Browsing by Subject "Drosophila"
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Item A molecular analysis of Src64 and its impact on cytoskeletal reorganization in the Drosophila embryo(2007-05) Strong, Taylor Clinton; Thomas, Jeffrey; Webster, Daniel R.; Williams, Simon C.Src-family kinases are involved in many processes related to development. As regulators of development, Src-family kinases are involved in cell motility, cell proliferation, and neo-angiogenesis. Src-family kinase activity has also been implicated in a number of cancers. Src64 in Drosophila encodes a non-receptor tyrosine kinase that is homologous to human Src family kinases. Src64 is involved in processes related to cell proliferation and apoptosis. Additionally, specific roles for Src64 have been identified in ring canal growth, dorsal closure, and salivary gland invagination. Our lab is specifically looking at the role of Src64 in microfilament ring constriction during cellularization. The Src64∆17 mutation deletes the first two exons of the gene and eliminates all but trace amounts of Src64 protein, suggesting that there is an alternate transcriptional start site that is unaffected by the Src64∆17 mutation. This alternate transcript may be responsible for the phenotypic differences observed between Src64 deficient flies and Src64∆17 flies. Semi-quantitative RT-PCR confirmed the presence of low levels of Src64 mRNA in Src64∆17 embryos. Using 5’ RACE analysis of Src64∆17 embryos we located the alternate transcriptional start site and identified a previously unknown exon located 13,100 base pairs downstream of the primary transcriptional start site, and 12,113 bp downstream of the two exons deleted in Src64∆17. To further investigate the role of Src64 in development we identified ten missense mutations in the Src64 coding region. This approach has allowed us to isolate several Src64 mutations in an unbiased manner that disrupt the SH2 and tyrosine kinase domains. We analyzed embryos affected by these Src64 mutations for defects related to Src64 activity. In our analysis we identified defects to the cellularization front and microfilament rings of varying strength depending on the specific Src64 mutation.Item Determination of how miRNAs mediate repression in Drosophila and the essential role of the oskar mRNA in egg chamber development(2009-12) Reich, John Curtis; Macdonald, Paul M.; Fischer, Janice; Stein, David; Sullivan, Christopher; Gross, JeffreymiRNAs are important regulators of gene expression. These small RNAs function throughout development and regulate translation of a number mRNAs. miRNAs exert their affect on translation as part of the RNP complex RISC. RISC can affect translation of transcripts at both the level of translation initiation, and post-initiation. Although mechanisms of repression mediated by miRNAs have been intensively studied, repression is not well characterized. In order to understand how miRNAs regulate translation in Drosophila, we first characterized miRNA-mediated repression in the ovary. We developed an ovarian assay sensitive to regulation by miRNAs and found that regulated transcripts localize to cytoplasmic puncta distinct from sponge bodies, cytoplasmic RNP structures consisting of proteins implicated in miRNA-mediated regulation. In addition, we devised a genetic screen to identify genes involved in miRNA-mediated regulation. Seven mutants were isolated from the screen, and two mutants were subsequently mapped to separate 1Mb genomic regions. Both these regions are devoid of genes implicated in miRNA-mediated regulation, suggesting our mutants identify novel components involved in repression. The oskar mRNA encodes for the Oskar protein, which is vital in establishing the posterior axis of the Drosophila embryo. In addition to its protein coding function, the osk mRNA has another essential role: it is required for egg chamber progression through oogenesis. This role of oskar is mediated by its 3ʼ UTR, but how it functions in this role is unknown. Here, we investigate the function of the 3ʼ UTR and discover that the well-defined BRE sequences are required for egg chamber progression through oogenesis. The BREs mediate translational repression of the highly regulated oskar mRNA and were previously defined by their ability to bind Bruno, which represses translation of the oskar mRNA. We also provide evidence that the osk BREs sequester Bruno, potentially inhibiting Bruno from binding and misregulating other mRNAs. Our results suggest a novel regulatory loop, where oskar sequesters and inhibits Bruno from misregulating mRNAs, and Bruno, in turn, regulates translation of the oskar mRNA.Item Elucidating the Functions of the Sialylation Pathway in Drosophila melanogaster(2012-10-19) Carnahan, MindySialylation is an important carbohydrate modification of glycoconjugates, which introduces sialic acids (SA). The relatively large nine-carbon, negatively charged sugars are typically located at the termini of carbohydrate chains. SA's are often required for functionally important molecular and cellular interactions including virus-host interactions, tumor progression and malignancy, immune system development and function, and nervous system development and function. However, the study of sialylation in vertebrates, including man, encounters serious obstacles associated with the complexity of vertebrates' biology and limitations of available experimental approaches. Drosophila is a useful model system with many advantages including quick generation time, a large number of progeny, simplified glycosylation and neurophysiology, and ease of genetic manipulations. The primary focus of this thesis is on the functions of