Esta Tese possui dois enfoques como objetivo geral. Primeiramente, nossos dados contribuem para o conhecimento do padrão de evolução do elemento de transposição micropia dentro do gênero Drosophila, também verificando a atuação desses elementos como fonte de variabilidade genética. Em segundo lugar geramos dados citogenéticos e moleculares inéditos a respeito de espécies do grupo cardini de Drosophila, bastante freqüente nas assembléias de Drosophilidae na região Neotropical e que havia sido muito pouco estudado do ponto de vista genético até então. Com isso, no Capítulo II verificamos a presença e a alta similaridade de sequências deste retroelemento em diferentes populações de D. cardinoides, D. neocardini e D. polymorpha. Ainda, quando comparadas com a sequência presente em uma espécie do grupo repleta, D. hydei, as sequências daquelas três espécies também apresentaram alta similaridade (97%). O grupo repleta e o grupo cardini do gênero Drosophila parecem ter divergido há 45 milhões de anos atrás. Portanto, para explicar os dados obtidos sugerimos a atuação de transmissão horizontal entre as espécies analisadas. Ampliando as análises, no Capítulo III a presença de micropia foi identificada nas demais espécies do grupo cardini com exceção das espécies do grupo que apresentam distribuição geográfica restrita às ilhas caribenhas. As comparações com sequências presentes em outras espécies do grupo repleta, assim como nos 12 genomas de espécies do gênero Drosophila disponíveis em bancos de dados públicos, verificamos que a história evolutiva do elemento micropia provavelmente inclui polimorfismo ancestral e transmissão tanto vertical quanto horizontal com mecanismos de introgressão entre as espécies potencialmente atuando na geração desses padrões. 11 Com a finalidade de estudar a possível atuação deste retroelemento como fonte de variabilidade genética através da geração de inversões nas espécies do grupo cardini, o primeiro passo foi a identificação de potenciais sítios de inserção nos cromossomos politênicos dessas espécies. No Capítulo IV estimamos o número de cópias de micropia no genoma de seis espécies do grupo cardini (D. cardini, D. cardinoides, D. neocardini, D. neomorpha, D. parthenogenetica e D. polymorpha) verificando que varia entre seis e 18 cópias. Ainda, observamos a presença de potenciais cópias em pontos de quebra para inversões cromossômicas em três espécies. No Capítulo IV discutimos o significado desses dados com base nos dados já obtidos para outras espécies. O polimorfismo cromossômico dos cromossomos politênicos e a análise comparada dessas estruturas para as seis espécies do grupo cardini previamente citadas, foi estudado no Capítulo V. Neste capítulo identificamos a presença de uma inversão nas populações estudadas de D. cardini e D. neocardini, duas inversões nas populações de D. cardinoides e D. parthenogenetica e quatro inversões nas populações de D. polymorpha. Das 10 inversões identificadas, sete são descritas pela primeira vez neste capítulo. Nenhuma inversão heterozigota foi encontrada na população de D. neomorpha aqui analisada, portanto apresentando um padrão homocariotípico. Ainda no Capítulo V apresentamos os primeiros fotomapas para as espécies D. cardini e D. parthenogenetica, assim como a reconstrução dos fotomapas das espécies D. cardinoides, D. neocardini e D. polymorpha. Também foi realizada a comparação par a par de cada cromossomo politênico entre todas as espécies com a finalidade de encontrar regiões similares úteis no estabelecimento de relações de parentesco através deste marcador. No Capítulo V esses dados são discutidos baseados no enfoque evolutivo para o grupo cardini. / This Thesis possesses two approaches as mainly objectives. First, our data contribute for the knowledge of the evolutionary pattern of the transposable element micropia in the Drosophila genus, and also to the role of these elements as source of genetic variability. Second, we generate cytogenetic and molecular new data regarding the cardini group species of Drosophila genus; these species are frequent in the Neotropical assemblies of Drosophilidae, and they had been very little studied of the genetic point of view until then. In Chapter II we verify the presence and the high sequence similarity of this retroelement in different populations of D. cardinoides, D. neocardini and D. polymorpha. Furthermore, when these sequences are compared with the sequence present in the genome of a repleta group species, D. hydei, they show also a high similarity (97%) among them. The repleta group and the cardini group of the Drosophila genus seem to have diverged 45 million years ago. Therefore, to explain the obtained data we suggest the action of horizontal transmission events between the species. Extending this analysis, in Chapter III the presence of micropia was identified in other species of the cardini group, with exception of the group species with geographic distribution restricted to the Caribbean islands. The comparisons with sequences present in other species of the repleta group as well as in the 12 Drosophila genomes available in public data bases, we verify that the evolutionary history of the micropia element probably includes ancestral polymorphism, vertical and horizontal transmission with introgression mechanisms among species potentially acting in the generation of this evolutionary pattern. 13 Aiming the analysis of micropia as source of genetic variability through the inversions generation in the cardini group species, the first step was the identification of potential insertion sites in the polytene chromosomes of these species. In Chapter IV we estimate the micropia copies number in the genome of six cardini group species (D. cardini, D. cardinoides, D. neocardini, D. neomorpha, D. parthenogenetica and D. polymorpha) verifying that it varies between six and 18 copies. Furthermore, we observed the presence of copies in the break points of chromosomal inversions for three species. In Chapter IV we argue the meaning of these data on the basis of the data already obtained for other species. The chromosomal polymorphism observed in the polytene chromosomes and the comparative analysis of these structures for the six species of the cardini group previously cited were studied in Chapter V. In this chapter we identified the presence of only one chromosomal inversion in the analyzed populations of D. cardini and D. neocardini, two inversions in the D. cardinoides and D. parthenogenetica populations, and four inversions in the D. polymorpha. From these 10 identified inversions, seven are described for the first time in this chapter. No heterozygous inversion was found in the D. neomorpha population analyzed here, therefore presenting a homokaryotipic pattern. Besides this, in Chapter V we also present the first reference photomaps for Drosophila cardini and D. parthenogenetica, as well as the photomaps reconstruction of D. cardinoides, D. neocardini and D. polymorpha. We also performed a pairwise analysis of the polytene chromosomes between the species aiming to find useful regions for the establishment of evolutionary relationships based in this marker. In Chapter V these data are argued based on evolutionary approaches for the cardini group.

