Genetic screens provide information on phenotype interactions as a step to uncovering functional interactions in vivo. In this study, I conducted genetic modifier screens in Drosophila that might reveal genes expressed in the embryonic CNS that interact genetically with slit. These screens include a blind genetic screen, stable line double mutant analysis, transheterozygote interactions and a dosage-sensitive dominant modifier screen. Differences in CNS phenotypes between the blind screen and the double mutant analysis were uncovered for some of the crosses, indicating background effects of genotype can influence phenotypes observed. A group of candidate genes were chosen based on motif composition for potential interaction with slit, and spatial and temporal expression patterns coinciding with Slit. The majority of these genes show a mutant CNS phenotype on their own. slit-interacting genes were identified by their ability to alter midline axon guidance, as assayed with antibodies specific to CNS-expressing proteins. The interacting genes include those encoding receptor and second messengers thought to function in growth cones, integrins and extracellular matrix proteins. From these interactions, I could then propose models of function in axon guidance. The first model I propose adds to a currently known repressor-derepressor model of axon guidance at the midline of the CNS, involving the Netrin, Commissureless and Robo signaling molecules, whereby Slit is locally suppressed by Netrin function to allow axons to cross the midline via Comrnissureless-mediated intemalisation of Robo. In my second model, Slit may also be involved in integrin signaling, especially through the αPS3 and βPS integrins, in concert with the Laminin molecule. Included in this model is the intersection of cytoplasmic Dock signaling within the growth cone. Thirdly, I demonstrate a genetic interaction with other molecules expressed by the midline glia, Masquerade, Toll and Neurexin. Masquerade and Toll may function in a common pathway, but in parallel to Slit function. Likewise, Neurexin may function in a parallel pathway to Slit. Slit has shown to interact genetically with a number of molecules that appear to be involved in different aspects of axon guidance. This study was meant to provide a survey of molecules that may functionally interact with Slit and provides a good basis upon which to explore the interactions in detail in future work. / Thesis / Master of Science (MSc)

Understanding the molecular composition of the synapse is one of the ongoing problems in neuroscience. The synaptic proteome consists of about 1100 proteins, most of which are organised into complexes. Understanding the composition and function of these complexes is important for understanding the mechanisms and pathways of diseases which these protein complexes are involved in determining the sub-cellular localisation of these proteins and also their spatial distribution in the nervous system may lead to important insights into the function of such protein complexes thus furthering our understanding of the function and diseases of the nervous system. Neuro-proteomics has led to identification of an NMDA receptor complex (also called MAGUK-associated signalling complex (MASC)) which has been extensively studied in mouse. It consists of ~186 proteins in mouse and represents 10 different functional classes of proteins. Drosophila offers an opportunity to study this complex in more detail because it is 50% less complex while still maintaining all the functional classes of proteins. Since the fundamental structure and principles of neuronal function are conserved between vertebrates and Drosophila the physiology and molecular biology of disease can be better resolved in this simple organism. Here I present an effort to characterise the MASC complex in Drosophila and present the molecular composition of the MASC complex in Drosophila. I will present an anatomical map of the complex in the fly brain and also show that there is significantly high conservation of this complex between Drosophila and mouse. Two different approaches have been used in this thesis to study the molecular composition of this complex give results which are in partial agreement with each other. Importantly it provides an evaluation of these two methods and indicates that they can be used to study molecular complexes in Drosophila.