Drosophila melanogaster CMP sialic acid synthetase (DmCSAS) and sialyltransferase (DSiaT) in the central nervous system (CNS). A combination of genetic, immunostaining, and neurobiology approaches were used to characterize the functions of DmCSAS and DSiaT in Drosophila. This investigation revealed the expression of DmCSAS and suggested that it plays an important role in a specialized and developmentally regulated process in the nervous system of Drosophila. Further experiments examined sub-cellular localization of DmCSAS revealing that this protein has a complex mostly Golgi-associated distribution within the cell in vivo. I discovered a novel link between Drosophila sialylation and circadian rhythm regulation. I also characterized the electrophysiological phenotypes of DmCSAS mutants and compared them to the corresponding defects associated with DSiaT mutations. My experiments also revealed that the relationship between DmCSAS and DSiaT are more complex than originally thought; these genes may have independent functions while also participating in the same pathway. Taken together, these results elucidate the sialylation pathway in Drosophila and shed more light on the role of sialylation in the nervous system. My experiments provide a unique evolutionary perspective on the sialylation pathway in animals and suggest that the neural function of SA in Drosophila can be conserved in vertebrates, including humans.Item Ethanol dependence in Drosophila larvae(2013-08) Robinson, Brooks Gregory; Atkinson, Nigel (Nigel S.)Addiction to alcohol is a disease of changed behavior that is uniquely human in it's complexity. Because of this, researchers have strived to develop animal models of individual endophenotypes of alcoholism in hopes that the larger picture will eventually come into focus. Recent studies in Drosophila have shown that many complex alcohol-related behaviors are conserved in this genetic model system. The series of projects presented in this dissertation outline the first account of physiological ethanol dependence in Drosophila. We first show that Drosophila larvae are able to form conditioned associations between an aversive heat stimulus and an attractive odor. We then show that an acute, low-dose ethanol exposure disrupts this learning ability. Finally, we present data that demonstrate that larvae adapt to the presence of chronic ethanol to the point that they only perform normally in the learning assay when ethanol is present in the animal. We then propose that the major mechanism for this dependence involves ethanol regulating the acetylation level and therefore expression level of a large number of genes by inhibiting histone deacetylase enzymes. These experiments set the groundwork for the analysis of a network of genes, connected through interactions with histone deacetylase enzymes, that are involved in producing ethanol dependence.Item Fitting It All Together: How Courtship- and Mating-Responsive Genes Affect Drosophila melanogaster Male Behavior(2011-10-21) Ellis, Lisa LynnBehavior is a complex process resulting from the integration of genetic and environmental information. Thus, the genetically tractable Drosophila melanogaster was utilized to better understand the interplay between these factors since Drosophila males and females exhibit sex-specific courtship behaviors that are innate yet modifiable. These sex-specific behaviors, as well as sexually dimorphic development, are regulated, in part, by the somatic sex-determination hierarchy. Since reproductive behaviors rely on the rapid integration of multiple sensory cues, it is likely that the perception and integration of such cues and mating-induced physiological changes are mediated in part by changes in gene expression. Therefore, it was hypothesized that assaying gene expression changes in response to courtship or mating in Drosophila males would uncover new targets of the sex-determination hierarchy and other behaviorally important loci. We took a novel approach to find these behaviorally-responsive loci by utilizing microarray technology to assess courtship- or mating-induced gene expression changes in Drosophila male whole bodies or heads. Mutations in candidate loci were tested for effects on reproductive behaviors and present the first data showing that egghead (egh) and female-specific independent of transformer (fit) affect male reproductive behavior. egh is up regulated in male heads 20 min after courting and is required post-developmentally in a subset of neurons for robust male courtship behavior. fit, a fat body-expressed sex-determination hierarchy target gene, is up regulated in male whole bodies after 5 min of courtship. fit is also up regulated in male heads after 20 min of courtship or 2 hrs after mating. Mutations in fit result in male-male courtship; more specifically, fit mutants direct courtship towards males and also elicit courtship from wild-type males. By analyzing fit's role in courtship behavior, we also shed light on the role the fat body plays in modulating behavior. These studies provide the first pieces of evidence that gene expression changes occur in Drosophila males performing reproductive behaviors. This novel approach identified behaviorally important loci that are expressed in the nervous system and the fat body, indicating that both tissues modulate behavior. Also identified were sex-determination hierarchy target genes and it is likely that further analysis of the remaining candidates will reveal more members of this genetic cascade.Item Functional characterization of the role of Imp, a Drosophila mRNA binding protein, during oogenesis(2006-12) Geng, Cuiyun; Macdonald, Paul M.Establishment of cell polarity