The Notch receptor is a key signaling protein that also acts as a transcriptional co-activator in numerous cell fate decisions in animals, including Drosophila melanogaster. Like many other transcriptional activators interacting on the DNA, the Notch protein carries a polyglutamine tract encoded by the opa repeats of the Notch gene. Here, I show that considerable variation exists within this tract across populations from the United States and Malawi. This variation is distributed asymmetrically across the range of possible alleles, with a peak in each population at opa31 (typically encoding Q₁₃HQ₁₇) and/or opa32 (typically encoding Q₁₃HQ₁₈) and a tendency towards a large tail of longer alleles and few shorter alleles. Variation in this pattern between populations may be a result of certain tracts being less harmful in certain backgrounds, or it may be due to the ancestry of these populations. This variation has real effects, such that lines bearing alleles longer or shorter than the common 31- and 32-codon alleles exhibit abnormal phenotypes, gene expression disruption, and decreased viability, effects that persist even when the Notch gene is outcrossed or recombined into other backgrounds. I also describe lines bearing CRISPR-Cas9-edited opa repeats and highlight a potential interaction between Notch and the transcriptional activator Dorsal. Dorsal also exhibits variation in the length of its polyglutamine tract, and short tracts at Dorsal most frequently appear alongside short tracts at Notch, while long tracts of each also frequently co-occur.

