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Identification of differentially expressed proteins in obese rats fed different high fat diets using proteomics and bioinformatics approachesGabuza, Kwazikwakhe January 2013 (has links)
Philosophiae Doctor - PhD / Obesity is a medical condition in which an energy imbalance leads to excessive
accumulation of body fat. Obesity leads to a reduction in life expectancy through its
association with chronic diseases of lifestyle. The prevalence of obesity is rapidly
increasing throughout the world. It is now accepted that most cases of obesity result
from an interaction between genetic and environmental factors. This rapid increase in obesity generally leads to an increase in morbidity and mortality from chronic diseases such as cardiovascular disease, type 2 diabetes, osteoarthritis and cancer of which obesity is a risk factor. There is a lack of information in molecular research to explain how obesity predisposes individuals to these diseases. Proteomics is a molecular tool and a set of techniques used to identify changes at protein level from a diseased state. This study aims to identify differentially expressed proteins in serum of obese rats fed different isocaloric diets using proteomics.
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Experimentos de microarrays e teoria da resposta ao item / Microarryas experiments and Iten Response TheoryNeves, Carlos Eduardo 25 February 2010 (has links)
Recentemente desenvolvida, a biotecnologia denominada por Microarrays permite o monitoramento simultâneo dos valores de expressão gênica de centenas de milhares de genes, fator este que traz uma nova interpretação aos resultados obtidos em pesquisas desenvolvidas nas mais diversas áreas do conhecimento incluindo, por exemplo, a Farmacologia e Medicina, uma vez que os resultados obtidos são interpretados ao nível molecular. Contudo, apesar de muita tecnologia ser empregada à técnica de Microarrays, sua aplicação ainda ocasiona algumas complicações decorrentes, por exemplo, das inúmeras fontes de variação existentes, da escala das respostas ou da natural dificuldade de se analisar uma grande quantidade de fragmentos genéticos avaliados sob poucas unidades experimentais. Frente a estas complicações, atualmente, muitas são as propostas metodológicas de análises estatísticas para atenuar ou eliminar os problemas inerentes à técnica de Microarrays e propiciar a extração de resultados mais confiáveis a partir dos valores de expressão gênica, porém muitos desafios ainda persistem. Sob esta colocação, o presente trabalho procurou explorar duas metodologias de análise estatística alternativas no que diz respeito a seus conceitos, embora ambas tenham sido contextualizadas ao problema de Microarrays e aplicadas para se atingir o mesmo objetivo: possibilitar a identificação dos genes diferencialmente expressos sob distintas condições experimentais. A primeira metodologia consistiu da aplicação de Modelos de Análise de Variância de efeitos fixos com a adoção de modificações nas estatísticas de teste, metodologias de correções para múltiplos testes e a construção de gráficos vulcão. Já, a segunda metodologia consistiu da contextualização e aplicação da Teoria da Resposta ao Item TRI aos experimentos de Microarrays, abordagem esta pouco explorada na análise deste tipo de dado, mas a qual possibilita a seleção de genes diferencialmente expressos a partir de uma medida latente estimada para cada gene e a construção de uma escala para as categorias de resposta de expressão gênica. A motivação para este trabalho originou de um experimento de Microarrays com ratos congênicos disponibilizado pelo Laboratório de Cardiologia e Genética Molecular do Instituto do Coração (InCor-USP) cujo objetivo é identificar genes associados à hipertensão. / Recently developed, the biotechnology denominated Microarrays permits a simultaneous monitoring of the gene expression values of hundred thousands of genes; fact that introduces a new interpretation of the results obtained in researches developed in many distinct areas including, for example, Pharmacology and Medicine, once the obtained results are read according to the molecular level. However, despite the fact that much technology is used in the Microarrays technique, its application still causes some implications, for example, the countless sources of existing variance, the scale of answers or the natural difficulty in analyzing a large number of genetic fragments measured by few experimental units. Facing such complications, a lot of methodologies were suggested in order to reduce or eliminate the problems caused by the Microarrays technique and also foster the obtainment of more reliable results from the gene expression values, yet many challenges still persist. Under this perspective, the present work aimed at exploring two alternative methodologies regarding concepts, despite both were contextualized according to the Microarrays problem and applied with the same objective: enabling the identification of the genes differently expressed under different experimental conditions. The first methodology was composed by the application of Analysis of Variance Models of fixed effects with changes in the test statistics, correction methodologies for multiple tests and volcano plot. The second methodology consisted of the contextualization and application of the Item Response Theory IRT towards the Microarrays experiments, being this one not much explored in analysis that use this kind of data, but enabling the selection of genes differently expressed from an estimated latent trait for each gene and the construction of a scale for the categories of gene expression answers. The motivation for the present work came from an experiment of Microarrays with congenic mice made available by the Cardiology and Molecular Genetics Laboratory of the Heart Institute (InCor-USP) that aimed at identifying genes associated with hypertension.