requires the involvement of several posttranscriptional regulatory mechanisms, including mRNA localization and translational control. A family of highly conserved RNA binding proteins in vertebrates, VICKZ (V̲g1RBP/V̲era, I̲MP-1, 2, 3, C̲RD-BP, K̲OC, Z̲BP-1) proteins, has been shown to act in these two processes. Previous studies of the posttranscriptional mechanisms mediated by VICKZ family members have been largely limited by the lack of genetic approaches in certain vertebrate systems. Identification of Imp, the Drosophila member of the VICKZ family, opened the possibility to use genetic approaches to investigate the roles of a VICKZ family member in mRNA localization and translational control. In this dissertation, we show that Imp is associated with Squid and Hrp48, two heterogeneous proteins (hnRNP) that complex with one another to regulate localized expression of gurken (grk). In addition, Imp binds grk mRNA with high affinity in vitro and is concentrated at the site of grk localization in midstage oocytes. Mutation of the Imp gene does not substantially alter grk expression, but does partially suppress the grk mis-expression phenotype of fs(1)k10 mutants. In contrast, overexpression of Imp in germ line cells results in mislocalization of grk mRNA and protein. The opposing effects of reduced and elevated Imp activities on grk expression suggest that Imp acts in regulation of grk expression, but in a redundant way. To further explore the mechanisms by which localized expression of grk is regulated by Imp, a deficiency screen was conducted to search for dominant modifiers of the dorsalized phenotype resulting from Imp overexpression. Twelve genomic regions were identified to contain dominant modifiers of the Imp overexpression phenotype. Further characterization of mutants of genes within these genomic regions led to identification of five modifiers, including cyclin E (cycE), E2f transcriptional factor 1 (E2f1), lingerer (lig), snail (sna) and mushroom body expressed (mub). E2f1 encodes a transcriptional factor that is involved in regulating the G1 to S phase transition during mitosis. Mutation of E2f1 results in altered grk mRNA and protein distribution within oocyte, revealing a role for this gene in regulation of grk expression.Item Group II intron and gene targeting reactions in Drosophila melanogaster(2011-08) White, Travis Brandon; Lambowitz, Alan; Bull, James J.; Macdonald, Paul M.; Paull, Tanya T.; Stevens, Scott W.Mobile group II introns are retroelements that insert site-specifically into double-stranded DNA sites by a process called retrohoming. Retrohoming activity rests in a ribonucleoprotein (RNP) complex that contains an intron-encoded protein (IEP) and the excised intron RNA. The intron RNA uses its ribozyme activity to reverse splice into the top strand of the DNA target site, while the IEP cleaves the bottom DNA strand and reverse transcribes the inserted intron. My dissertation focuses on the Lactococcus lactis Ll.LtrB group II intron and its IEP, denoted LtrA. First, I investigated the ability of microinjected Ll.LtrB RNPs to retrohome into plasmid target sites in Drosophila melanogaster precellular blastoderm stage embryos. I found that injection of extra Mg2+ into the embryo was crucial for efficient retrohoming. Next, I compared retrohoming of linear and lariat forms of the intron RNP. Unlike lariat RNPs, retrohoming products of linear intron RNPs displayed heterogeneity at the 5’-intron insertion junction, including 5’-exon resection, intron truncation, and/or repair at regions of microhomology. To investigate whether these junctions result from cDNA ligation by non-homologous end-joining (NHEJ), I analyzed retrohoming of linear and lariat intron RNPs in D. melanogaster embryos with null mutations in the NHEJ genes lig4 and ku70, as well as the DNA repair polymerase polQ. I found that null mutations in each gene decreased retrohoming of linear compared to lariat intron RNPs. To determine whether novel activities of the LtrA protein contributed to the linear intron retrohoming 5’ junctions, I assayed the polymerase, non-templated nucleotide addition and template-switching activities of LtrA on oligonucleotide substrates mimicking the 5’-intron insertion junction in vitro. Although LtrA efficiently template switched to 5’-exon DNA substrates, the junctions produced differed from those observed in vivo, indicating that template switching is not a significant alternative to NHEJ in vivo. Finally, I designed and constructed retargeted Ll.LtrB RNPs to site-specifically insert into endogenous chromosomal DNA sites in D. melanogaster. I obtained intron integration efficiencies into chromosomal targets up to 0.4% in embryos and 0.021% in adult flies. These studies expand the utility of group II intron RNPs as gene targeting tools in model eukaryotic organisms.Item In Pursuit of a Molecular Fountain of Youth: the Identification and Characterization of Lifespan Regulators in Drosophila(2012-07-20) Stenesen, Drew Stanness; Graff, Jonathan M.Over the past century, average human lifespan has experienced steady increase despite lack of substantial intervention or understanding of the aging process. In fact, many organisms have the latent potential to live much longer than they normally do. This indicates lifespan determination is an active process subject to regulation. Components of this impending longevity are beginning to unravel through dietary and genetic studies in model systems. To date, several pathways indicate human lifespan extension through direct molecular intervention may be feasible, however, important limitations persist. A common thread among these conserved lifespan regulators is