This study explores novelty choice, a behavioral paradigm for the investigation of visual pattern recognition and learning of the fly Drosophila melanogaster in the flight simulator. Pattern recognition in novelty choice differs significantly from pattern recognition studied by heat conditioning, although both paradigms use the same test. Out of the four pattern parameters that the flies can learn in heat conditioning, novelty choice can be shown for height (horizontal bars differing in height), size and vertical compactness but not for oblique bars oriented at +/- 45°. Upright and inverted Ts [differing in their centers of gravity (CsOG) by 13°] that have been extensively used for heat conditioning experiments, do not elicit novelty choice. In contrast, horizontal bars differing in their CsOG by 13° do elicit novelty choice; so do the Ts after increasing their CsOG difference from 13° to 23°. This indicates that in the Ts the heights of the CsOG are not the only pattern parameters that matter for the novelty choice behavior. The novelty choice and heat conditioning paradigms are further differentiated using the gene rutabaga (rut) coding for a type 1 adenylyl cyclase. This protein had been shown to be involved in memory formation in the heat conditioning paradigm. Novelty choice is not affected by mutations in the rut gene. This is in line with the finding that dopamine, which in olfactory learning is known to regulate Rutabaga via the dopamine receptor Dumb in the mushroom bodies, is dispensable for novelty choice. It is concluded that in novelty choice the Rut cAMP pathway is not involved. Novelty choice requires short term working memory, as has been described in spatial orientation during locomotion. The protein S6KII that has been shown to be involved in visual orientation memory in walking flies is found here to be also required for novelty choice. As in heat conditioning the central complex plays a major role in novelty choice. The S6KII mutant phenotype for height can be rescued in some subsets of the ring neurons of the ellipsoid body. In addition the finding that the ellipsoid body mutants ebo678 and eboKS263 also show a mutant phenotype for height confirm the importance of ellipsoid body for height novelty choice. Interestingly some neurons in the F1 layer of the fan-shaped body are necessary for height novelty choice. Furthermore, different novelty choice phenotypes for different pattern parameters are found with and without mushroom bodies. Mushroom bodies are required in novelty choice for size but they are dispensable for height and vertical compactness. This special circuit requirement for the size parameter in novelty choice is found using various means of interference with mushroom body function during development or adulthood. / Diese Studie untersucht Novelty Choice, ein Verhaltens-Paradigma für die Untersuchung der visuellen Mustererkennung und des Lernens der Fliege Drosophila melanogaster im Flugsimulator. Mustererkennung in Novelty Choice unterscheidet sich deutlich von Mustererkennung durch heat conditioning, obwohl beide Paradigmen den gleichen Test verwenden. Von den vier Muster-Parametern, die die Fliegen im heat conditioning für die Musterunterscheidung lernen kann, lernt sie in Novelty Choice nur die Höhe (horizontale Balken in unterschiedlicher Höhe), Größe und vertikale Kompaktheit, nicht dagegen die schrägen Balken im Winkel von +/- 45°. Aufrechte und umgekehrte Ts [hinsichtlich ihrer Schwerpunkte (CsOG) um 13° voneinander verschieden], die bisher weitgehend für heat conditioning Experimente verwendet werden, lösen kein Novelty Choice aus. Im Gegensatz dazu reagiert die Fliege auf horizontale Balken, die sich in ihren CsOG um 13° unterscheiden, mit Novelty Choice. Auch die Ts lösen Novelty Choice aus, wenn ihre CsOG-Differenzen von 13° auf 23° erhöht wird. Dies deutet darauf hin, dass in den Ts die Höhen der CsOG nicht die einzigen relevanten Musterparameter für Novelty Choice Verhalten sind. Die Novelty Choice und heat conditioning Paradigmen unterscheiden sich darüber hinaus in der Rolle des Gens rutabaga (rut), das eine Typ-1-Adenylylcyclase codiert. Für dieses Protein wurde gezeigt, dass es bei der Gedächtnisbildung in der heat conditioning beteiligt ist. Novelty Choice wird nicht durch Mutationen im Gen rut beeinflusst. Dies steht im Einklang mit der Erkenntnis, dass Dopamin, das bei olfaktorischem Lernen bekanntermaßen Rutabaga über den Dopamin-Rezeptor Dumb in den Pilzkörpern reguliert, entbehrlich für die Novelty Choice ist. Die Schlussfolgerung ist, dass der Rut cAMP Signalweg bei der Novelty Choice nicht beteiligt ist. Novelty choice erfordert kurzfristigen Arbeitsgedächtnisspeicher, wie in der räumlichen Orientierung während der Fortbewegung beschrieben wurde. Das Protein S6KII, für welches gezeigt wurde, dass es am visuellen Orientierungsgedächtnis laufender Fliegen beteiligt ist, wird hier als ebenso notwendig für Novelty Choice entdeckt. Wie in heat conditioning spielt der Zentralkomplex eine wichtige Rolle in Novelty Choice. Der S6KII Mutantenphänotyp für Höhe kann in einigen Untergruppen der Ring-Neuronen des Ellipsoidkörpers gerettet werden. Weiterhin kann festgestellt werden, dass die Ellipsoidkörper-Mutanten ebo678 und eboKS263, welche ebenfalls einen Mutantenphänotyp für Höhe zeigen, die Bedeutung des Ellipsoidkörpers für die Novelty Choice hinsichtlich des Höheparameters bestätigen. Interessanterweise sind einige Neuronen in der F1-Schicht des Fächerförmigen Körpers notwendig für Novelty Choice (für Höhe). Darüber hinaus werden mit und ohne Pilzkörper unterschiedliche Phänotypen für verschiedene Musterparameter bei Novelty Choice gefunden. Die Pilzkörper sind in der Novelty Choice für Größe erforderlich, aber für Höhe und vertikale Kompaktheit sind sie entbehrlich. Diese spezielle Schaltungsvoraussetzung für den Größenparameter in Novelty Choice wird unter Verwendung verschiedener Mittel der Interferenz mit Pilzkörperfunktionen während der Entwicklung oder im Erwachsenenalter gefunden.

