Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the cell cycle proceeds. The Chk2 family of kinases plays a central role in mediating responses to DNA damage or DNA replication blocks in various organisms. My functional analysis of the Drosophila serine/threonine kinase Loki/Chk2 shows that fly chk2 monitors double-strand breaks caused by irradiation during S and G2 phases and induces cell cycle arrest in embryonic cells around cellularization. / loki is also required for the normal number of germ line cells to form in the embryo, and for normal modification of Vasa, a crucial factor in germ cell formation. However, during normal oogenesis loki expression is suppressed by orb. Another group described the involvement of Drosophila loki/chk2 in the meiotic pachytene checkpoint. Using our loki·null mutant, I obtained the opposite result: loki/chk2 does not have an essential function in this process. / The second part of my thesis deals with the question of how cells are instructed about their identity in a developing organism. (Abstract shortened by UMI.)

The clk-1 gene was found in the screen for the maternal effect viable mutants in Caenorhabditis elegans. It is believed to be involved in controlling timing of various biological processes in the nematode including lengthening it's life span, and, as such, it belongs to the Clock group of genes. The CLK-1 homologue in yeast, COQ7, was shown to be involved in ubiquinone biosynthesis and gluconeogenic gene activation. The Drosophila clk-1 gene has been cloned. As a means to assay probable correlation between the timing mechanism of clk-1 and the circadian clocks, well studied in Drosophila, the clk-1 circadian mRNA expression has been monitored. At the middle of the dark phase, the level of clk-1 transcript decreases by approximately 30%. The Drosophila clk-1 mRNA expression during developmental stages was also analyzed. The amount of clk-1 mRNA is doubled during the larvae stage, the most metabolically active stage in the early Drosophila development.

Drosophila Lasp contains a LIM domain, two nebulin repeats, and a SH3 domain, and exhibits high homology with mammalian Lasp family proteins. Vertebrate Lasp localizes to focal adhesions and to the leading edge of migrating cells and binds filamentous actin. To investigate Drosophila Lasp in vivo, we generated a Lasp null mutant, named Laspl, and showed that Laspl is male sterile. We observed two major functions of Lasp during Drosophila spermatogenesis. First, in the stem cell niche, hub cells fail to localize to the apical end of Drosophila testis in Laspl mutant. Hub cell anchoring is dependent on cell adhesion between cells and extracellular matrix (ECM), which is mediated by integrins. Lasp genetically interacts with betaPS integrin showing complete hub cell mislocalization. This indicates that Lasp is involved in an integrin-dependent process. However, hub cell anchoring is not required for fertility or stem cell maintenance. Secondly, we observe that actin cones, a unique actin structure during spermatid individualization, are perturbed in Laspl. Our data for Lasp expression in actin cones and incomplete individualization indicate that Lasp may play a role in tethering actin to the plasma membrane.

This thesis reports the results of genetic and molecular analysis of polytene region 38 of D. melanogaster. / A detailed genetic map of the region has been constructed through the complementation analysis of 22 genes with 37 deficiency stocks, providing 48 breakpoints. 44 of the breakpoints have been precisely mapped and separate the complementation groups into 16 distinct intervals. / Molecular characterisation was achieved using a combination of computational sequence analysis and experimental techniques. In this manner, 46 new transcription units and their developmental expression patterns were identified and the physical location of 7 deficiency breakpoints revealed. Links have been established between the genetic and the physical maps using STSs and sequence information from previously cloned genes. This allowed the possible correlation of 4 previously genetically defined genes to their transcription units.

We report the cloning and characterization of valois (vls), a posterior group gene of Drosophila melanogaster , which was initially identified in a screen for female steriles. Three EMS alleles of vls contain premature stop codons in the open reading frame. Sequence analyses show the presence of WD domains in Vls and find significant similarity with the human MEP50 protein which is involved in the assembly of the splicing machinery. We did not find evidence that this function is conserved in flies yet. / We created a null mutant for vls, which shows a maternal effect lethal phenotype accompanied by posterior polarity defects in the embryos. Hemizygous vlsEMS females show a weaker, partially maternal-effect lethal and a fully penetrant grandchildless phenotype. The posterior localization of Vasa is disrupted in vlsnull ovaries, but the initial distribution of Oskar protein and mRNA appear normal. However, levels of the Short Oskar isoform responsible for pole plasm assembly are greatly reduced and Vasa appears to be differently modified post-translationally. Furthermore, a Vls::GFP fusion protein is detected all throughout oogenesis in the nurse cell and oocyte cytoplasm. Taken together, these data suggest that Vls is a cytoplasmic protein involved in the transport or activation of Vasa at the posterior of the oocyte essential for the accumulation of Short Osk.