metabolism. Therefore, further insight into lifespan extending mechanisms may lie within tissues governing important metabolic processes. Here we describe a multi-tiered, strategy to identify Drosophila melanogaster mutants with extended lifespan based upon enrichment for insertions in genes that are expressed in metabolic tissues. Our results indicate metabolically relevant tissues are a rich source of genetic longevity regulation. We identified a regulator of G protein signaling (RGS) domain containing sorting nexin, termed snazarus (sorting nexin lazarus, snz). Flies with insertions into the 5’ untranslated region of snz live up to twice as long as controls. Transgenic expression of UAS-Snz from the snz Gal4 enhancer trap insertion, active in metabolic tissues, rescued lifespan extension. Notably, old snz mutant flies remain active and fertile indicating that snz mutants have prolonged youthfulness, a goal of aging research. Since mammals have snz-related genes, it is possible that the functions of the snz family may be conserved to humans. Next, we identified the two key adenosine monophosphate (AMP) biosynthetic pathways as regulators of Drosophila longevity. We found that heterozygous mutation of anabolic components of the de novo as well as the salvage AMP biosynthesis pathways extend lifespan. These pathway mutations, and caloric restriction, increased adenosine mono- and diphosphate to adenosine triphosphate (ATP) ratios. Consistent with the altered ratios, lifespan extension was dependent on functional adenosine monophosphate-activated protein kinase (AMPK). Supplementing the diets of adult mutants with adenine restored adenosine nucleotide ratios and rescued lifespan extension. These data establish de novo and salvage AMP biosynthesis as determinants of adult lifespan. The dosage sensitivity and enzymatic nature of de novo and salvage AMP biosynthesis, and the conserved aspects of adenosine nucleotide derivatives and lifespan extension, indicate that these pathways are potentially amendable drug targets worth continued exploration. [Keywords: longevity, Drosophila, genetics, aging, metabolism]Item The innate immune system and alcohol responses in Drosophila(2016-05) Troutwine, Benjamin Robert; Atkinson, Nigel (Nigel S.); Pierce-Shimomura, Jon; Aldrich, Richard; Mihic, S. John; Stein, DavidA growing body of evidence has shown that alcohol alters the activity of the innate immune system and that changes in innate immune system activity can influence alcohol-related behaviors (Cui et al., 2014; Vetreno & Crews, 2014). Here we show that the Toll innate immune signaling pathway modulates the level of alcohol resistance in Drosophila. In humans, a low level of response to alcohol is correlated with increased risk of developing an alcohol use disorder (Schuckit, 1994). The Toll signaling pathway was originally discovered in, and has been extensively studied in Drosophila. The Toll pathway is a major regulator of innate immunity in Drosophila, and mammalian Toll-like receptor signaling has been implicated in alcohol responses. Here, we use Drosophila-specific genetic tools to test eight genes in the Toll signaling pathway for effects on the level of response to ethanol. We show that increasing the activity of the pathway increases ethanol resistance while decreasing pathway activity reduces ethanol resistance. Furthermore, we show that gene products known to be outputs of innate immune signaling are rapidly induced following ethanol exposure. The interaction between the Toll signaling pathway and ethanol is rooted in the natural history of Drosophila melanogaster.Item Investigating the role of Bruno interactions with oskar regulatory proteins(2014-08) Kim, Goheun; Macdonald, Paul M.; Browning, Karen; Fischer, Janice; Stein, David; Stevens, ScottOskar (Osk) is a posterior body-patterning determinant in Drosophila melanogaster and is highly concentrated at the posterior pole of the oocyte. osk mRNA is translationally repressed until it reaches the posterior of the oocyte where Osk protein is made. Bruno (Bru) represses translation during osk mRNA localization by direct binding, but how Bru-mediated repression is relieved at the posterior of the oocyte is unknown. Two types of Bru protein interactions are implicated in repression of osk: Bru-Cup interaction and Bru dimerization. By mapping the Bru domains that are important for these interactions, I found that the amino-terminal domain of Bru contributes to both interactions, and deletion of this domain caused a defect in translational repression. However point mutations, within the amino-terminal domain, that disrupt both types of interaction in vitro did not interfere with translational repression in vivo. The difference may be due to other factors stabilizing the Bru-Cup interaction in vivo, as the mutant Bru still associates with Cup in vivo. My work supports the model of repression that relies on Bru interaction with Cup. I also build a new model in which Bru dimerization promotes translational activation of osk, based on my unexpected results: dimerization-defective Bru only weakly accumulated Osk::GFP fusion protein encoded by an osk::GFP reporter RNA bearing a Bru-binding region, while dimerization-competent Bru showed the opposite effect. This suggests that dimerization may contribute to switching Bru from a repressor to an activator, with dimerization controlled via a post-translational modification. Consistent with this, I found that a small fraction of Bru in ovaries is phosphorylated. PKA is a positive regulator of osk expression and phosphorylates Bru in vitro. To test if PKA regulation of osk is mediated through Bru, I examined the effect of altering PKA activity