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Combinatoire du polynôme de Tutte et des cartes planaires / Combinatorics of the Tutte polynomial and planar mapsCourtiel, Julien 03 October 2014 (has links)
Cette thèse porte sur le polynôme de Tutte, étudié selon différents points de vue. Dans une première partie, nous nous intéressons à l’énumération des cartes planaires munies d’une forêt couvrante, ici appelées cartes forestières, avec un poids z par face et un poids u par composante non racine de la forêt. De manière équivalente, nous comptons selon le nombre de faces les cartes planaires C pondérées par TC(u + 1; 1), où TC désigne le polynôme de Tutte de C. Nous commençons par une caractérisation purement combinatoire de la série génératrice correspondante, notée F(z; u). Nous en déduisons que F(z; u) est différentiellement algébrique en z, c’est-à-dire que F satisfait une équation différentielle polynomiale selon z. Enfin, pour u ≥ -1, nous étudions le comportement asymptotique du n-ième coefficient de F(z; u). Nous observons une transition de phase en 0, avec notamment un régime très atypique en n-3 ln-2(n) pour u ϵ [-1; 0[, témoignant d’une nouvelle classe d’universalité pour les cartes planaires. Dans une seconde partie, nous proposons un cadre unificateur pour les différentes notions d’activités utilisées dans la littérature pour décrire le polynôme de Tutte.La nouvelle notion d’activité ainsi définie est appelée Δ-activité. Elle regroupe toutes les notions d’activité déjà connues et présente de belles propriétés, comme celle de Crapo qui définit une partition (adaptée à l’activité) du treillis des sous-graphes couvrants en intervalles. Nous conjecturons en dernier lieu que toute activité qui décrit le polynôme de Tutte et qui satisfait la propriété susmentionnée de Crapo peut être définie en termes de Δ-activités. / This thesis deals with the Tutte polynomial, studied from different points of view. In the first part, we address the enumeration of planar maps equipped with a spanning forest, here called forested maps, with a weight z per face and a weight u per non-root component of the forest. Equivalently, we count (with respect to the number of faces) the planar maps C weighted by TC(u + 1; 1), where TC is the Tutte polynomial of C.We begin by a purely combinatorial characterization of the corresponding generating function, denoted by F(z; u). We deduce from this that F(z; u) is differentially algebraic in z, that is, satisfies a polynomial differential equation in z. Finally, for u ≥ -1, we study the asymptotic behaviour of the nth coefficient of F(z; u).We observe a phase transition at 0, with a very unusual regime in n-3 ln-2(n) for u ϵ [-1; 0[, which testifiesa new universality class for planar maps. In the second part, we propose a framework unifying the notions of activity used in the literature to describe the Tutte polynomial. The new notion of activity thereby defined is called Δ-activity. It gathers all the notions of activities that were already known and has nice properties, as Crapo’s property that defines a partition of the lattice of the spanning subgraphs into intervals with respect to the activity. Lastly we conjecture that every activity that describes the Tutte polynomial and that satisfies Crapo’s property can be defined in terms of Δ-activity.
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The Performance of the Differentially Coherent DS/SS Code Synchronization with Different Adaptive LMS Filtering SchemesChang, Yu-Chen 02 August 2005 (has links)
The efficiency of direct sequence spread spectrum (DS/SS) receiver is highly dependent on the accurate and fast synchronization between the incoming and locally generated PN (pseudo-noise) codes. The code synchronization is processed in two steps, acquisition (coarse alignment) and tracking (fine alignment), to estimate the delay offset between the two codes. In general, the schemes for code acquisition and tracking processes are performed, separately, and implemented with different structure. Recently, an alternative approach, with the adaptive LMS filtering scheme, has been proposed for performing both code acquisition and tracking with the identical structure, where the coherent receiver was employed. With this approach, dramatically, hardware complexity reduction could be achieved, especially, when long PN code is considered.
