Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 102 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 86-100).

Bibliography: p. 75-84. Identifies key interactions between Polycomblike and other members of the Polycomb group and suggests a model for the role of Polycomblike within the group.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Apoptosis, or programmed cell death, is a genetically controlled form of cell suicide used to rid an organism of superfluous or damaged cells and serves as one of the major mechanisms for patterning during the development of complex animal structures. The antenna and ovary of the fruitfly, Drosophila melanogaster, were chosen as model systems in which to examine the molecular mechanisms of developmental apoptosis. The caspases are a family of cysteine proteases required for the execution of cell death, while the inhibitor of apoptosis protein Diap-1 is responsible for repressing the function of caspases. Diap-1, also known as Thread, was discovered in 1922 with the isolation of thread^1, a homozygous viable mutant whose molecular nature is unknown. thread was given its name due to its branchless or thread-like arista, the featherlike structure at the tip of the antenna. thread^1 antennal imaginal discs show excessive cell death during a brief period of larval development, which corresponds to a significant decrease in levels of Thread protein. Caspase activity is found more broadly than apoptotic DNA fragmentation in thread1 imaginal discs, suggesting that the mutant fails to inhibit caspases in many cells, but only a fraction succumb to apoptosis. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the formation of the arista. Additionally, a candidate mutation has been discovered in a transcriptional regulatory region of the thread gene. Proof that this mutation is responsible for the branchless aristal phenotype may have important implications for understanding tissue-specific gene regulation during organogenesis. In the ovary, apoptotic roles for the DP subunit of the E2F transcription factor and the actin binding protein Profilin were investigated. While mutants for DP affect the regulation of Cytochome c and caspase activity in nurse cells, Profilin mutants show milder effects. To further characterize the role of apoptosis in the ovary, an ethylmethane sulphonate (EMS) mutagenesis screen was conducted and has led to the identification of several new genes. Future study of the apoptotic pathway will assist in developing treatments for diseases associated with its misregulation. / 2031-01-02