on Bru phosphorylation and Bru-mediated repression. Modulating PKA activity caused small, yet detectable changes in Bru phosphorylation and Bru-dependent translational repression using an osk::GFP reporter. However, while the studies with Bru mutants suggest that phosphorylation promotes repression by Bru, these studies argue for a role in promoting activation. Further work will be required to explain these phenomena.Item Investigation of the role of VML in the establishment of Drosophila embryonic dorsal-ventral polarity(2012-08) Zhang, Yuan, M.A. in Cellular and Molecular Biology; Stein, David S.; Fischer, Janice A.Drosophila embryonic dorsal-ventral (D-V) polarity is defined by the expression of the gene pipe in the ventral cells of the follicular epithelium surrounding the developing oocyte. pipe encodes a sulfotransferase that transfers sulfate groups to several protein components of the vitelline membrane layer of the eggshell, including Vitelline Membrane-Like protein (VML). These sulfated proteins represent a ventral cue embedded in the eggshell, which, during embryogenesis, leads to the spatially-restricted activation of a serine protease cascade involving Gastrulation Defective (GD), Snake and Easter. Several important pieces of information missing from our understanding of Drosophila D-V patterning include the structures of the carbohydrates borne by the Pipe targets that represent the direct substrates for Pipe and how the Pipe-sulfated ventral cue triggers the spatially-regulated activation of the serine protease cascade. Two major goals of my studies on VML are to elucidate the structures of Pipe-sulfated carbohydrates associated with VML and to identify proteins that interact with VML in a Pipe-dependent manner. To achieve the first goal, I explored different purification systems to isolate VML and eventually found a way of partially purifying VML from Drosophila ovaries. Mass spectrometric analysis of the purified VML is underway to determine the carbohydrate structures on VML and the sites of Pipe-mediated sulfation. Future experiments will involve identification of putative enzymes responsible for the glycosylation of VML and examination of their requirements in D-V patterning. To identify interacting partners of VML, a strategy combining in vivo biotinylation of VML, reversible protein crosslinking and Streptavidin purification of crosslinked complexes will be used. In the second section of my studies, I have demonstrated that VML bearing a biotin acceptor peptide (BAP) tag can be efficiently biotinylated in vivo by co-expressing it with a biotin protein ligase BirA in the follicle cells. As an extension of the application of the approach, I also show that the Torso-like protein, which is localized to the poles of the vitelline membrane and whose polar localization is crucial for Drosophila terminal patterning, can also be biotinylated in vivo when the BAP-tagged protein is co-expressed with BirA in the follicle cells.Item Mathematical modeling of Drosophila larvae crawling(2013-12) Guenther, Maximilian Norbert; Shubeita, George T.; Florin, Ernst-LudwigThe fruit fly Drosophila melanogaster is a widely used model organism for development and gene-based diseases. The flies’ genetic homology to humans, tractable genetics and straightforward manipulability make them well suitable for studies of neurologic disorders and neural degeneration. Due to the close relation between the latter and the musculoskeletal system, locomotive impairment and behavioral changes can be used diagnostically for screening and characterization of such disease models. For this purpose, advanced methods to quantify behavioral phenotypes are crucial. Given the complications arising with studying adult flies on a population level as well as the lethality of some mutations before adulthood, studies at the larval stage are more suitable. However, a quantitative mathematical model of the crawling pattern has been lacking so far. In this thesis work I show that the development of such a model and appropriate analysis techniques enable quantification of the crawling behavior and extraction of intricate details that were previously missed. In my studies, Drosophila larvae were found to follow a bimodal persistent random walk pattern, switching between an actual forward crawling phase and events where the larvae rest and reorient. This enabled quantifying the larval behavior using a set of parameters within the framework of this mathematical model. I further used the analysis I developed to study larval model systems of Alzheimer’s disease and Fragile X mental retardation, which allowed identifying differences in the modes of locomotion that were previously missed. The novel ability to sensitively and robustly quantify behavior, as described in this work, opens up the possibility to employ these methods for future drug and genetic screens. Finally, I show that starting from the analysis of a small sample of crawling larvae we can robustly simulate the mutant-specific crawling in its quantitative and qualitative aspects. Using these simulations, predictions can be made on the feasibility of experiments that may require an impractically large number of individuals to reach statistical significance, and the outcome of laborious experiments can be pre-estimated by simulations.Item Measuring molecular motor forces to probe transport regulation in vivo(2012-05) Leidel, Christina Paulette; Shubeita, George T.The cell relies on molecular motor proteins for long range transport of vesicles and organelles to maintain the organization required within the cell as it changes over time. Cargos move bidirectionally along microtubules due to the presence