In this thesis, a new differentially coherent code synchronization scheme, based on a differential detector followed by an adaptive constrained LMS (CLMS) filtering algorithm with maximum tap weight (MTW) test scheme, is devised for performing both code acquisition and tracking with the identical structure. With a differential detector for code synchronization, the prior knowledge of the carrier phase is not required as the non-coherent techniques. Numerical analyses and simulation results verify that the proposed scheme has better acquisition performance, in terms of mean acquisition time, than the conventional LMS filtering algorithm with MTW test and mean square error (MSE) test schemes for the integer and non-integer time delay environments. At the same time, the proposed scheme has better tracking capability, in terms of mean hold-in time and mean penalty time, over the conventional LMS filtering schemes, for the variation of signal-to-noise ratio (SNR) and delay offset (delay difference).
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Caracterização molecular de genes preferencialmente expressos na fase leveduriforme patogênica de ´Paracoccidioides brasiliensis´ através das técnicas de ´Macroarray´ e de SSH (Suppression Substractive Hybridization) / Molecular characterization of preferentially expressed genes in the yeast pathogenic phase of Paracoccidioides brasiliensis through the techniques of Macroarray and SSH (Suppression Subtraction Hybridization)Everaldo dos Reis Marques 22 December 2005 (has links)
Paracoccidioides brasiliensis, um fungo termodimórfico, é o agente causador da paracoccidioidomicose (PCM), a micose sistêmica prevalente da América Latina. A patogenicidade aparenta estar intimamente relacionada com a transição dimórfica da forma de micélio para a de levedura, que é induzida pela mudança da temperatura do ambiente pela temperatura do hospedeiro mamífero. Há poucas informações disponíveis sobre genes de P. brasiliensis que são necessários durante a fase patogênica. Nós, então, realizamos as técnicas de SSH (Suppression Subtraction Hybridization) e de Macroarray com o objetivo de identificar genes que sejam preferencialmente expressos na fase leveduriforme do isolado Pb18. Genes identificados em ambos os procedimentos estão mais expressos na fase leveduriforme e estão envolvidos em metabolismo básico, transdução de sinal, crescimento e morfogênese e metabolismo do enxofre. Para testar se as mudanças observadas na expressão gênica refletem as diferenças entre as condições de crescimento usadas para obter as duas formas morfológicas preferivelmente às diferenças intrínsecas dos tipos celulares, nós realizamos experimentos com RT-PCR em tempo real utilizando preparações de RNA derivadas de ambas as fases, micélio e levedura, crescidas a 26°C e 37°C nos meios de cultura completos (YPD e Sabouraud) e meio mínimo. Vinte genes, incluindo AGS1 ( -1,3-glucan synthase) e TSA1 (thiol-specific antioxidant), foram mostrados como mais expressos na levedura patogênica em relação ao micélio. Embora a expressão de RNA mensageiro foi bastante diferente em relação aos meios completos e meio mínimo, mostramos uma tendência geral para que esses genes serem mais expressos nas células leveduriformes patogênicas de P.x brasiliensis. Além disso, mostramos a complementação dos genes METR e SCONC de P. brasiliensis e uma cepa com estes genes deletados de Aspergillus nidulans, sugerindo uma possível homologia entre eles. Mostramos também a análise de genes da via do metabolismo do enxofre foram mais expressos na levedura patogênica de P. brasiliensis em relação ao micélio saprofítico. / Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNA derived from both yeast cells and mycelia that had been cultured at 37 and 26°C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 ( 1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells. Moreover, complementation of P. brasiliensis METR and SCONC genes in strains of Aspergillus nidulans with these genes deleted suggested a possible homology between them. We show the analyses of genes involved in the xii sulphur metabolism pathway and these genes were more expressed in the pathogenic yeast than saprophytic mycelia of P. brasiliensis.