Memoria para optar el título de Bioquímico / La localización precisa de los componentes sinápticos durante el desarrollo requiere del ordenamiento de una red de proteínas entre las cuales participan proteínas andamio de 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 en complejos macromoleculares a través de sus dominios de interacción proteína-proteína. La sinapsis neuromuscular de la larva de Drosophila melanogaster es un excelente modelo de sinapsis centrales de mamíferos. En esta sinapsis glutamatérgica, la proteína andamio Discs large (Dlg), un miembro de la familia MAGUK, cumple un papel importante en la formación de la sinapsis y en la localización de proteínas involucradas en la transducción de señales. Se ha demostrado que Dlg determina la localización sináptica del canal de K+ tipo Shaker, Fasciclina II (FasII), una molécula de adhesión celular involucrada en el crecimiento y plasticidad de la sinapsis neuromuscular y Scribble (Scrib), una proteína involucrada en el mantenimiento de la concentración de vesículas sinápticas en los sitios de liberación durante la estimulación de alta frecuencia. Dlg, además participa en otros procesos, incluyendo el mantenimiento de la polaridad apicobasal de los epitelios, el control de la proliferación y la neurogénesis embrionaria. Todas estas funciones han sido atribuidas a una sola isoforma, DlgA. Sin embargo, en nuestro laboratorio se aislaron 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 trabajo se estudió el papel de las variantes del gen dlg con dominio S97N en la estructura y formación de la sinapsis de la unión neuromuscular de la larva. Mediante análisis de “western blot” se determinó la proteína DlgS97 de ∼116 kDa y al menos una variante de DlgA de ∼97 kDa. Experimentos de ganancia y pérdida de función específicas para las variantes epitelial y neuronal muestran que las variantes que contienen el dominio S97N participan en el desarrollo de la sinapsis, afectando la morfología y el número de botones sinápticos, además de la expresión de la proteína Scrib y parcialmente la localización de FasII en la sinapsis. Esto difiere de lo observado para la pérdida de función de DlgA que no parece afectar la morfología o expresión de Scrib, aunque sí afecta el número de botones y la expresión de FasII. Mediante ensayos en matriz sólida e inmunoprecipitación se logró determinar que el dominio S97N es capaz de formar homodímeros, sin interactuar con dominios de DlgA. Estos resultados sugieren que ambas isoformas participan en la sinapsis pero con funciones diferentes, formando complejos macromoleculares de manera independiente / The precise location of the synaptic components during development requires the arrangement of a protein network which includes scaffold proteins of the MAGUK family. The members of this family of proteins recruit receptors, ionic channels, signal transduction components and proteins of the cytoskeleton to macromolecular complexes through protein-protein interaction domains. The neuromuscular synapse of the Drosophila melanogaster larva has been used as a model of mammalian central synapses. In this glutamatergic synapse, the scaffold protein Discs large (Dlg), a member of the MAGUK (membrane-associated guanylate kinases) family of proteins, has an important role in the formation of the synapse and in the localization of proteins involved in signal transduction. It has been demonstrated that Dlg determines the synaptic localization of the Shaker K+ channel, of Fasciclin II (FasII), an adhesion molecule involved in the growth and plasticity of the neuromuscular synapse and of Scribble (Scrib), a protein involved in the maintenance of the synaptic vesicle pool in the release sites during high frequency stimulation. Dlg also participates in other processes, including the maintenance of the epithelial apico-basal polarity, the control of proliferation and embrionic neurogenesis. All these functions have been attributed to a single isoform, DlgA. In our laboratory, however, a number of transcripts has been isolated that represent alternatively processed products of the gene dlg, these present a 5' region that encodes a segment of 65 amino acids called S97N that is absent in DlgA. The variants S97N-containig as protein denominated DlgS97 are only expressed in nervous system and muscle, unlike DlgA that is also expressed in epithelial cells. In this work, the role of Dlg variants with S97N domain in the structure and development of the neuromuscular junction was studied. Western blot analysis indicates the presence of the protein DlgS97 of 116 kDa and at least one variant of DlgA of 97 kDa. Loss of function and gain of function experiments specific for the epithelial and neuronal variants show that variants containing the S97N domain participate in the development of the synapse, affecting the morphology and number of synaptic boutons, together with the expression of Scrib and the partial localization of FasII at the synapse. This is different from the DlgA loss of function where the morphology or the expression of Scrib does not change, although the number of boutons and the expression of FasII is affected. By pulldown and inmunoprecipitation assays it was possible to determine that the S97N domain is able to undergo homodimerization, but does not interact with domains of DlgA. These results suggest that both isoforms participate in the synapse but with different functions, forming macromolecular complexes in an independent way

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-02-26T12:26:42Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE - Adiles Paulo de Lima.pdf: 1609682 bytes, checksum: 889f967461ad04b505907e00ea91cfab (MD5) / Made available in DSpace on 2016-02-26T12:26:42Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE - Adiles Paulo de Lima.pdf: 1609682 bytes, checksum: 889f967461ad04b505907e00ea91cfab (MD5) Previous issue date: 2015-03-12 / REUNI / CAPES / PT-31 [3-(2-cloro-6-fluorobenzil)-imidazolidina-2,4-diona] é um composto descrito com atividade analgésica e o elixir SANATIVO® (SAN e extrato de SAN, E. SAN) é um fitoterápico comum no Nordeste do Brasil. Esses produtos terapêuticos necessitam de testes adicionais antes de ter aplicabilidade humana, inclusive ensaios genéticos. Assim, dois testes de danos genéticos foram escolhidos para o presente trabalho: o Teste de Mutação e Recombinação Somática (SMART) em Drosophila melanogaster que detecta tipos diferentes de mutações e recombinação, e mutantes de recombinação e reversão de Saccharomyces cerevisiae, ambos apresentam baixo custo, confiabilidade e rapidez, visando a análise genética para o uso mais seguro dos compostos. O fármaco PT-31 foi avaliado através da técnica SMART e pelo teste com os mutantes de S. cerevisiae; com o elixir SANATIVO® foi formada uma curva de sobrevivência larval utilizando os dois tipos de cruzamentos (ST e HB) do SMART, em seguida este ensaio foi empregado; e o extrato de SAN foi analisado pelo SMART e através das linhagens de S. cerevisiae. Notou-se a possibilidade de SAN ter reduzido a toxicidade do álcool por meio da sobrevivência larval. Enquanto que o SMART de SAN e de E. SAN revelou efeito mutagênico na maioria das concentrações testadas, com ativação metabólica e efeito recombinogênico ausentes, sem alteração identificável nos testes em S. cerevisiae. O PT-31 mostrou efeito mutagênico e atividade recombinogênica no teste SMART, aumentando o efeito mutagênico pela atividade metabólica do sistema P450, e nenhuma alteração fenotípica no teste em S. cerevisiae. Esses dados contribuem para a ampliação das informações sobre os compostos terapêuticos na literatura, para que futuros experimentos possam elucidar melhor a ação em organismos vivos. / PT-31 [3-(2-chloro-6-fluorobenzyl)-imidazolidine-2,4-dione] is a compound described with analgesic activity and SANATIVO® elixir (SAN e SAN extract, E. SAN) is a common phytotherapic in the Northeast of Brazil. Those therapeutic products require additional tests before human applicability, including genetic assays. The Mutation and recombination Somatic Test (SMART) in Drosophila melanogaster provides different sorts of mutations and recombination, and recombination and reversion mutants of Saccharomyces cerevisiae, both present low cost, reliability and speed, in order the genetic analysis for a safer use of the compounds. The PT-31 drug was evaluated by SMART technique and by the test with S. cerevisiae mutants; with the SANATIVO® elixir was constructed a larval survival curve using two types of crosses (ST and HB) of SMART, then this assay was used; and the SAN extract was assessed by SMART and by S. cerevisiae strains. It was noticed the possibility of SAN have reduced toxicity of the alcohol by larval survival. While the SMART of SAN and of E. SAN revealed mutagenic effect in most of the tested concentrations, with metabolic activation and absent recombinogenic effect, without identifiable modification in the tests in S. cerevisiae. The PT-31 showed mutagenic effect and recombinagenic activity in the SMART test, increasing of mutagenic effect by the metabolic activity of P450 system, and none phenotypic change in the test in S. cerevisiae. These data contribute to the expansion of information on the therapeutic compounds in the literature, so that future experiments will be able to elucidate the action in living organisms.

