Feeding is one of the most important behaviors for an animal’s survival. At a gross level, it is known that the nervous system plays a major role in the expression of this complex behavior, yet a detailed understanding of the neural circuits directing feeding behavior remains unknown. Here we identify a command neuron in Drosophila melanogaster whose artificial activation, using dTrpA1, a heat-activated cation channel, induces the appearance of complete feeding behavior. We use behavioral, genetic, cellular and optical imaging techniques to show that the induced behavior is composed of multiple motor programs and can function to uptake exogenous, even noxious, material. Furthermore, we resolve the neuron’s location to the subesophageal ganglion, characterize its pre and post-synaptic sites, and determine its responsiveness to sucrose stimulation. Interestingly, the neuron’s dendritic field is proximal to sweet sensing axon terminals and its baseline activity corresponds to the fly’s satiation state, suggesting a potential point of integration between sensory, motor and motivational systems. The identification of a command neuron for feeding in a genetically tractable organism provides a useful model to develop a deeper understanding of the neural control of this ubiquitous and evolutionarily ancient behavior.

The function of homologous DNA recombination in human mitochondria has been a topic of ongoing debate for many years, with implications for fields ranging from DNA repair and mitochondrial disease to population genetics. While genetic and biochemical evidence supports the presence of a mitochondrial recombination activity, the purpose for this activity and the proteins involved have remained elusive. The work presented in this thesis was designed to evaluate the mitochondrial localization of the major recombinase protein in human cells, Rad51, as well as determine what function it plays in the maintenance of mitochondrial DNA (mtDNA) copy number that is critical for production of chemical energy through aerobic respiration. The combination of subcellular fractionation with immunoblotting and immunoprecipitation approaches used in this study clearly demonstrates that Rad51 is a bona fide mitochondrial protein that localizes to the matrix compartment following oxidative stress, where it physically interacts with mtDNA. Rad51 was found to be critical for mtDNA copy number maintenance under stress conditions. This requirement for Rad51 was found to be completely dependent on ongoing mtDNA replication, as treatment with the DNA polymerase gamma (Pol ϒ) inhibitor, ddC, suppresses both recruitment of Rad51 to the mitochondria following the addition of stress, as well as the mtDNA degradation observed when Rad51 has been depleted from the cell. The data presented here support a model in which oxidative stress induces a three-part response: (1) The recruitment of repair factors including Rad51 to the mitochondrial matrix, (2) the activation of mtDNA degradation systems to eliminate extensively or persistently damaged mtDNA, and (3) the increase in mtDNA replication in order to maintain copy number. The stress-induced decrease in mtDNA copy number observed when Rad51 is depleted is likely the result of failure to stabilize or repair replication forks that encounter blocking lesions resulting in further damaged to the mtDNA and its eventual degradation.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active players in the development and integrity of the nervous system. The formation of functional neuronal circuits requires the precise addition of new synapses. Mounting evidence implicates glial function in synapse remodeling and formation. However, the precise molecular mechanisms governing these functions are poorly understood. My thesis work begins to define the molecular mechanisms by which glia communicate with neurons at the Drosophila neuromuscular junction (NMJ). During development glia play a critical role in remodeling neuronal circuits in the CNS. In order to understand how glia remodel synapses, I manipulated a key component of the glial engulfment machinery, Draper. I found that during normal NMJ growth presynaptic boutons constantly shed membranes or debris. However, a loss of Draper resulted in an accumulation of debris and ghost boutons, which inhibited synaptic growth. I found that glia use the Draper pathway to engulf these excess membranes to sculpt synapses. Surprisingly, I found that muscle cells function as phagocytic cells as well by eliminating immature synaptic ghost boutons. This demonstrates that the combined efforts of glia and muscle are required for the addition of synapses and proper growth. My work establishes that glia play a crucial role in synapse development at the NMJ and suggests that there are other glial-derived molecules that regulate synapse function. I identified one glial derived molecule critical for the development of the NMJ, a TGF-β ligand called Maverick. Presynaptically, Maverick regulates the activation of BMP pathway confirmed by reducing the transcription of the known target gene Trio. Postsynaptically, it regulates the transcription of Glass bottom boat (Gbb) in the muscle suggesting that glia modulate the function of Gbb and consequently the activation of the BMP retrograde pathway at NMJ. Surprisingly, I also found that glial Maverick regulates the transcription of Shaker potassium channel, suggesting that glia potentially could regulate muscle excitability and consequently modulate synaptic transmission. Future work will elucidate such hypothesis. My work has demonstrated two novel roles for glia at the NMJ. First is that glia engulfing activity is important for proper synaptic growth. Second is that the secretion of glial-derived molecules are required to orchestrate synaptic development. This further supports that glia are critical active players in maintaining a functional nervous system.