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Absor??o, assimila??o e remobiliza??o de nitrog?nio em arroz, sob nutri??o n?trica. Avalia??o da express?o g?nica diferencial. / Nitrogen uptake, assimilation and remobilization in rice under supply of nitrate. Evaluation of differential genic expressionSantos, Andr? Marques dos 27 February 2007 (has links)
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Previous issue date: 2007-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Rice is one of the most important cereals for the humanity, because it is primary source of
nutrition for more than half of the world s population. Nitrogen is the most important element
for plants and is predominantly present in aerated soils in the nitrate form. As a result of the
poor quality of the cultivated areas, the usage of nitrogen fertilizers intensified, contributing to
the increase of pollution, since this nutrient has high mobility in the soil. With the objective of
evaluating physiologic, biochemical and molecular mechanisms responsible for the efficiency
of the application of nitrogen in rice, experiments were conducted at the Soils Department of
Federal Rural University of Rio de Janeiro. Two rice varieties were used, Piaui (traditional
rice) and IAC-47 (improved rice). In the beginning, both varieties were cultivated in nutrition
solutions and submitted to treatments simulating the seasonal fluctuation of nitrate,
characteristic of the tropical region. Under this condition an evaluation was done in order to
define the soluble sugar levels, nitrogen fractions, and the Nitrate Reductase Activity (NRA)
as well as Glutamine Synthetase Activity. Piaui rice was more efficient in the acquisition and
usage of nitrogen compared to IAC-47, accumulating nitrate in its tissue during the initial
phase of growth for future consumption. Later, these same rice varieties were cultivated in a
growth chamber with the objective of studying the effect of increased nitrate concentrations
over nitrogen fraction levels, NRA, relative expression of genes for nitrate transporters
(NRT1 and NRT2) as well as a nitrate reductase (Nia2). From the results obtained it was
observed that the traditional rice variety contained less NRA, higher levels of nitrogen and
free amino-N. Further, traditional variety presented higher relative expression of the genes
which codify nitrate transporters of low affinity (NRT1) and those of high affinity (NRT2).
These plants seemed to adopt a differentiated control of NRA, because even having a higher
relative expression of the genes, the NRA maintained practically without influence from the
different concentrations of nitrate in the solution. In the last experiment, genes in the tips of
rice roots were identified, induced by different concentrations of nitrate (0.1 and 10 mM).
Several genes were sequenced, were the most important data was the subunit E1 of the
dehydrogenase pyruvate complex, and phosphofructokinase, suggesting the need for a better
understanding of the complexity of the interaction between carbon and nitrogen metabolism.
There were also found sequences homologous to the alpha actin, myosin, tropomyosin 1,
creatin kinase, and glyoxalase I genes. / O arroz ? um dos mais importantes cereais para o homem, porque ? fonte prim?ria na
alimenta??o de mais da metade da popula??o do mundo. As plantas necessitam de nitrog?nio
para seu desenvolvimento, sendo este presente em solos aerados, na forma de nitrato. Em
fun??o do desgaste das ?reas cultivadas, tem sido intensificada a utiliza??o de fertilizantes
nitrogenados, o que contribui para o aumento da polui??o, visto que, esse composto possui
grande mobilidade no solo. Objetivando-se avaliar os mecanismos fisiol?gicos, bioqu?micos e
moleculares respons?veis pela efici?ncia no uso de nitrog?nio em arroz foram conduzidos tr?s
experimentos no Departamento de Solos da Universidade Federal Rural do Rio de Janeiro,
com duas variedades de arroz, uma tradicional (Piau?) e outra melhorada (IAC-47).
Primeiramente, ambas as variedades foram cultivadas em solu??o nutritiva e submetidas a
tratamentos simulando o fluxo sazonal de nitrato, caracter?stico das regi?es tropicais. Nessa
condi??o, foram avaliados os teores de a??cares sol?veis, fra??es nitrogenadas e a atividade
das enzimas Nitrato Redutase (ANR) e Glutamina Sintetase. A variedade Piau? foi mais
eficiente na aquisi??o e uso do N quando comparada a IAC-47, acumulando nitrato em seus
tecidos durante as fases iniciais de crescimento. Depois, essas mesmas variedades foram
cultivadas em c?mara de crescimento para estudar o efeito de concentra??es crescentes de
nitrato sobre os teores das fra??es nitrogenadas, ANR e sobre o perfil de express?o relativa
dos genes dos transportadores de nitrato (NRT1 e NRT2) e da Nitrato Redutase (Nia2). Dos
resultados obtidos, foi observado que a variedade tradicional possui menor ANR, maiores
teores de nitrato e N-amino livre. Al?m disso, esta apresentou maior express?o relativa dos
genes que codificam os transportadores de nitrato de baixa (NRT1) e os de alta (NRT2)
afinidade, sendo esse na parte a?rea. Estas plantas pareceram adotar um controle diferenciado
da ANR, pois mesmo tendo maior express?o relativa desse gene, a atividade desta enzima se
mant?m praticamente sem sofrer influ?ncia das concentra??es de nitrato na solu??o. No
?ltimo experimento, foram identificados os genes induzidos por duas concentra??es de nitrato
(0,1 e 10 mM) em ?pices radiculares de arroz. Diversos genes foram seq?enciados, dentre
eles, destacou-se a subunidade E1 do complexo piruvato desidrogenase e fosfofrutoquinase,
indicando a necessidade de se melhor entender a complexidade da intera??o entre o
metabolismo de carbono e nitrog?nio. Foram encontradas tamb?m seq??ncias hom?logas aos
genes da alfa actina, miosina, tropomiosina 1, creatina cinase e glioxalase I.