No description available.

Small non-coding RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) play a big role in regulating gene expression in cells. In my work, I focus primarily on miRNAs, which represses the expression of the mRNA targets post-transcriptionally. For Drosophila melanogaster, I predicted the tissue-specific expression of several miRNAs based on the expression levels of the predicted mRNA targets in those tissues. The computational predictions are then followed up by quantitative PCR validation of miRNA expression levels in dissected fly tissues. For Stylophora pistillata (a species of coral found in the Red Sea) and Symbiodinium sp. (a photosynthetic, symbiotic algae present in the coral cell), my collaborators and I strived to study the genome, transcriptome and proteome of both organisms. At present, there is another coral genome available — from Acropora digitifera — but the large evolutionary distance between both corals (about 240 million years apart) warrants in-depth study of our coral of interest. On the other hand, our Symbiodinium genome will be the first of its kind for any dinoflagellate. My role in the project was to investigate the small RNAome of both organisms via small RNAseq. As the presence of a thick cell wall in Symbiodinium sp. poses a unique challenge to RNA extraction, and compounded by the dearth of literature regarding RNA extraction from the dinoflagellate, we optimised a procedure that consistently produced high quality RNA for downstream sequencing. From our draft proteome, I showed that the RNA interference (RNAi) machinery is very likely to be present in both organisms. Based on our short RNAseq data, I predicted miRNAs in both organisms. Two of the predicted miRNAs in S. pistillata have been identified in other organisms, while all of the predicted miRNAs in Symbiodinium sp. were novel.