Pancreatic cancer is the 4th leading cause of cancer related death in the United States with a median survival time of less than 6 months. Pancreatic ductal adenocarcinoma (PDAC) accounts for greater than 85% of all pancreatic cancers, and is marked by early and frequent mutation of the KRAS oncogene, with activating KRAS mutations present in over 90% of PDAC. To date, though, targeting activated KRAS for cancer treatment has been very difficult, and targeted therapies are currently being sought for the downstream effectors of activated KRAS. Activation of KRAS stimulates multiple signaling pathways, including the MEK-ERK and PI3K-AKT signaling cascades, but the role of downstream effectors in pancreatic tumor initiation and progression remains unclear. I therefore used primary pancreatic ductal epithelial cells (PDECs), the putative cell of origin for PDAC, to determine the role of specific downstream signaling pathways in KRAS activated pancreatic tumor initiation. As one third of KRAS wild type PDACs harbor activating mutations in BRAF , and KRAS and BRAF mutations appear to be mutually exclusive, I also sought to determine the effect of activated BRAF (BRAF V600E ) expression on PDECs and the signaling requirements downstream of BRAF. I found that both KRAS G12D and BRAF V600E expressing PDECs displayed increased proliferation relative to GFP expressing controls, as well as increased PDEC survival after challenge with apoptotic stimuli. This survival was found to depend on both the MEK-ERK and PI3K-AKT signaling cascades. Surprisingly, I found that this survival is also dependent on the IGF1R, and that activation of PI3K/AKT signaling occurs downstream of MEK/ERK activation, and is dependent on signaling through the IGF1R. Consistent with this, I find increased IGF2 expression in KRAS G12D and BRAF V600E expressing PDECs, and show that ectopic expression of IGF2 rescues survival in PDECs with inhibited MEK, but not PI3K. Finally, I showed that the expression of KRAS G12D or BRAF V600E in PDECs lacking both the Ink4a/Arf and Trp53 tumor suppressors is sufficient for tumor formation following orthotopic transplant of PDECs, and that IGF1R knockdown impairs KRAS and BRAF-induced tumor formation in this model. In addition to these findings within PDECs, I demonstrate that KRAS G12D or BRAF V600E expressing tumor cell lines differ in MEK-ERK and PI3K-AKT signaling from PDECs. In contrast to KRAS G12D or BRAF V600E expressing PDECs, activation of AKT at serine 473 in the KRAS G12D or BRAF V600E expressing tumor cell lines does not lie downstream of MEK, and only the inhibition of PI3K alone or both MEK and the IGF1R simultaneously results in loss of tumor cell line survival. However, inhibition of MEK, PI3K, or the IGF1R in KRAS G12D or BRAF V600E expressing tumor cell lines also resulted in decreased proliferation relative to DMSO treated cells, demonstrating that all three signaling cascades remain important for tumor cell growth and are therefore viable options for pancreatic cancer therapeutics.