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Expressão de Ultrabithorax e o Desenvolvimento Casta-Específico de Apêndices Torácicos de Apis mellifera / Ultrabithorax Expression and Development of Caste-Specific Thoracic Appendages in Apis melliferaAna Durvalina Bomtorin 12 April 2013 (has links)
A diferenciação morfo-fisiológica entre rainhas e operárias de Apis mellifera decorre da alimentação recebida durante o desenvolvimento larval. Dentre as diferenças morfológicas entre as duas castas encontram-se estruturas especializadas para a coleta de pólen e própolis, localizadas nas pernas metatorácicas das operárias, ausentes nas pernas de rainhas. Os padrões de expressão de Ultrabithorax (Ubx) durante o desenvolvimento de operárias e rainhas estão associados aos padrões de cerdas das pernas de fêmeas adultas. Pernas mesotorácicas de operárias apresentam estruturas descritas como importantes na coleta de pólen, ausentes em rainhas. Por outro lado, as asas não possuem estruturas casta-específicas. No presente trabalho, análises globais de transcrição gênica por hibridação de lâminas de microarrays a partir de RNA total de pernas metatorácicas de operárias e rainhas em três estágios do desenvolvimento mostram 1952 genes diferencialmente expressos. Discos de pernas metatorácicas de larvas no início do quinto estágio larval, quando comparados aos de estágios mais tardios no desenvolvimento, têm alto níveis de transcritos de ativadores de Ubx, o qual está 25 vezes mais expresso em estágios subseqüentes do desenvolvimento. Buscas por motivos de ligação de fatores de transcrição nos promotores dos grupos de genes diferencialmente expressos revelam que os motivos para ligação de Ubx, Zeste e Twist estão super-representados em um dos conjuntos analisados. Dentro deste grupo, estão presentes genes cujos ortólogos em Drosophila são controlados por Ubx, como o caso do gene lola. Análises do processamento do mRNA de Ubx em pernas e asas de ambas as castas mostram que são produzidas três isoformas diferentes quanto à presença de dois microéxons (m1 and m2), que contêm 42 nt and 53 nt, respectivamente. A isoforma IIa-like, que contém o m2, entretanto, parece não ser capaz de produzir uma proteína Hox, já que possui um códon de terminação antes do homeodomínio. O perfil de transcrição diferencial de Ubx entre as castas está associado a apêndices que apresentam diferenças morfológicas, sendo Ubx mais transcrito em pernas meso e metatorácicas de operárias que rainhas. Quando analisadas as porcentagens de expressão de cada isoforma nos apêndices, claramente a isoforma IVa-like, sem microéxons, é a mais transcrita em todos os tecidos. Entretanto, nota-se que nas asas anteriores, onde há menos Ubx, a isoforma IIa-like é proporcionalmente mais transcrita que II nos outros apêndices. Destaca-se uma tendência à inclusão do microéxon m1 (isoforma IIIa-like) ao mRNA de Ubx transcrito em asas posteriores e pernas de rainhas em comparação a operárias, em detrimento da isoforma IVa-like. Análises do uso da região 3UTR em pupas de operárias mostram que há um microssatélite transcrito na porção distal da região 3UTR deUbx. A estrutura secundária predita agrupa separadamente as regiões codificadora e as regiões 3UTR proximal e distal. Análises de seqüenciamento de última geração revelaram que oito dos 51 microRNAs com sítios-alvo preditos na região 3UTR de Ubx estão mais expressos em asas anteriores, e outros dois em asas posteriores. Assim, nossos resultados mostram que o controle da expressão diferencial de Ubx é dada pela ativação desse gene por fatores de transcrição que se ligam ao promotor, controle do splicing alternativo, e expressão de microRNAs diferencial em cada casta e apêndice, controlando, assim, a morfogênese diferencial dos apêndices de fêmeas observada em A. mellifera. / Along with differences in physiological and behavioral characteristics, workers and queens of Apis mellifera also differ in appendage morphology. Some appendage specializations in