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 46-56). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99354

Vertebrate development requires the activity of multiple members of the myocyte enhancer factor 2 (mef2) gene family for muscle cell specification and subsequent differentiation. Additionally, it is thought that several muscle-specific functions of MEF2 family proteins require binding additional co-factors including members of the Transcription Enhancing Factor-1 (TEF-1) and Vestigial-like protein families. In Drosophila there is a single mef2 (Dmef2) gene as well single homologues of TEF-1 and vestigial-like; sd and vg, respectively. To help clarify the role(s) of these factors, we examined the requirements for Vg and Sd during Drosophila muscle specification. Analysis of loss of Vg or Sd function mutations confirms that both are required for muscle differentiation, as loss of sd or vg leads to a reproducible loss of a subset of cardiac or somatic muscle cells in developing embryos. However, the requirement for Sd or Vg is cell specific, as over-expression of each of these proteins in other muscle cells also has a deleterious effect on muscle differentiation. Finally, I determined that Sd, Vg and Dmef2 can interact directly. Thus, the muscle specific phenotypes associated with loss or ectopic Vg or Sd expression may be a consequence of alternative binding of Vg and Sd to Dmef2 to form alternative protein complexes that modify Dmef2 activity. The somatic muscles of Drosophila develop in a complex pattern that is repeated in each embryonic hemi-segment. Initial communication between somatic muscles and the epidermal tendon cells is critical for formation of this muscle pattern. However, later establishment of attachments between longitudinal muscles at the segmental borders is largely independent of the muscle-epidermal attachment signals, and relatively little is known about how this event is regulated. Here I show that expression of the transcription factor Vg is required in ventral longitudinal muscles (VL1-4) to make them competent to form stable inter-muscular attachments. Further, the cell-specific differentiation events induced by Vg in two muscles fated to form attachments appear to be coordinated by Drosophila Epidermal Growth Factor (DER) signalling. / Molecular Biology and Genetics

Egg-laying decisions can have significant fitness consequences. In female Drosophila melanogaster, egg-laying involves foraging-like behaviour. Natural allelic variation in foraging (for) underlies the rover/sitter foraging behaviour polymorphism found in D. melanogaster. for encodes a cGMP-dependent protein kinase (PKG) where rovers have higher for-PKG transcript levels and PKG activity than sitters. Interestingly, the orthologue of for in nematodes (egl-4) affects both egg-laying and foraging behaviours. When given a choice between low- and high-nutrient patches, rovers preferentially lay more eggs on the low-nutrient patches while sitters and a sitter mutant prefer high-nutrient patches. Using the neuronal driver elav-GAL4, rover-like preferences were rescued in sitter flies. Compared to sitters, rovers have higher fitness on a sub-optimal substrate which may explain the observed egg-laying preferences. By studying the link from genes to behaviour, this study provides insight to the evolutionary basis and maintenance of behaviour.

Egg-laying decisions can have significant fitness consequences. In female Drosophila melanogaster, egg-laying involves foraging-like behaviour. Natural allelic variation in foraging (for) underlies the rover/sitter foraging behaviour polymorphism found in D. melanogaster. for encodes a cGMP-dependent protein kinase (PKG) where rovers have higher for-PKG transcript levels and PKG activity than sitters. Interestingly, the orthologue of for in nematodes (egl-4) affects both egg-laying and foraging behaviours. When given a choice between low- and high-nutrient patches, rovers preferentially lay more eggs on the low-nutrient patches while sitters and a sitter mutant prefer high-nutrient patches. Using the neuronal driver elav-GAL4, rover-like preferences were rescued in sitter flies. Compared to sitters, rovers have higher fitness on a sub-optimal substrate which may explain the observed egg-laying preferences. By studying the link from genes to behaviour, this study provides insight to the evolutionary basis and maintenance of behaviour.

The zinc-finger transcription factor Senseless is co-expressed with basic helix-loop -helix (bHLH) proneural protein in Drosophila sensory organ precusors and is required for their normal development. Recently, we analysed Senseless protein sequence with bioimformatics and found many SUMOs consensus sites. In this study, we successfully performed in vitro sumoylation of Senseless proteins and observed the colocalisation between Senseless and SUMOs protein.

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Biochemistry." Includes bibliographical references (p. 92-99).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 131-147).