Fabry disease (FD) is an X-linked recessive lysosomal storage disorder (LSD) known to affect approximately 1 in every 40,000 males, and a smaller number of females. FD results from a deficiency of functional α-galactosidase (α-GAL), which leads to the accumulation of terminally α-galactosylated substrates in the lysosome. The predominant treatment is Enzyme Replacement Therapy (ERT), requiring the regular infusion of recombinant human α-GAL. More than half of individuals receiving ERT experience a range of adverse infusion reactions, and it has been reported that as many as 88% of patients receiving ERT develop neutralizing IgG antibodies against the drug. In aim of designing a non-immunogenic treatment candidate for Fabry disease ERT, we have engineered the active sites of α-GAL and another homologous family 27 exoglycosylase named α-N-acetylgalactosaminidase (α-NAGAL) to have interconverted substrate specificities. 11 of 13 active site residues are conserved between these two enzymes, and we have shown that their substrate specificities can be interconverted by mutating the two non-conserved active-site residues. We report the kinetic properties of these two mutants along with wild type controls, and use western blotting to show that both mutant enzymes retain their respective wild type enzyme antigenicity. Structural data obtained by X-ray crystallography on the α-GAL mutant (called α-GALSA ) reveals the mechanism by which substrate specificity is dictated between these two proteins, and provides explanations for the mutant’s reduced catalytic efficiency.

Machine learning is applied to a challenging and biologically significant protein classification problem: the prediction of flavonoid UGT acceptor regioselectivity from primary protein sequence. Novel indices characterizing graphical models of protein residues are introduced. The indices are compared with existing amino acid indices and found to cluster residues appropriately. A variety of models employing the indices are then investigated by examining their performance when analyzed using nearest neighbor, support vector machine, and Bayesian neural network classifiers. Improvements over nearest neighbor classifications relying on standard alignment similarity scores are reported.

Microsporidia are spore-forming, obligate intracellular pathogens typically associated with opportunistic infections in immunocompromised individuals. Treatment options for microsporidia infections in humans are limited and additional research is necessary to create better therapeutic agents. For many pathogenic organisms, adhesion to the host cell surface is a prerequisite for tissue colonization and invasion. Our previous research has demonstrated a direct relationship between adherence of microsporidia spores to the surface of host cells and infectivity in vitro. In an effort to better understand adherence, we have turned our attention to determining what proteins may be involved in this process. Examination of the Encephalitozoon cuniculi genome database revealed a gene encoding a protein with sequence homology to members of the ADAM (a disintegrin and metalloprotease) family of type I transmembrane glycoproteins. The microsporidia ADAM (MADAM) protein is of interest because ADAMs are known to be involved in a variety of biological processes including cell adhesion, proteolysis, cell fusion, and signaling. The objectives for this study were to examine the localization of MADAM, analyze its potential involvement during adherence and/or host cell infection, and to identify potential binding partners or substrates. Through the use of immunoelectron transmission microscopy, we demonstrated that MADAM is localized to the surface exposed exospore, plasma membrane, and the polar sac-anchoring disk complex (a bell-shaped structure at the spore apex involved in the infection process). Location of MADAM within the exospore and polar sac-anchoring disk suggests that MADAM is in a position to facilitate spore adherence or host cell infection. Thus far, we have been unable to conclusively demonstrate that MADAM is involved in either event. Through the use of a yeast two-hybrid system, we were able to identify polar tube protein 3 (PTP3) as a potential binding partner or substrate for the MADAM protein. The interaction between MADAM and PTP3 was confirmed by in vitro co-immunoprecipitation. PTP3 is hypothesized to be involved in the process of polar tube extrusion by stabilizing the interaction between PTP1-PTP2 polymers. Further analysis of the interaction between MADAM and PTP3 may lead to a better understanding of the events that occur during polar tube extrusion.