the hind legs of honeybee workers, which are highly specialized pollinators, deserve special attention. The hind tibia of the worker has an expanded bristle-free region used for carrying pollen and propolis, the corbicula. In queens, this structure is absent. Although these morphological differences have been well characterized, the genetic inputs triggering the development of this alternative morphology have remained unknown. Through microarray analysis, we detected 1,952 genes that are differentially expressed during worker versus queen hind leg development. The gene expression signatures of the two castes have similar patterns of genes controlling development. At the beginning of the last larval instar, Ultrabithorax (Ubx) activators are more strongly expressed than in prepupae and early pupae; at this time Ubx expression is approximately 25 times higher. Within the gene expression signature, we identified a cluster formed by genes in which Ubx, Twist and Zeste binding sites are over-represented. This cluster includes genes for which Drosophila orthologs are known to be bound by Ubx, as in the case of lola. We also tested the extent of Ubx mRNA processing during wing and leg development. Unexpectedly, we found Ubx alternative splicing in both workers and queens; there were two microexons (m1 and m2) encoding 42 nt and 53 nt, respectively, arguing against the hypothesis that alternative splicing occurs exclusively within the Diptera. Inclusion of the m2 exon inserts a stop codon upstream from the exon containing the homeodomain, producing a truncated protein. Moreover, these bee microexons conserve the nucleotides known to be important for alternative splicing in Drosophila. During bee wing development, Ubx mRNA isoforms are transcribed in similar amounts in both castes; however, during leg development, queens produce 60% of the Ubx levels transcribed by workers. Analysis of 3UTR usage during bee development revealed a microsatellite region transcribed within the Ubx 3UTR. The predicted secondary structure locations separated the coding region into three branches and the proximal and the distal 3UTR regions. Deep-sequencing analysis revealed that eight out of 51 miRNAs predicted to target the Ubx mRNA are more highly expressed in worker forewings and two are more expressed in the hindwings. Therefore, we conclude that Ubx differential expression is activated by transcription factors that bind to its promoter, by control of alternative splicing, and moreover by microRNAs differentially expressed according to tissue and caste, resulting in differential morphogenesis of the hind leg in honeybee females.
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Desenvolvimento de marcadores SSR e SNP em maracujá-doce a partir de uma biblioteca enriquecida com genes de resposta à Xanthomonas axonopodis / Development of SSR and SNP markers in sweet passion fruit from a library enriched for genes induced in response to Xanthomonas axonopodisZirlane Portugal da Costa 15 July 2014 (has links)
Um dos desafios atuais da pesquisa em frutíferas tropicais é incorporar abordagens baseadas em marcadores moleculares nos programas convencionais de melhoramento. O maracujá-doce (Passiflora alata) é uma espécie diploide, de fecundação cruzada e pouco explorada. Recentemente, nosso grupo construiu um mapa de ligação de P. alata composto de diferentes tipos de marcadores moleculares. Além disso, dispõe-se de um conjunto de transcritos de Passiflora edulis, obtidos a partir de duas bibliotecas de expressão: forward e reverse onde foram isolados transcritos diferencialmente expressos na planta inoculada com Xanthomonas axonopodis (Xap) (672) e na planta controle, não inoculada (310), respectivamente. Assim, neste estudo, este conjunto de transcritos foi explorado visando ao desenvolvimento de marcadores SSR e SNP com o intuito de enriquecer, posteriormente, o mapa de ligação de P. alata com marcadores funcionais putativos. Para o desenvolvimento dos marcadores SSRs, as 