It has been suggested (Suzuki, 1965a) that the extrinsic and intrinsic factors which increase crossing over in the centromere regions of Drosophila do so by inactivating these regions at the time of crossing over; the inactivation resulting in altered chromosome structure which permits the intimate synapsis necessary for crossing over. This hypothesis predicts that the 3L heterochromatic marker w⁺ carried in Dp(wm)264.58a, which exhibits position effect variegation, would tend to be inactivated by chromosome aberrations, known to increase crossing over. The reversed acrocentric (RA) compound X chromosome, the X chromosome inversions sc⁴sc⁸ and sc⁸, the autosomal inversions Cy, Sb, and Ubx, and the Minute mutants M(2) and M(3) were tested for their effects on the expression of w⁺ in various coisogenic stocks. The amount of pigment in each eye was visually scored into twelve classes. Crossover analyses of the centromere regions of chromosomes 2 and 3 were performed to confirm the effects of sc⁴sc⁸, Cy, Ubx, and Sb on crossing over. The RA, sc⁴sc⁸, Cy, and Ubx all increase crossing over and significantly depress the activity of w⁺. The degree of pigment reduction is correlated with the amount of increase in crossing over. Combination of these aberrations produces an effect on pigmentation and crossing over greater than that produced by either considered singly. Sb has a slight effect in increasing crossing over and decreasing pigmentation. M(3), known to increase crossing over, significantly decreases pigmentation whereas M(2) does not. The effect of the X chromosome inversion sc⁸ is doubtful. Expression of w⁺ is affected similarly in males and females, an observation suggesting that the chromosome physiology of both sexes is similar although the actual factor(s) mediating crossing over are absent in the males. These results lend support to the proposed hypothesis but, with fluctuating parental source effects and variations found within a stock testifying to the general lability of the system, further tests under stringently controlled conditions are necessary before such experiments can be considered critical. / Science, Faculty of / Zoology, Department of / Graduate

During the screening process 5,20 8 X chromosomes of -Drosophila melanogaster were analyzed for the presence of temperature-sensitive (ts) lethal mutations (i.e. mutants which die at 29°C but are viable at 22°C) in short proximal and distal segments of the chromosome. Seven ts and 16 non-ts lethals were recovered in both regions combined. A new class of mutations (class-3), which failed to survive at 29°C with either proximal or distal duplication and showed ts lethality with one, was found and extensively analyzed. These mutants were initially interpreted to be dominant ts's, although the heterozygotes of each mutant showed this not to be so. It was decided that these might more probably be chromosomes carrying a lethal mutation covered by the duplication, and a ts lethal mapping elsewhere. By masking the non-conditional lethal with a duplication, developmental studies of the ts mutant were made. The temperature-sensitive period (TSP) and lethal phase (LP) were characterized for each. All TSP’s spanned the early pupal interval, though an individual TSP might extend to either side of this interval. The pattern of temperature-sensitivity of C3-3 suggested that once formed at permissive temperature, its product was not affected by 29°C. The experiments suggest that the temperature-sensitive process occurs at transcription or translation. A lethal allele of the dor locus was recovered, and, in analysis of this mutant with other dor alleles and several variegating duplications, dor itself was found to be a ts lethal. "Warped" wing, a new phenotype of the dor locus which occurred only with the variegating duplications, was described. This paper further describes a method for developmental analysis of non-ts lethal mutations, involving the use of variegating rearrangements. / Science, Faculty of / Zoology, Department of / Graduate

Mitants of Prosophila melanogaster which are paralysed by exposure to one temperature, but recover mobility at another temperature may aid in the investigation of the neural and muscular components which govern motor activity. With the help of a mechanical screening method, a recessive sex-linked temperature-sensitive paralytic mutant (parats -53°9) was discovered among the progeny of ethyl methane- sulphonate-treated males and attached-X females. Parats flies which had been raised at 22°C were paralysed within 5 seconds after transfer to 29°C, but quickly regained mobility when returned to 22°C. When left at 29°C for prolonged periods, the flies gradually regained mobility. Further studies indicated that the time required for recovery following an increase in temperature was directly related to the magnitude and rate of the temperature rise. Temperature-sensitive paralysis was seen only in adult flies. The abnormal movements which are characteristic of the behavioural mutants Hk1P, Hk2T , Sh⁵, when linked to parats in males,were quickly stopped and started by temperature shifts from 22°C to 29°C and 29°C to 22°C, respectively. The possible significance of these observations is discussed. parats/M(1)0 females exhibited temperature-sensitive paralysis. The possibility that the chromosome bearing the M(1)0 mutation might also carry a deletion or mutant allele of parats has not yet been investigated. A sex-linked dominant mutation which caused abnormal movements of the head and appendages under ether anaesthesia as well as shuddering movements in unetherised flies was also discovered. The mutation was called Shuddering (Shu – 55.1). The shuddering movements could be temporarily suppressed by feeding the flies media containing LiCl but not NaCl, NH₄Cl or KCl. The evidence presented in this and other studies suggests that the effects of parats and Shu mutations upon motor activity are mediated through their effects upon the nervous system. / Medicine, Faculty of / Medical Genetics, Department of / Graduate