of multiple copies of opposite polarity motors. Individual motor properties have been teased out in vitro, but understanding how multiple motors cooperate in vivo has thus far been limited by many obstacles. The goal of this work is to study how multiple similar and dissimilar motors operate together in vivo. Since the function of motors is to generate force to haul cargos, I designed a novel optical trapping system capable of precisely measuring the forces exerted by molecular motors in their native environment, a living cell. Using this system, I find evidence that motors do not fight against each other, supporting the regulation model over the tug-of war model for bidirectional transport. I then study motor regulation in axons in the context of Alzheimer’s disease. I find that GSK-3, a kinase found in abnormal amounts in Alzheimer’s brains, is a negative regulator of transport. I show that GSK-3 regulates motor activity rather than cargo binding. Finally, I also use the optical trap to probe the viscosity of cytosol in vivo and investigate its implications on the cooperation of multiple motors.Item Mechanisms of benzyl alcohol tolerance in Drosophila melanogaster(2009-12) Alhasan, Yazan Mahmoud; Atkinson, Nigel (Nigel S.); Zakon, Harold H.; Gonzales, Rueben A.; Singer, Michael C.; Bergeson, Susan E.Proper neuronal function requires the preservation of appropriate neural excitability. An adaptive increase in neural excitability after exposure to agents that depress neuronal signaling blunts the sedative drug effects upon subsequent drug exposure. This adaptive response to drug exposure leads to changes in drug induced behaviors such as tolerance, withdrawal and addiction. Here I use Drosophila melanogaster to study the cellular and neuronal components which mediate behavioral tolerance to the anesthetic benzyl alcohol. I demonstrate that rapid tolerance to benzyl alcohol is a pharmacodynamic mechanism independent of drug metabolism. Furthermore, tolerance is a cell autonomous response which occurs in the absence of neural signaling. Using genetic and pharmacological manipulations I find the synapse to play an important role in the development of tolerance. In addition, the neural circuits that regulate arousal and sleep also alter benzyl alcohol sensitivity. Beyond previously described transcriptional mechanisms I find a post-translational role of the Ca2+-activated K+-channel, slowpoke in the development of tolerance. Finally, I explore a form of juvenile onset tolerance, which may have origins that differ from rapid tolerance. The implications of this study go beyond tolerance in Drosophila melanogaster to benzyl alcohol and can shed light on human drug tolerance, withdrawal and addiction.Item Persistence of Adh gene in the environment, in Drosophila pupae, and in the mite Proctolaelaps regalis(Texas Tech University, 1997-05) Chasteen, Leslie A.There is substantial evidence supporting the horizontal gene transfer of DNA (P mobile elements) from Drosophila willistoni to Drosophila melanogaster. A potential vector of this transfer is the mite F^octolaelaps regalis. The mite is hypothesized to acquire DNA while feeding on flies and to carry and transmit that DNA to subsequent prey. This research concerns the persistence and longevity of DNA in the respective environmental components. Separate experiments were run testing: (1) the persistence of DNA in the environment using agar blocks as the substratum, (2) the persistence of DNA in Drosophila pupae after microinjection of the DNA, and (3) the persistence of DNA in the mite, Proctolaelaps regalis after feeding. A surrogate for the P element was used in the experiments in the form of a recombinant plasmid with a mammaUan alcohol dehydrogenase insert (Adh-V). The Adh-V signal was detected in the environment up to and including 14 days, in developing pupae up to the time of emergence and in emerged adults, and in the mite up to and including 60 hours. The data generated from these experiments provides estimates of the time frame in which a mite can potentially acquire, retain, and be competent to transfer genetic material among eukaryotes upon which it feeds.Item Regulation of DIAP1 function by Dropsophila Omi and the N-end rule pathway(2007-12) Malladi, Madhavi, 1976-; Bratton, Shawn B.The molecular mechanisms of apoptosis are evolutionarily-conserved with caspases being the chief executioners of this process. Though key regulators of apoptosis, including caspases, inhibitor of apoptosis (IAP) proteins, and IAP antagonists exist in both mammals and flies, there are reportedly mechanistic differences in the way the apoptotic process is executed. One of the differences pertains to the importance of mitochondrial permeabilization for caspase activation. Herein, we demonstrate that dOmi, a Drosophila homologue of the serine protease Omi/HtrA2, is a developmentallyregulated mitochondrial intermembrane space protein that undergoes processive cleavage in situ to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the pro-apoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. Thus, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis, but also results in the release of a bona fide pro-apoptotic protein. DIAP1, in addition to being regulated by dOmi, is also regulated by RINGdependent autoubiquitination and by the N-end rule degradation (NERD) pathway. Despite decreasing the cellular levels of DIAP1, the NERD pathway enhances its antiapoptotic function through an unknown mechanism(s). Herein, we show for the first time that the NERD pathway facilitates trans-ubiquitination and degradation of IAP antagonist bound to DIAP1. Indeed, Grim is trans-ubiquitinated in an Ubr1-dependent