672 sequências da biblioteca forward foram investigadas e em 91 delas foram encontrados 115 SSRs. Como esperado, a classe de repetições trinucleotídicas foi a mais abundante, sendo o motivo (AG)n o mais comum entre as repetições dinucleotídicas. Desenhou-se primers para amplificar 42 desses SSRs. Dois acessos de P. edulis e seis indivíduos da população de mapeamento de P. alata foram usados nos testes de transferibilidade e avaliação do polimorfismo. Trinta e quatro pares de primers apresentaram bom padrão de amplificação, porém apenas 10 deles revelaram polimorfismo em P. alata. Para o desenvolvimento dos marcadores SNPs, 118 sequências selecionadas das bibliotecas de expressão forward e reverse foram usadas para o desenho de primers; 37 delas foram usadas para avaliar o polimorfismo no mesmo set de indivíduos de P. alata. Foram encontrados 34 locos contendo SNPs bialélicos em 16 fragmentos gênicos, cujas sequencias variaram em tamanho de 332 a 872 pb. Considerando todos os fragmentos gênicos (16), foi analisado um total de 10.003 pb; a frequência de SNPs foi estimada como sendo 1 a cada 294 pb. Observou-se a mesma ocorrência de SNPs (50%, 17/34) em regiões codantes e não-codantes. Uma função putativa pôde ser atribuída a todos os fragmentos gênicos de P. alata, sendo que 82% mostraram homologia com as mesmas proteínas das sequências de origem, isoladas de P. edulis. No geral, os locos marcadores apresentaram baixo nível de polimorfismo molecular. Este é o primeiro trabalho sobre o desenvolvimento de locos marcadores funcionais putativos em Passiflora usando transcritos expressos em resposta à Xap. / One of the current challenges of tropical fruit research is to incorporate molecular marker-based approaches into conventional breeding programs. The sweet passion fruit (Passiflora alata) is a diploid, outcrossing and underexploited species. Recently, our group has constructed a P. alata linkage map consisted of different types of molecular markers. Moreover, we have a set of transcripts of Passiflora edulis obtained from two expression libraries: the forward and the reverse where differentially expressed transcripts were isolated from a plant inoculated with Xanthomonas axonopodis (Xap) (672), and from the control plant, uninoculated (310), respectively. Thus, in this study, this set of transcripts were exploited aiming at the development of SNP and SSR markers for future enrichment of the P. alata linkage map with putative functional markers. For the development of SSR markers, the 672 sequences from the forward library were investigated and 91 of them were found to have 115 SSRs. As expected, the trinucleotide class of repeats was the most abundant, and the (AG)n motif was the most common among the dinucleotide repeats. Primers were designed to amplify 42 of these SSRs. Transferability tests and polymorphism investigation were carried out using two accessions of P. edulis and six individuals of the mapping population of P. alata. Thirty-four primer pairs showed a good amplification pattern but only 10 loci revealed polymorphism in P. alata. For the development of SNP markers, 118 sequences selected from forward and reverse expression libraries were used for designing primers; 37 were used to assess the polymorphism in the same set of individuals of P. alata. Thirty-four biallelic SNPs were found in 16 gene fragment sequences that ranged in size from 332 to 872 bp. Considering all gene fragments, a total of 10,003 bp was obtained; the frequency of SNPs was estimated to be 1 every 294 bp. The same prevalence of SNPs (50%, 17/34) was observed within coding and non-coding regions. A putative function was assigned to all gene fragments of P. alata; 82% of them have shown homology to the original protein sequences isolated from P. edulis. Overall, the marker loci showed a low level of molecular polymorphism. This is the first report on the development of putative functional marker loci in Pasiflora using transcripts induced in response to Xap.
