under current climate change predictions. My research focuses on the evolutionary genetics involved in cold tolerance and plasticity of cold tolerance using both artificially selected and naturally segregating populations, while tying the genes of interest to their physiological components. First I address cross-tolerance of stress traits following artificial selection to a non-lethal cold tolerance metric, chill-coma recovery. Using these artificial selection populations, we found that stress traits such as desiccation tolerance, starvation tolerance, acclimation, and chronic and acute cold tolerance do not correlate with level of cold tolerance as defined by chill-coma recovery time. We next assessed lifetime fitness of these different cold tolerance lines and found that only at low temperatures did fitness differ among cold tolerance levels. We then analyzed gene expression differences between resistant and susceptible populations at three time points to understand where selection pressures are hypothesized to act on genomic variation. Our gene expression analyses found many differences between resistant and susceptible lines, primarily manifesting themselves in the recovery period following cold exposure. We next utilized a community resource, the Drosophila melanogaster reference panel, to identify naturally segregating variation in genes associated with cold acclimation and fitness. We specifically asked if long- and short-term acclimation ability had overlapping genetic regions and if plasticity values from constant rearing environments were associated with demographic parameters in fluctuating environments. We found that long- and short-term acclimation are under unique genetic control and functionally tested several genes for acclimation ability. We also found that acclimation ability in constant environments and fitness in fluctuating environments do not correlate, but that genotypes are constrained in their fitness abilities between a warm and cool environment. Our analyses describe several novel genes associated with cold tolerance selection and long- and short-term acclimation expanding our knowledge of the complex relationship between demographic components and survivorship as well as a unique investigation of the change in gene expression during cold exposure.

Tesis entregada a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al grado de Doctor en Ciencias Biológicas con mención en Biología Molecular, Celular y Neurociencias. / Lysosomal physiology has been shown to be important for the correct functioning of immune cells, including their migratory and their phagocytic properties. In this work, we aimed to explore the migratory process of immune cells and understand the connection between cytoskeletal dynamics and lysosomal degradative capacity. Specifically, we characterized the role of the calcium permeable channel Trpml in the process of cell migration, as well as that of the immune response and its connection with lysosomal physiology, using as model of study the Drosophila melanogaster macrophages, called hemocytes. Hemocytes are the cellular component of the immune response in Drosophila, performing key functions during development, as well as in immune defense in response to bacterial or parasitic infection. These cells are highly migratory in vivo and, importantly, present a substantial degree of functional and structural conservation with mammalian innate immune cells. Conveniently, the Drosophila genome contains only one gene coding for the Trpml channel, and its mutant phenotype resembles that of Mucolipidosis type IV patients, which bear a loss of function mutation in MCOLN1, the human homolog of the Drosophila trpml gene. Results presented in this thesis demonstrate that Trpml plays a fundamental role in phagolysosomal degradation and in vivo hemocyte migration, and that it participates in these processes through partially independent mechanisms. The most likely scenario is that Trpml is important for the degradative process by favoring phagolysosomal fusion and further phagocytic degradation. This is supported by the fact that calcium ions are needed for the fusion of phagosomal and lysosomal membranes. On the other hand, for the migratory process, localized Trpml activation from the lysosomes in the rear of polarized hemocytes, would promote the local activation of non-muscle myosin-II, which is necessary event to trigger cell body translocation during movement. / La fisiología lisosomal ha mostrado ser importante para el correcto funcionamiento de células inmunes, abarcando tanto sus propiedades migratorias como fagocíticas. En este trabajo, se quiso ahondar en el proceso migratorio de células del sistema inmune para comprender el nexo existente entre la dinámica del citoesqueleto y la capacidad degradativa de los lisosomas. En particular, se caracterizó el rol del canal lisosomal permeable a calcio, Trpml, en el proceso de migración celular, al igual que la respuesta inmune y su vínculo con la fisiología lisosomal, usando como modelo de estudio, los macrófagos de Drosophila melanogaster denominados hemocitos. Los hemocitos, son el componente celular de la respuesta inmune de Drosophila, cumpliendo funciones claves durante el desarrollo, así como durante la defensa ante infección por microorganismos o parásitos. Estas células son altamente migratorias in vivo, e importantemente, presentan un sustancial grado de conservación funcional y estructural con las células del sistema inmune innato de mamíferos. Convenientemente, Drosophila posee sólo un gen que codifica para el canal Trpml, cuyo fenotipo mutante semeja al de pacientes con Mucolipidosis tipo IV, quienes presentan pérdida de función del gen homólogo que codifica Trpml en humanos, MCOLN1. Los resultados presentados en esta tesis demuestran que Trpml cumple un rol fundamental en la degradación fagolisosomal, y la migración de hemocitos in vivo, y que estaría participando en estos dos procesos mediante mecanismos parcialmente independientes. A partir de los resultados obtenidos podemos proponer que Trpml es fundamental para el procesamiento degradativo, mediante el favorecimiento de la fusión fagolisosomal, y consecuente degradación fagocítica. Esto posiblemente se sustenta en la necesidad de iones Ca2+ para la fusión de las membranas del fagosoma con el lisosoma. Por su parte, en el proceso migratorio, la activación localizada de Trpml desde los lisosomas en la región posterior de células polarizadas, promovería la activación de Miosina-II no muscular en esta región, evento necesario para la translocación del cuerpo celular. / Conicyt 21140289, Proyectos Redes 140004, Fondecyt 1140522, Anillo ACT 1401 y Fondap 15090007.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy. / This thesis reports on the investigation of two genetic aspects of Southern African populations of Drosophila Melanogaster: the " family of transposable elements and the allelic variation present in several enzyme systems. (Abbreviation abstract) / Andrew Chakane 2018