Memoria para optar al Título Profesional de Médico Veterinario / La correcta ubicación de todos los componentes sinápticos durante el desarrollo requiere del ordenamiento preciso de una red de proteínas, este ordenamiento es provisto por proteínas andamio, pertenecientes a la familia MAGUK (membrane-associated guanylate kinases). Los miembros de esta familia, reclutan receptores, canales iónicos, componentes de cascadas de transducción de señales y proteínas del citoesqueleto a través de sus dominios de interacción proteína-proteína, formando complejos macromoleculares multiproteicos asociados a membrana. Se ha descrito que el gen discs-large, de Drosophila melanogaster, codifica para, al menos, dos proteínas andamio de la famila MAGUK. La sinapsis neuromuscular de la larva de Drosophila es una sinapsis glutamatérgica. En ella la proteína andamio Discs large cumple un papel importante en la formación de la sinapsis y en la localización de proteínas involucradas en la transducción de señales. Gracias a la conservación de los procesos básicos de transmisión sináptica, la sinapsis neuromuscular de la larva de Drosophila melanogaster ha demostrado ser un excelente modelo de estudio de sinapsis centrales de mamíferos. Numerosos estudios han demostrado que Dlg determina la localización sináptica del canal de potasio tipo Shaker y de Fasciclina II (FasII), una proteína de adhesión celular involucrada en el crecimiento y plasticidad de la sinapsis neuromuscular. Además, Dlg participa en múltiples procesos, incluyendo el mantenimiento de la adhesión y polaridad apicobasal de los epitelios y la neurogénesis embrionaria. Dlg también participa en el control de la proliferación en tejido epitelial por lo que se le considera un gen supresor de tumores. Todas estas funciones, están asociadas a una única isoforma, DlgA. Sin embargo, en la sala de moscas del Laboratorio de Neurobiología Celular Molecular de la Facultad de Medicina Norte de la Universidad de Chile se aisló una serie de transcritos que corresponden a variantes de procesamiento alternativo del gen dlg que presentan en su extremo 5’ una región que codifica un segmento de 65 aminoácidos llamado S97N y que está ausente en DlgA. Las variantes de dlg que contienen S97N como la proteína denominada DlgS97, sólo se expresan en sistema nervioso y músculo, a diferencia de DlgA, que también se expresa en células epiteliales. En este estudio se describen los defectos ocasionados por el estrés térmico tanto agudo como crónico en individuos de Drosophila melanogaster que expresan variantes del gen dlg con dominio S97N, para parámetros como viabilidad, fertilidad, conducta locomotora de la larva y del adulto, y en la estructura de la sinapsis de la unión neuromuscular de la larva

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viii, 104 leaves : ill., photos., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1979

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 3 uppsatser.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Medicine, 2005? / "December 2004" Amendments inside back cover. Bibliography: leaves 131-146.

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Biological Sciences. / Includes bibliographical references (leaves 75-82).

Many species of Drosophila have an X chromosome inversion polymorphism: the Standard arrangement and the Sex-Ratio (SR) arrangement. Male carriers of SR transmit predominantly X-bearing sperm and hence produce nearly all-female progeny. In the absence of strong counteracting selection, SR would be meiotically driven to fixation, causing population extinction. In PART I, the role of virility selection in maintaining the SR polymorphism in D. pseudoobscura is examined by partitioning it into two components. The SR males are found to suffer substantial virility reduction which would not be detected if the differences between the two components are not heeded. The significance of this finding is discussed in light of two specific observations usually associated with the Sex-Ratio polymorphism in this species. One is the absence of suppressor modifiers of "sex-ratio" expression and the other is the temperature-dependent distribution of the sex-ratio trait. In PART II, a model is proposed to describe the behavior of the autosomal suppressor modifiers of "sex-ratio" meiotic drive. These modifiers, if neutral in other respects, should increase because they tend to be associated with the rare sex (males). However, selection operating on the sex-linked drive locus will sometimes act against autosomal modifiers as well because the two loci are always in gametic phase disequilibrium. Conditions under which modifiers will not increase are presented in terms of the fitnesses of different genotypes at the sex-linked drive locus. To prevent an increase in modifier alleles, the fitness of Sex-Ratio males relative to Standard males has to be no greater than 0.3. This result is in good agreement with that of PART I. PART III deals with "sex-ratio" genes, tightly linked within the Sex-Ratio inversion. By taking advantage of the fact that the Sex-Ratio chromosome of D. persimilis (SR(B)) is homosequential to the Standard chromosome of D. pseudoobscura (ST(A)), two reciprocal introgression experiments were carried out. Individual segments of SR(B) or ST(A) were introgressed into the genome of D. pseudoobscura or D. persimilis, respectively. Males possessing a hybrid SR(B)-ST(A) X chromosome and a genetic background derived from either of the two species were tested for fertility and "sex-ratio" expression. It was found that, in terms of the meiotic drive genes, the Sex-Ratio chromosome differs extensively from the Standard chromosome. Because recombinations of these genes result in a complete loss of sex-ratio expression, this finding lends strong support to the hypothesis of gene coadaptation. Coadaptation, in this context, is the advantage of being transmitted preferentially. In light of this finding, evolution of the sex-ratio system in these two sibling species is discussed. Introgression experiments also yielded information about hybrid sterility. Four types of sterility interactions were identified; one of them involved at least three genetic elements. With reciprocal introgression, sterility interactions were found to be "asymmetric". The asymmetry is fully expected from the viewpoint of evolution of postmating reproductive isolation. / Medicine, Faculty of / Medical Genetics, Department of / Graduate