manner and requires its interaction specifically with the BIR1 domain of DIAP1. These results demonstrate that similar to RING domain-dependent ubiquitination, the NERD pathway regulates not only the levels of DIAP1, but also of the levels of IAP antagonists bound to it.Item The role of circadian genes in tolerance to ethanol in Drosophila melanogaster(2011-12) Pohl, Jascha Benjamin, 1981-; Atkinson, Nigel (Nigel S.); Harris, Adron; Singer, Michael C.; Pierce-Shimomura, Jonathan T.; Zakon, Harold H.Alcoholism is a devastating inheritable disease that causes a large fiscal and societal impact worldwide. The fruit fly, Drosophila melanogaster, has proven to be a useful model system in identifying genetic factors underlying ethanol-associated behaviors. Many genes have been identified in the fly that are involved in the acquisition of tolerance, or the reduced response of an effect of a drug caused by previous exposure. In this thesis, I investigated whether circadian genes are involved in tolerance to ethanol in the fly. Circadian genes had previously been implicated in cocaine sensitization in flies, as well as some ethanol responses in mammals. I developed a novel assay using a bootstrapping paradigm to analyze tolerance to ethanol in the fly that allows for the investigation of multiple components of tolerance. I then used this assay to test whether circadian genes were necessary for the acquisition of tolerance in the fly. Interestingly, only some circadian genes affect tolerance to ethanol. These results argues that circadian genes are involved in tolerance to ethanol, but because some mutants are arrhythmic but still acquire tolerance, that they are acting in a role outside of the circadian system. While in the course of this work, I intended to investigate if these mutations affected ethanol preference in a two-choice assay. Before employing this assay, however, we wished to determine if flies prefer ethanol for its pharmacological effect or for its value as a food. Weperformed experiments in which flies had a choice between food supplemented with ethanol and food supplemented with an isocaloric carbohydrate. When presented with the isocaloric alternative, flies no longer demonstrated preference for ethanol. Flies will even stop showing preference for ethanol when switched to a balanced assay after preference has already been attained. We conclude that the flies prefer ethanol not because of its effect as a drug, but as a food source.Item The role of Dropsophila auxilin in Notch signaling(2007-12) Eun, Suk Ho, 1973-; Fischer, Janice AnnThe goal of my graduate study is to understand the role of endocytosis for signaling receptor activation during development, especially ligand endocytosis for Notch activation. Notch is a transmembrane receptor which is conserved in metazoans. I am using the Drosophila model system. Notch is required in almost every developmental context and abnormality in Notch signaling components is related to many human diseases. Delta, one of the Notch ligands, is also a transmembrane protein. To activate Notch, endocytosis of Delta in the signaling cells is essential. However, the exact mechanism of how Delta endocytosis regulates Notch activation is not known. Liquid facets (Lqf) is an endocytic protein, called epsin in vertebrates, which is required only in the signaling cells for Delta endocytosis and Notch activation. Overexpression of Lqf in the eyes results in malformed eyes. Using this phenotype as a background, an EMS-mutagenesis screen was performed and auxilin mutants were isolated as enhancers of the eye phenotype. Auxilin is a J-domain protein involved in fission and uncoating of clathrin-coated vesicles. Mosaic clonal analysis showed that auxilin functions in Notch activation and that auxilin is required only in the signaling cells. The auxilin mutant phenotype was suppressed by addition of a clathrin heavy chain transgene. This result suggests that the auxilin phenotype is at least partly caused by clathrin depletion and that auxilin generates a pool of free clathrin which is required for Delta endocytosis. Auxilin is a multi-domain protein. Two C-terminal domains, the clathrin-binding and the J domains, are sufficient to function as auxilin in Drosophila. One of the popular models to explain why Delta endocytosis is required in the signaling cells is the 'recycling model' in which inactive Delta is endocytosed and recycled to the plasma membrane in active form. Rab11 is a small GTPase that regulates recycling. If the recycling model is correct, rab11 mutants may show a phenotype similar to auxilin, lqf and Delta mutants. The rab11 hypomorphs or expression of rab11 dominant negative result in fewer photoreceptor cells and less Delta protein in the eye. These phenotypes are the opposite of typical mutant phenotypes of Notch components. The rab11 mutant phenotype argues against the recycling model.Item The role of Fragile X mental retardation protein in Drosophila cleavage furrow formation(2009-12) Monzo, Kate Frances; Sisson, John Charles; Macdonald, Paul M.; Fischer, Janice A.; Johnson, Arlen W.; Wallingford, John B.Reduced activity of Fragile X mental retardation protein (FMRP) in brain neurons results in the most common form of heritable mental retardation in humans, Fragile X Syndrome (FXS). FMRP is a selective RNA-binding protein that is implicated in the translational regulation of specific mRNAs in neurons. Although very few direct targets of FMRP have been identified and verified in vivo, FXS is thought to result from the aberrant regulation of potentially hundreds of mRNAs causing defects in neuron morphology and synapse function. Identifying additional targets will