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Etude de la convection naturelle turbulente en cavité verticale différentiellement chauffée : Analyse des structures et des transferts turbulents / Study of the Turbulent Natural Convection in a Differential Heated Cavity : Analysis of the Turbulent structures and TransfersBelleoud, Pierre 05 July 2016 (has links)
Les écoulements de convection naturelle en espace confiné sont généralement turbulents (grandes dimensions et/ou écarts de température importants). L'approfondissement de la connaissance de ce régime et des transferts qui y sont associés semble donc essentielle. Cette étude expérimentale concerne les mécanismes de transferts thermiques turbulents en s'appuyant sur des mesures couplées vitesse/température dans un écoulement de convection naturelle turbulente à haut nombre de Rayleigh (Ra1-1= 1,2x1011) au sein d'une cavité différentiellement chauffée de rapport de forme vertical (hauteur/largeur) égale à 4. Une chaîne de mesure PIV permet les acquisitions de vitesse tandis que la température est mesurée par micro-thermocouple de type K (0=12,7 μm). Les deux mesures étant synchronisées via un générateur de pulses type BNC®. Une attention particulière a été portée à la détermination des conditions aux limites en température des parois adiabatiques et au post traitement des données de PIV à l'aide d'une décomposition orthogonale basée sur l'énergie cinétique des champs de vitesse (POD). Cela a rendu possible, d'une part le calcul expérimental de quantités liées à la turbulence comme les flux d'enthalpie et des nombres adimensionnés pour la turbulence (Prandtl, diffusivité ... ) et, d 'autre part, l'évaluation de la contribution des autres termes des équations de Navier-Stokes (gradient de pression et terme source volumique). Enfin, une discussion sur les échelles caractéristiques de l'écoulement est menée et une comparaison avec des résultats de simulations numériques est apportée. / Natural convection flows in confined spaces are often turbulent (large dimensions and/or temperature difference ). Improving the knowledge of this type of regime and of the associated heat and mass transfers seems, therefore, to be essential. This experirnental work studies heat transfer mechanisms using coupled and synchronized measurernents of temperature and velocity in a turbulent natural convection flow at high Rayleigh number (Ra1-1=I.2 x1011) in a differentially heated cavity with a vertical aspect ratio (height/width) of 4. Velocity measurernents are acquired by PIV and temperature is measured with K-type micro-thermocouple (0=12.7 μm). Both measurementsystems are synchronized using a BNC® pulse generator. Thermal boundary conditions of the adiabatic walls and post treatment of PIV data using an orthogonal decomposition based on the kinetic energy of the flow - POD - have been considered with caution. It makes possible, on the one hand, the experimental assessment of turbulent quantities like enthalpy fluxes and dimensionless nurnber (Prandtl, diffusivity ... ) and, on the other hand the estimation of the contribution of the other terms of Navier-Stokes equations (pressure gradient and source term). Finally a discussion on characteristic scales of the flow and a comparison with numerical simulations are provided.
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Flots géodésiques et théorie des modèles des corps différentiels / Geodesic Flows and Model Theory of Differential FieldsJaoui, Rémi 30 June 2017 (has links)
Le travail de cette thèse a pour objet les interactions entre deux approches d'étude des équations différentielles: la théorie des modèles des corps différentiellement clos d'une part et l'étude dynamique des équations différentielles réelles d'autre part. Dans le premier chapitre, on présente un formalisme d'algèbre différentielle, en termes de D-schémas à la Buium au-dessus du corps des nombres réels (muni de la dérivation triviale), qui permet de rendre compte de ces deux approches d'étude en même temps. Le résultat principal est un critère d'orthogonalité aux constantes pour le type générique d'une D-variétés réelle absolument irréductible, basé sur la dynamique topologique de son flot réel analytique associé. Le deuxième chapitre est consacré aux équations différentielles algébriques décrivant le flot géodésique de variétés algébriques réelles munies de 2-formes symétriques non-dégénérées. A l'aide du critère précédent, on démontre un théorème d'orthogonalité aux constantes "en courbure strictement négative'', s'appuyant sur les résultats d'Anosov et de ses successeurs concernant la dynamique topologique - la propriété de mélange topologique faible - du flot géodésique d'une variété riemannienne compacte à courbure strictement négative. En dimension 2, on conjecture en fait une description plus précise - son type générique est minimal de prégéométrie triviale - de la structure associée aux équations différentielles géodésiques unitaires. On présente, dans le troisième chapitre, des motivations et des résultats partiels concernant cette conjecture. / This thesis is dedicated to studying the interactions between two different approaches regarding differential equations: the model-theory of differentially closed fields on the one side and the dynamical analysis of real differential equations, on the other side. In the first chapter, we present a formalism from differential algebra, in terms of D-varieties à la Buium over the field of real numbers (endowed with the trivial derivation), that allows one to realise both approaches at the same time. The main result is a criterion of orthogonality to the constants, based on the topological dynamic of its associated real analytic flow. The second chapter is dedicated to the algebraic differential equations describing the (unitary) geodesic flow of a real algebraic variety endowed with an algebraic, non-degenerated symmetric 2-form. Using the previous criterion, we prove a theorem of orthogonality to the constants "in negative curvature'', that relies on the results of Anosov and of his followers, regarding the topological dynamic - the weakly mixing topological property - for the geodesic flow of a compact Riemannian manifold with negative curvature. In dimension 2, we conjecture a more precise description - its generic type is minimal and has a trivial pregeometry- for the structure associated to the unitary geodesic equation. In the third chapter, we present some motivations and partial results on this conjecture.
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