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, May 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 100-111).

Undergraduate honors paper--Mount Holyoke College, 2005. Dept. of Biological Sciences. / Includes bibliographical references (leaves 74-79).

No description available.

Orientador: Lilian Madi-Ravazzi / Banca: Andréa Regina Baptista Rossit / Banca: Claudia Márcia Aparecida Carareto / Resumo: Uma questão central da evolução da resistência a inseticidas é o custo adaptativo do organismo contendo um genótipo que confere a resistência. O custo adaptativo refere-se à sobrevivência e à habilidade reprodutiva de um organismo. As mutações que conferem resistência aos inseticidas são preditas trazerem um custo na ausência dos mesmos e, consequentemente não se fixam nas populações. Entretanto, a resistência ao DDT em Drosophila melanogaster (DDT-R) está se aproximando de uma fixação global, mesmo após ter sido abolido o uso do DDT. Existem evidências consideráveis na literatura apoiando a hipótese que a resistência ao DDT baseada no metabolismo é poligênica em Drosophila e está parcialmente associada à superexpressão de genes do citocromo P450. Alguns trabalhos, entretanto consideram que a superexpressão de um único gene, Cyp6g1, associado com a inserção de um elemento transponível, Accord em D. melanogaster e Doc em D. simulans, é suficiente para promover a resistência, enquanto outros discutem que a superexpressão desse gene não confere necessariamente a resistência em D. melanogaster. O presente trabalho avaliou sete componentes do valor adaptativo (fertilidade, fecundidade, viabilidade ovo-pupa, viabilidade pupa-imago, viabilidade ovoimago, longevidade e o tempo de desenvolvimento) em quatro experimentos que diferiram quanto à metodologia. Essa análise teve por objetivo avaliar o custo dos fenótipos resistentes de linhagens recém coletadas e de laboratório de D. melanogaster e de D. simulans. Além disso, foi realizada uma análise do polimorfismo dos exons 1 e 2 do gene Cyp6g1, de linhagens resistentes e suscetíveis de laboratório, e resistentes recém coletadas dessas espécies, com o objetivo de examinar a relação entre o nível de suscetibilidade ao DDT e o polimorfismo. Foi verificado que, mesmo após um período de dois anos em... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: A central matter of the evolution in insecticide resistance is the adaptive cost of the organism carrying a genotype that confers resistance. The adaptive cost refers to the survival and reproductive capacity of an organism. Mutations that confer pesticide resistance are predicted to carry a cost in the absence of the pesticide and consequently they are not fixed in the population. However, DDT resistance in Drosophila melanogaster (DDT-R) is closer to a global fixation, even after the suppression of DDT use. There is considerable evidence in the literature supporting the hypothesis that DDT resistance in Drosophila, based on metabolism, is poligenic and partially associated with overexpression of CYP genes. However, a recent study concluded that a single P450 gene, Cyp6g1, associated with the insertion of Accord element in the upstream region, in D. melanogaster, is sufficient to promote resistance. In a striking example of parallel evolution, the insertion of a different transposon, Doc element, is correlated with overtranscription of Cyp6g1 homolog in D. simulans and also show to be related to resistance. Other studies disagree that the overexpression of this gene necessarily confers resistance in D. melanogaster. Seven fitness components (fertility, fecundity, viability - egg-pupa, pupa-imago, egg-imago - developmental time and longevity) were evaluated in the present study, in four experiments that differed in methodology. This analysis aimed at evaluating the cost of the resistant phenotype in both species. In addition, the analysis of the polymorphism of exons 1 and 2 of Cyp6g1 gene of the susceptible and resistant strains of D. melanogaster and D. simulans was performed with the aim of examining the relationship between the degree of polymorphism and the susceptibility to DDT. D. melanogaster and D. simulans strains showed high intra and interspecific variability... (Complete abstract click electronic access below) / Mestre