be important for elucidating the mechanism of FMRP regulation as well as the etiology of FXS. Drosophila melanogaster offers a unique and powerful system for studying the function of FMRP. Flies with loss of FMRP activity have neuronal and behavioral defects similar to those observed in humans with FXS. Importantly, FMRP regulates common target mRNAs in neurons in both mice and flies. Here, I will describe our discovery of a previously unknown requirement for Drosophila FMRP (dFMRP) during the cleavage stage of early embryonic development. First, we identified a requirement for dFMRP for proper cleavage furrow formation and found that dFMRP functions to regulate the expression of specific target mRNAs during the cleavage stage. Among these is trailer hitch (tral) mRNA, which encodes a translational regulator as well, and represents a new in vivo target of dFMRP translational regulation. In addition, I have identified twenty-eight proteins that change in expression in the absence of dFMRP using a comparative proteomics based screen for dFMRP targets. One of these is the Chaperonin containing tcp-1 complex (CCT), a previously unidentified target, which I found is itself also required for cleavage furrow formation. Finally, we have identified a new dFMRP protein-binding partner, Caprin, and found that together dFMRP and Caprin are required for the proper timing of the MBT. This set of work has led to a better understanding of the mechanism of dFMRP-dependent regulation of cellular morphogenesis in early embryos and has the potential to lead to a better understanding of the etiology of FXS.Item The role of liquid facets-Related in Drosophila development(2011-12) Lee, Ji-Hoon, active 2011; Fischer, Janice A.The goal of my graduate research is to find the role of a Drosophila gene, liquid facets-Related, encoding an ENTH (Epsin N-term homology) domain protein using developing Drosophila eye as a model system. The ENTH domain is a well-conserved globular domain with affinity to phosphoinositides, and found in endocytic Epsins and Golgi Epsins. With the ENTH domain and peptide motifs, such as clathrin binding motifs and other protein binding motifs, endocytic Epsins and Golgi Epsins are localized to the plasma membrane and the Golgi membrane, respectively. The main function of Epsins is to facilitate clathrin-dependent vesicle formation. An interesting finding from endocytic Epsin research using Drosophila is that this seemingly generic factor has in fact a specific role in the Notch signaling pathway by mediating ligand endocytosis which is crucial for receptor activation in the adjacent cell. The role of Golgi Epsin, on the other hand, has not been understood in a multi-cellular context. A former graduate student in our lab, Erin Overstreet, generated loss-of-function mutants of liquid facets-Related and found that this gene is essential for viability and important for cell growth and patterning in Drosophila eye development. Her finding suggests that liquid facets-Related has a specific role in development. She also found that the ENTH domain is dispensable for the function of liquid facets-Related. This is an interesting result because studies using other model organisms show that ENTH domain directly recognizes the cargos, suggestive of essential function it in Golgi Epsin. Therefore, I aimed to figure out what is the function of liquid facets-Related in a multi-cellular context using the Drosophila eye as a model system. To address this, I further characterized the mutant phenotype, screened for dominant modifiers of the hypomorphic eye phenotype, and performed structure/function assays that helped me to generate specific hypotheses and then I tested them. My graduate research contributed to understanding the role of liquid facets-Related by providing the in vivo function, identifying genetic interactions, and specifying the domain necessary and sufficient for its function. First of all, characterization of the mutant phenotype indicated that liquid facets-Related is crucial for proliferation, suppression of apoptosis, insulin receptor-independent cell growth, and progression of the morphogenetic furrow at the D/V midline in the developing Drosophila eye. Secondly, from a forward genetic screen, I found Delta, neuralized, polychaetoid (ZO-1), string (cdc25), and altered disjunction (Mps1) as dominant enhancers of the hypomorphic eye phenotype, suggestive of the role of liquid facets-Related in the Notch signaling pathway and cell cycle regulation. I also found that wingless and armadillo dominantly enhance the hypomorphic phenotype of liquid facets-Related, which suggests that liquid facets-Related has a role in the Wingless signaling pathway. Indeed, the expression of a transcriptional target of the Wingless signal, dachsous, is reduced in liquid facets-Related null cells. Baso-lateral levels of E-cadherin and Armadillo are increased in the liquid facets-Related null cells, which is consistent with the fact that E-cadherin antagonizes the Wingless signal activity. Finally, an unexpected result from structure/function analysis is that exon 6 of the liquid facet-Related gene is necessary and sufficient to rescue all visible morphological defects of null mutants. This is interesting because exon 6 is conserved in Golgi Epsin gene only in several insects but not in most other species including yeast, nematode, mouse, and human. In fact, exon 6 is a homolog of a recently studied gene known as tel2. Evidence suggests that liquid facets-Related is Drosophila tel2. As the function of Tel2 is not clearly understood, this study may contribute to better understand the essential role of Tel2 in Drosophila and other model systems.