Polycystic ovary syndrome is an endocrine disorder with symptoms of hyperandrogenism, insulin-resistance and anovulation in women which creates a disposition for other disorders, like Type 2 Diabetes. Drosophila Melanogaster flies have short generation, low cost-management and share several signaling pathways with humans and are therefore an excellent tools to study disease aetiology – yet no such model exists for PCOS. This thesis aimed to create such PCOS-like disease model through treatment of Drosophila Melanogaster adults and larvae with 5-α Dihydrotestosterone and/or high sugar diet. The samples were studied by measuring weight, fecundity, total glycerol concentration and Real-Time quantitative Polymerase Chain Reaction (RT qPCR) using PCOS-related orthologue genes. While previous studies have frequently studied the effects of high sugar diets on Drosophila Melanogaster – there are no previous studies that have studied the effects of 5-α Dihydrotestosterone, with or without high sugar diet, on the weight, fecundity and gene expression of Drosophila Melanogaster. Results showed that larva raised on high sugar diet had traits resembling an insulin-resistant phenotype while the fecundity of flies treated with high sugar diet was significantly lower than those treated with standard sugar diet and DHT. RT qPCR showed overexpression of the standard sugar diet and DHT groups in the investigated genes EcR, Med, TOLL, THADA and FOXO. The conclusion, based on the results, is that insulin-resistance was produced but that more studies are required before a complete PCOS-like disease model is possible in Drosophila Melanogaster.

As the prevalence of type 2 diabetes has increased globally, so has the need to further investigate the disorder’s underlying features and a potential target for treatment. Drosophila melanogaster has proven to be an excellent model organism to study type 2 diabetes (T2D). To see whether it can serve as a model organism to identify the treatment of T2D, a type 2 diabetes like model were created in Drosophila through high sugar diet (HSD). The aim of the study was to investigate the therapeutic effects of six different probiotic bacteria strains on T2D characteristics markers. Longevity, size, and weight measurement were performed. Followed by verification of HSD effects on these phenotypes. It was demonstrated that probiotics could improve weight and lifespan. Treatment using probiotics showed statistically increased body weight in both 3rd instar larvae and adult flies (p-value <0.05). There was no statistically significant difference in length between any of the groups including controls (p-value 0.25). However, the triglyceride assay showed a slightly significant difference between control low sugar diet and few of the treatment groups (L. paracasei with p-value 0.037, and L. acidophilus with p-value 0.025) fed larvae/adult flies, and there was no statistically significant difference between controls (high and low sugar diets), and probiotics treatment groups (L. plantarum, B. animalis and B. breve) fed larvae/adult flies with a p-value >0.05. To investigate the effect on gene expression of four genes (FASNCG3523, FASNCG3524, FASNCG17374 & dsREBP) important in lipid metabolism, qPCR was performed using the Taqman method. All probiotic treatment groups had significantly decreased gene expression of FASN compared to the control groups. Findings of this study suggest that Drosophila melanogaster can be utilized as a model organism to study T2D and that further studies concerning the effects of probiotic treatment in Drosophila are required to fully understand the interactions and mechanism of action.