Huntington’s disease is an incurable, progressive neurological disorder characterized by loss of motor control, psychiatric dysfunction, and eventual dystonia leading to death. Despite the fact that this disorder is caused by a mutation in one single gene, there is no cure. The mutant Huntingtin (mHtt) protein is expressed ubiquitously throughout the brain but frank cell death is limited to the striatum. Recent work has suggested that Rhes, Ras homolog enriched in striatum, which is selectively expressed in the striatum, may play a role in Huntington’s disease neuropathology. In vitro studies have shown Rhes to be an E3 ligase for the post-translational modification protein SUMO. Rhes increases binding of SUMO to mHtt which competes for the same binding site as Ubiquitin. SUMOylation of mHtt leads to disaggregation and cellular death, whereas ubiquitination leads to aggregation and cellular protection. In a previous study we showed that deletion of Rhes caused a decrease in the Huntington’s disease phenotype in mice. We hypothesized that mice lacking Rhes would also show increased aggregation in the striatum and this increased aggregation would correlate in a rescue of behavioral symptoms. Despite the prior in vitro and in vivo evidence, deletion of Rhes in vivo did not alter the aggregation of mHtt in the striatum of mice however deletion of Rhes still showed a rescue from the diseased phenotype. This result would indicate that deletion of Rhes alters the neurobehavioral phenotype of Huntington’s disease through a different pathway than promoting aggregation in striatal cells.

The insulin-like growth factor-1 receptor (IGF-1R) and many of its downstream signaling components have long been implicated in tumor progression and resistance to therapy. The insulin receptor substrate-1 (IRS-1) and IRS-2 adaptor proteins are two of the major downstream signaling intermediates of the IGF-1R. Despite their considerable homology, previous work in our lab and others has shown that IRS-1 and IRS-2 play divergent roles in breast cancer cells. Signaling through IRS-1 promotes cell proliferation, whereas signaling through IRS-2 promotes cell motility and invasion, as well as glycolysis. Moreover, using a mouse model of mammary tumorigenesis, our lab demonstrated that IRS-2 acts as a positive regulator of metastasis, while IRS-1 cannot compensate for this function. The focus of my thesis research is to understand how IRS-2, but not IRS-1, promotes breast carcinoma cell invasion and metabolism to support metastasis. In preliminary studies, I have found that IRS-1 and IRS-2 exhibit different expression patterns in both cell lines and human tumors with correlations to patient survival, which provides a potential mechanism for their distinct functions. The localization of IRS-1 and IRS-2 within separate intracellular compartments would determine their access to downstream effectors and substrates, and this would result in unique cellular outcomes. Specifically, I have observed that IRS-2, but not IRS-1, co-localizes with microtubules in breast carcinoma cell lines with implications for signaling through AKT and mTORC2. The goal of this research is to determine how the localization of IRS-2 contributes to its regulation of breast cancer progression and response to therapy and how this information could be used to better predict patient outcomes.

The development of early diagnostics and prevention strategies for preterm birth is an important global health challenge with the potential to impact over 15 million children annually, by improving health outcomes and reducing economic burden. Advances in microbial sequencing technology have opened the door to 16S rRNA gene survey, whole metagenomics, and whole transcriptomics, providing molecular evidence that the composition of the vaginal microbiome affects pregnancy outcomes in women, particularly those of African descent. A current gap in our molecular level understanding of the vaginal microbiome as it relates to healthy pregnancies is the metaproteome which comprises proteins from both the woman and colonizing microorganisms. Herein, I describe the development of a label-free mass spectrometry-based workflow for preparing and analyzing the vaginal metaproteome as sampled from vaginal swab extracts. The workflow was applied to two longitudinal cohorts of vaginal swab samples collected during the VCU MOMS-PI study. The work presented herein demonstrates for the first time that sufficient vaginal-specific biomaterial can be extracted from swabs for metaproteomics analysis as evidenced by high proteome coverage (>1790 human and >1609 microbial proteins), quantitative readouts for over 37% of identified proteins, and the identification of candidate protein biomarkers that change with gestational age and parturition status.