Neuronal cell adhesion molecules of L1 family play a critical role in proper nervous system development. Various mutations on human L1-CAM that lead to severe neurodevelopmental disorders like retardation, spasticity etc. termed under L1 syndrome. The vertebrr their roles in axon pathfinding, neurite extension and cell migration, howeverate L1CAM and its homolog in Drosophila, neuroglian (nrg) have been well studied fo, much less is known about the mechanisms by which they fine tune synaptic connectivity to control the development and maintenance of synaptic connections within neuronal circuits. Here we characterized the essential role of nrg in regulating synaptic structure and function in vivo in a well characterized Drosophila central synapse model neuron, the Giant Fiber (GF) system. Previous studies from our lab revealed that the phosphorylation status of the tyrosine in the Ankyrin binding FIGQY motif in the intracellular domain of Nrg iscrucial for synapse formation of the GF to Tergo-Trochanteral Motor neuron (TTMn) synapse in the GF circuit. The present work provided us with novel insights into the role of Nrg-Ank interaction in regulating Nrg function during synapse formation and maintenance. By utilizing a sophisticated Pacman based genomic rescue strategy we have shown that dynamic regulation of the Neuroglian–Ankyrin interaction is required to coordinate transsynaptic development in the GF–TTMn synapse. In contrast, the strength of Ankyrin binding directly controls the balance between synapse formation and maintenance at the NMJ. Human L1 pathological mutations affect different biological processes distinctively and thus their proper characterization in vivo is essential to understand L1CAM function. By utilizing nrg14;P[nrg180ΔFIGQY] mutants that have exclusive synaptic defects and the previously characterized nrg849 allele that affected both GF guidance and synaptic function, we were able to analyze pathological L1CAM missense mutations with respect to their effects on guidance and synapse formation in vivo. We found that the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation and not for axon guidance while L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Aging is a biological process that has many detrimental effects due to the accumulation of oxidative damage to key biomolecules due to the action of free radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each form has been shown to reduce a specific enantiomer of bound and free oxidized methionine. Effects of Msr have yet to be studied in the major developmental stages of Drosophila melanogaster despite the enzymes elevated expression during these stages. A developmental timeline was determined for MsrA mutant, MsrB mutant, and double null mutants against a wild type control. Results show that the Msr double mutant is delayed approximately 20 hours in the early/mid third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon is due to an issue gaining mass. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2013. / FAU Electronic Theses and Dissertations Collection

Anoxia is characterized by an absence of oxygen supply to a tissue (Dawson- Scully et al., 2010). Unlike humans, Drosophila melanogaster is an organism that can survive low oxygen levels for hours without showing any pathology (Lutz et al., 2003) Under anoxia, the fruit fly loses locomotive activity, resulting in an anoxic coma (Haddad et al., 1997). In this study we investigate the influence of five variables for anoxic tolerance in adult Drosophila: 1) anoxic environment (gas vs. drowning), 2) anoxia duration, 3) temperature (cold [3ÀC] or room temperature [21ÀC]), 4) age (young 2-9 days and old 35-39 days), and 5) PKG variation. Tolerance to anoxia is measured by the time of recovery and survival of the fruit fly from the anoxic coma. The results from this study show that short stress, low temperature, young age, and low PKG activity increased anoxic tolerance. Our findings will lay the foundation to investigate different variables, genes or pharmacological compounds that can modulate neuronal anoxic tolerance. / by Raquel Benasayag Meszaros. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

The human brain functions within a narrow range of temperatures and variations outside of this range incur cellular damage and death and, ultimately, death of the organism. Other organisms, like the poikilotherm Drosophila melanogaster, have adapted mechanisms to maintain brain function over wide ranges in temperature and, if exposed to high temperatures where brain function is no longer supported, these animals enter a protective coma to promote survival of the organism once the acute temperature stress is alleviated. This research characterized the role of different neuronal cell types, including glia, in the protection of brain function during acute hyperthermia, specifically looking at two protective pathways: the heat shock protein (HSP) pathway and the cGMP-dependent protein kinase G (PKG) pathway. Whole animal behavioral assays were used in combination with tissue-specific genetic manipulation of protective pathways to determine the specific cell types sufficient to confer protection of neuronal function during acute hyperthermia. Using the neuromuscular junction (NMJ) preparation, calcium imaging techniques were combined with pharmacological and genetic manipulations to test the hypothesis that alterations in ion channel conductance via endogenous mechanisms regulating the cellular response to high temperature stress alter neuronal function. Expression of foraging RNAi to inhibit PKG expression in neurons or glia demonstrated protection of function during acute hyperthermia measured behaviorally through the extension of locomotor function. This extension of function with the tissue-specific inhibition of PKG was also confirmed at the cellular level using the genetically encoded calcium indicator (GECI), GCaMP3, to image calcium dynamics at the NMJ, where preparations expressing foraging RNAi could continue to elicit changes in calcium dynamics in response to stimulation. Over the course of this study, the mechanism underlying a novel glial calcium wave in the peripheral nervous system was characterized in order to elucidate glia’s role in the protection of neuronal function during acute hyperthermia. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection