Orientador: Lúcia Elvira Alvares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T20:53:58Z (GMT). No. of bitstreams: 1 Peterlini_DennerJefferson_M.pdf: 3626461 bytes, checksum: 754caba3e8964b3c91965dc6f4912b32 (MD5) Previous issue date: 2011 / Resumo: Os membros de vertebrados representam uma aquisição importante do grupo, os quais possibilitaram a expansão destes pela Biosfera. As bases moleculares do desenvolvimento dos membros estão sob intensa investigação, e o papel de diversos genes e moléculas de sinalização começam a ser bem compreendidos tanto no contexto do estabelecimento de seus eixos quanto da padronização dos tecidos e estruturas. A família dos genes Dapper (Dpr) tem sido associada a diversos processos da embriogênese de vertebrados, desde a coordenação de movimentos morfogenéticos durante a gastrulação à morfogênese de estruturas tão distintas quanto encéfalo, olhos e coração. No entanto, pouco se sabe sobre o papel destes genes no desenvolvimento dos membros, um sítio marcante de sua expressão durante a embriogênese de vertebrados. Resultados preliminares obtidos com emprego de hibridação in situ em embrião de galinha no nosso laboratório já haviam mostrado a expressão destes genes nos membros, e isto sugeriu que eles pudessem desempenhar um papel importante na ontogênese destas estruturas. assim, os padrões de expressão dos genes Dpr1 e Dpr2 entre os estádios HH24 e HH34 da ontogênese de aves foram caracterizados por meio de hibridação in situ neste trabalho. Também foram avaliadas a expressão dos marcadores moleculares MyoD para desenvolvimento de músculo esquelético, e Sox9 para desenvolvimento de cartilagem, bem como foi feita coloração dos membros com alcian blue, que evidência matriz extra-celular de tecido cartilaginoso. Os resultados obtidos revelaram que, no estádio HH24, a expressão de Dpr1 está presente no mesênquima proximal e medial dos membros anterior e posterior, e ausente da região distal (autópode). Neste estádio, a expressão de Dpr2 é claramente associada à agregação das células mesenquimais em condensações pré-condrogênicas. No estádio HH25, transcritos de Dpr1 e Dpr2 foram localizados pela primeira vez no autópode, delimitando uma região com o formato dos moldes cartilaginosos dos dígitos em formação. No estádio HH28, o padrão de expressão de Dpr1 ainda acompanha o contorno dos dígitos, além de serem observados altos níveis de expressão nos precursores dos tarsos e carpos. Por sua vez, Dpr2 é expresso fortemente nos dígitos 1 e 5 dos membros anterior e posterior, bem como nos blastemas dos dígitos posteriores. Finalmente, no estádio HH34, transcritos Dpr1 e Dpr2 estão concentrados nas regiões das articulações dos membros em desenvolvimento, enquanto Dpr2 é expresso também em tendões e em anexos ectodérmicos em formação. Este estudo suporta fortemente a hipótese de que os genes Dpr1 e Dpr2 desempenham um papel no processo de condrogênese que antecede a formação dos ossos dos membros de aves, bem como no desenvolvimento de outras estruturas, como articulações, tendões e anexos cutâneos / Abstract: The acquisition of vertebrates limbs represents an important novelty for this group and allowed the expansion of vertebrates through the Biosphere. The molecular basis of limbs development are under intense investigation, and the role of several genes and signaling molecules begin to be understood both within the context of axis determination as well as in the patterning of tissues and structures. The Dapper (Dpr) gene family has been associated with different processes of vertebrates embryogenesis, from the coordination of morphogenetic movements during gastrulation to morphogenesis of structures as different as brain, eyes and heart. However, nothing is known about the role of these genes in limb development, am important domain of Dpr expression during the embryogenesis of vertebrates. Preliminary results of in situ hybridization in chicken embryo obtained in our laboratory had already shown the expression of these genes in limb, suggesting that they could play an important role in the ontogeny of these structures. Thus, in this study the Dpr1 and Dpr2 expression pattern was characterized by in situ hybridization between stages HH24 and HH34 of chicken development. We also determined the expression of the molecular markers MyoD (skeletal muscle) and Sox9 (cartilage) and stained limbs at the different stages with alcian blue, that labels the extracellular matrix of cartilage. The results revealed that, at stage HH24, Dpr1 expression is observed in the proximal and medial mesenchyme in the fore and hindlimb buds but avoids the autopod. At this stage, the expression of Dpr2 is clearly associated with mesenchymal condensations of pre-chondrogenic cells. At stage HH25, Dpr1 and Dpr2 transcripts were found for the first time in the autopod, delimiting a region with the shape of the cartilaginous templates of the developing digits. At stage HH28, Dpr1 is still expressed around the developing digits, and transcripts are found at high levels in the tarsi and carpi precursors. In turn, Dpr2 is expressed strongly in the first and fifth digits of the forelimbs and hindlimbs, as well as in the digit blastemas. Finally, at stage HH34, Dpr1 and Dpr2 transcripts are concentrated in the developing joints, while Dpr2 is also expressed in ectodermal tendons and developing skin appendages. This study strongly supports the hypothesis that Dpr1 and Dpr2 play a role in the process of chondrogenesis before the formation of the limb bones in birds as well as the development of other structures such as tendons and skin appendages / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Thesis advisor: Eric S. Folker / Thesis advisor: David R. Burgess / Skeletal muscle is a multinucleated cell type in which the many nuclei are precisely positioned to maximize the distance between adjacent nuclei. In order to reach this final positioning, nuclei undergo an elaborate set of movements during muscle development. The disruption of this process is evident throughout muscular dystrophies and myopathies. However, the contribution of aberrant nuclear positioning toward disease progression is unclear and the mechanisms regulating nuclear movement and positioning are poorly defined. The goal of this thesis is to determine the contribution of disease-linked genes to the regulation of nuclear movement and positioning and how these mechanisms are coordinated in skeletal muscle. In this thesis, we utilize Drosophila melanogaster skeletal muscle as an in vivo model system to investigate nuclear positioning throughout muscle development and correlate aberrant nuclear positioning with a decrease in muscle function. We provide the first evidence of distinct mechanisms that are independently regulated by genes that are associated with two different muscle diseases, Emery-Dreifuss muscular dystrophy and Centronuclear myopathy (Chapter 2). We also provide evidence that Emerin-dependent regulation of the LINC complex is a critical determinant of nuclear positioning and for the first time demonstrate a division of Emerin functions among the two Drosophila emerin homologs, bocksbeutel and otefin (Chapter 3). Finally, we conduct a proof-of-concept screen to identify novel regulators of muscle development and function (Chapter 4). Together, the work presented in this thesis provides a framework to further our understanding of the mechanisms regulating nuclear movement and positioning as well as muscle development as a whole. Using the tools and techniques developed throughout this thesis, we provide novel insight into the mechanisms regulating nuclear movement and positioning and strengthen Drosophila as an in vivo model for investigating muscle development and function. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

A redução no desenvolvimento muscular de filhotes cujas mães foram submetidas ao consumo de dietas baseadas no padrão ocidental pode ser, ao menos em parte, explicada pela resistência periférica à insulina, condição na qual a atividade de proteínas relacionadas à via de sinalização intracelular sensível a esse hormônio encontra-se reduzida. A regulação positiva dessa via resulta no aumento da atividade do Alvo da Rapamicina em Mamíferos (mTOR) que atua como efetor positivo da taxa de tradução de RNAm e, consequentemente, da síntese proteica. Estudos que avaliam a atividade dessa proteína frente ao consumo crônico de dietas hiperlipídicas são escassos e controversos e, até o momento, não são conhecidos trabalhos que avaliaram esses marcadores em animais neonatos ou desmamados, provenientes de mães alimentadas com dieta hiperlipídica gestacional e pós-gestacional. O presente estudo objetiva avaliar o efeito do consumo de uma dieta hiperlipídica por ratas adultas sobre a morfologia e sobre a expressão e a fosforilação das proteínas que compõem a via de sinalização intracelular do mTOR no músculo esquelético da prole em dois momentos: nascimento e desmame. Para isso, inicialmente, 39 ratas foram distribuídas em dois grupos, de acordo com a dieta oferecida: controle (n=19) e hiperlipídica (n=20). Após o nascimento, cerca de seis filhotes por mãe foram eutanasiados para coleta de amostras e análise dos marcadores investigados. Os filhotes selecionados para dar continuidade ao experimento foram dispostos junto às mães que, por sua vez, foram distribuídas em outros quatro grupos, segundo a dieta gestacional e pós-gestacional: CON/CON (n=8); CON/HL (n=9); HL/HL (n=8); HL/CON (n=7). Ao final da lactação, os filhotes foram eutanasiados e amostras foram coletadas para análise. Os resultados obtidos indicam que, em relação aos animais neonatos, há redução das concentrações séricas de leptina e de IGF-I e aumento da fosforilação da Akt e do mTOR musculares, em resposta ao consumo materno da dieta hiperlipídica. Por sua vez, nos animais lactentes, observamos influência da dieta hiperlipídica materna pós-gestacional sobre a promoção de fenótipo obesogênico, com concomitante redução do desenvolvimento muscular e da fosforilação de proteínas alvo do mTOR em estado pós-prandial. Com base nos resultados obtidos, concluímos que a dieta hiperlipídica materna afeta a atividade do mTOR, sendo, esse efeito, dependente da idade e da condição fisiológica dos animais. / The decrease in muscle development of offspring whose mothers consume a typical Western diet can be partly explained by the progression of peripheral insulin resistance, a condition in which the activity of proteins related to the intracellular signaling pathway sensitive to this hormone is reduced. The positive regulation of this pathway results in increased activity of the Mammalian Target of Rapamycin (mTOR) that acts as a positive regulator of the rate of mRNA translation and protein synthesis. Studies that assess the activity of this protein in response to chronic consumption of high fat diets are scarce and controversial and, to date, studies that evaluated these markers in the offspring of mothers fed a high fat diet during gestational and lactation are not known. This study aims to evaluate the effect of consuming a high fat diet for female adult rats in morphology and expression and phosphorylation of proteins that comprise the intracellular signaling pathway of mTOR in skeletal muscle of offspring in two stages: birth and weaning. Therefore, initially, 39 rats were divided into two groups, according to the available diet: control (n = 19) and diet (n = 20). After birth, around six pups per mother were killed for sample collection and analysis of the markers investigated. The pups selected to continue the experiment were placed with the mothers who, in turn, were divided into four groups according to gestational and post-gestational diets: CON/CON (n = 8), CON/HL (n = 9), HL/HL (n = 8), HL/CON (n = 7). At the end of lactation, the pups were euthanized and samples were collected for analysis. The results indicate that, for the newborn animals, there is a reduction of serum leptin and IGF-I concentrations and increased phosphorylation of Akt and mTOR in muscle in response to maternal consumption of high fat diet. In turn, we found that maternal high-fat diet during lactation promoted an obese phenotype in weaned animals, with concomitant reduction of muscle development and mTOR target proteins phosphorylation in the postprandial state. Based on these results, we conclude that maternal high-fat diet affects the activity of mTOR, depending on age and physiological condition of the animals.

Resumo: A divergência genética entre Bos taurus taurus e Bos taurus indicus é estimada em ~ 250.000 anos mas, apesar dessa proximidade filogenética, estes dois grupos de bovinos apresentam diferenças marcantes em relação à taxa de crescimento muscular e a maciez da carne. De maneira que este estudo foi delineado para avaliar perfil da expressão gênica diferencial no musculo Longissimus dorsi das raças Nelore e Aberdeen Angus com o objetivo de identificar conjuntos de genes cuja expressão está associada à manifestação das características acima mencionadas. Vinte bezerros machos de cada raça foram criados e terminados em regime de confinamento, metade dos quais foi abatida aos 15 e a metade restante aos 19 meses de idade. Nossos resultados sugerem fortemente que a ação autócrina e/ou parácrina do IGF-1 produzido no tecido muscular deve exercer um papel crucial na modulação da taxa de crescimento muscular e na maciez da carne de bovinos. Por outro lado, nossa análise das redes de interação gênica mostrou um grande número de genes que codificam proteínas que agem na proteólise muscular, pequeno número de inibidores e reguladores de proteases, assim como diversos genes relacionados com a morte celular programada nos animais que apresentaram maior maciez da carne, reforçando a hipótese do envolvimento de um complexo multi-enzimático (incluindo as calpaínas e outras proteases) no processo de amaciamento da carne, o qual seria semelhante ao processo de apoptose. / Abstract: The genetic divergence between Bos taurus taurus and Bos taurus indicus is estimated to be ~ 250.000 years but, in despite of their phylogenetic proximity, this two bovine groups show remarkable differences in regard to the muscle growth rate and meat tenderness. Thus, this study was aimed to evaluate the differential gene expression profile in Longissimus dorsi muscle of Nellore and Aberdeen Angus breeds in order to identify set of genes whose expression is associated with the manifestation of the above-mentioned traits. Twenty male calves of each breed were grown and finished in the feedlot system, half of which was slaughtered at 15 and the remaining half at 19 months of age. Our results strongly suggest that the autocrine and/or paracrine action of the IGF-1 produced in the muscle might play a crucial role in the modulation of muscle growth rate and meat tenderness in beef cattle. On the other hand, our gene interaction network analysis revealed a large number of genes encoding proteins acting in the skeletal muscle proteolysis, a small number of inhibitors and regulators of proteases, as well as several genes related to the programmed cell death in animals that had greater tenderness of meat, reinforcing the hypothesis of the involvement of a multi-enzymatic complex (including calpains and other proteases) in the meat tenderization process, which resembles the apoptosis process. / Orientador: Luiz Roberto Furlan / Coorientadora: Lúcia Maria Carareto Alves / Banca: Leandro Marcio Moreira / Banca: Marcelo Luiz de Laia / Banca: Janete Apparecida Desidério Sena / Banca: Jesus Aparecido Ferro / Doutor

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-09-04T21:36:01Z No. of bitstreams: 1 2017 - Odair Scatolin Rossafa Garcia.pdf: 1812000 bytes, checksum: 37d6d5a375daf6bc6b0ef40d4687a7f7 (MD5) / Made available in DSpace on 2018-09-04T21:36:01Z (GMT). No. of bitstreams: 1 2017 - Odair Scatolin Rossafa Garcia.pdf: 1812000 bytes, checksum: 37d6d5a375daf6bc6b0ef40d4687a7f7 (MD5) Previous issue date: 2017-05-26 / Goat meat has lower levels of fat than those found in other types of meat such as beef, pork, sheep and deer, but the lack of selection criteria for slaughter, storage and commercialization of meat leads to a low level because of the lack of standardization of the product presenting unpleasant sensory characteristics. A study of polymorphism, variation in gene expression and the association of these variations with the desired phenotype allows to broaden the understanding of the physiological processes, which helps in the strategies aimed at improving the characteristic of interest, resulting in the expected final phenotype. The objective of the present study was to evaluate polymorphisms and gene expression among some of the most promising genotypes of pleiotropic genes, comparing the polymorphism and expression among groups of animals with greater and lesser weight at slaughter to verify if there is any relation with the weight difference Or softness of the flesh. For this purpose, genotypes of 40 goats from the Saanen and Alpina breeds for the growth hormone (GH) gene, diacylglycerol acyltransferase 1 gene (DGAT1), myostatin gene (MSTN), growth factor gene Similar to insulin 1 (IGF1), fatty acid carrier protein (FATP1) gene, nuclear factor 1 (NF1) gene, gamma peroxisome proliferator activated receptor (PPAR?) gene. After analyzing the association of the different genotypes with the slaughter weight (AP), carcass weight (PC) and meat softness expressed in shear force (FC), some genes were selected for the analysis of expression and association with them Variables. The GH, NF1 and PPAR genes were not evaluated for expression, the first for not having presented a good result for the efficiency analysis of the other two primers due to the lack of substantial data for the preparation of the primers. For the softness test, previously performed in another study by the same team, the longissimus lumborum muscle was used. Polymerase chain reaction (PCR) technique was used for the genotyping and later, for some genes, the digestion of the fragments amplified by restriction enzymes, a technique known as PCR-RFLP. Gene expression was conducted using the Real Time PCR technique (qPCR) and the meat tenderness phenotype was analyzed in a texturometer. The data were statistically related using the SAS GLM procedure. The UNIVARIATE procedure was used to verify the normality of residues of expression of the genes under study (expressed as 2-?Ct) and softness data. The averages were compared by the Tukey test and the Pearson correlations tested by the t test. The polymorphism already described in the GH was also detected in the population studied in the present study, the genotype heterozygous AB presented a mean 2.78kg at slaughter weight more than the AA individuals, for the MSTN the individuals with heterozygous M1M2 genotype presented higher scores for weight at slaughter, while for the IGF1 gene the heterozygous AB animals present less tender meat. The group with lower weight at slaughter showed higher expression of the DGAT1 and FATP genes, which may reflect a higher deposition of fat in the carcass and greater softness, in comparison with the group of higher weight. / A carne caprina apresenta teores de gordura abaixo dos encontrados em outros tipos de carne como a de bovino, su?no, ovino e veado. Entretanto, a falta de crit?rio de sele??o para o abate, estocagem e comercializa??o da carne, acaba por gerar um baixo n?vel de consumo, devido ? falta de padroniza??o do produto apresentando caracter?sticas sensoriais desagrad?veis. Estudo de polimorfismo, varia??o na express?o g?nica e associa??o destas varia??es com o fen?tipo desejado permite ampliar a compreens?o sobre os processos fisiol?gicos, al?m de auxiliar programas de melhoramento gen?tico animal para a sele??o de animais com fen?tipos superiores para a caracter?stica de interesse. O objetivo do presente trabalho foi avaliar a associa??o de polimorfismos e express?o g?nica com a caracter?stica peso ao abate e verificar se h? rela??o entre a diferen?a de peso e a maciez da carne. Para este prop?sito foram identificados inicialmente os gen?tipos de cabritos das ra?as Saanen e Alpina para os seguintes genes: horm?nio do crescimento (GH), diacilglicerol aciltransferase 1 (DGAT1), miostatina (MSTN), fator de crescimento semelhante ? insulina 1 (IGF1), prote?na transportadora de ?cidos graxos (FATP1), fator nuclear 1 (NF1), receptor ativado por proliferadores de peroxissomas gama (PPAR?). Ap?s a an?lise de associa??o dos diferentes gen?tipos com o peso ao abate (PA), peso da carca?a (PC) e maciez da carne expressa em for?a de cisalhamento (FC), foram selecionados alguns genes para a an?lises de express?o e associa??o com as mesmas vari?veis. Os genes GH, NF1 e PPAR n?o foram avaliados quanto a express?o, o primeiro por n?o ter apresentado um bom resultado para as analise de efici?ncia dos primers os outros dois devido ? problemas no genoma refer?ncia para a confec??o dos primers. Para o teste de maciez foi utilizado o m?sculo longissimus lumborum. Para a genotipagem foi utilizada a t?cnica da rea??o em cadeia pela polimerase (PCR) e posteriormente, para alguns genes, digest?o dos fragmentos amplificados por enzimas de restri??o (PCR-RFLP). A express?o g?nica foi conduzida utilizando a t?cnica de PCR em Tempo Real (qPCR) e o fen?tipo de maciez da carne foi analisado em textur?metro. Os dados foram analisados estat?sticamente utilizando o procedimento GLM do SAS. O procedimento UNIVARIATE foi utilizado para verificar a normalidade dos res?duos da express?o dos genes em estudo (expressos com 2-?Ct) e dados de maciez. As m?dias foram comparadas pelo teste de Tukey e as correla??es de Pearson testadas pelo teste de t. O polimorfismo j? descrito no GH foi tamb?m detectado na popula??o estudada no presente trabalho, o gen?tipo heterozigoto AB apresentou m?dia 2,78kg a mais de peso ao abate do que os indiv?duos AA, para a MSTN os indiv?duos com gen?tipo heterozigoto M1M2 apresentaram maiores escores para peso ao abate, enquanto para o gene IGF1 os animais heterozigotos AB apresentam carne menos macia. O grupo com menor peso ao abate apresentou maior express?o dos genes DGAT1 e FATP, o que pode refletir maior deposi??o de gordura de gordura na carca?a e maior maciez, em compara??o com o grupo de maior peso.

Domestic animals have become very different from their wild ancestors during domestication and animal breeding. This provides a good model to unravel the molecular mechanisms underlying phenotypic variation. In my thesis I have studied genes affecting two important traits, leanness in pigs and hair greying-associated melanoma in horses. In the first part of the thesis, I focused on an intronic mutation leading to more muscle growth and less fat deposition in domestic pigs to identify a transcription factor (TF) that binds to the regulatory element overlapping with the mutation. The aim has been to further study the function of the previously unknown TF in mouse myoblast cells and in insulin-producing cells (Paper I-III). We discovered a new TF ZBED6 binding to intron 3 of the IGF2 gene, in which a single nucleotide substitution in pigs abrogates the binding and causes increased leanness in domestic pigs. Silencing of ZBED6 expression in mouse myoblasts increased Igf2 expression, cell proliferation and migration, and myotube formation. This result is in line with the increased leanness phenotype in mutant pigs. Chromatin Immunoprecipitation-sequencing (ChIP-seq) using an anti-ZBED6 antibody identified 1200 ZBED6 target genes besides IGF2 and many are TFs controlling fundamental biological processes. In the first follow-up study we found ZBED6 mainly affected the expression of muscle protein genes by directly regulating Igf2 and Twist2 expression, in agreement with our previous observation of faster myotube formation in ZBED6-silenced cells. ChIP-seq with antibodies against six different histone modifications revealed that ZBED6 preferentially binds to active promoters and modulates transcriptional activity by a novel mechanism rather than by recruiting repressive histone modifications. The second follow-up study revealed that ZBED6 affects the morphology and insulin content and release in pancreatic ß cells. In the second part (Paper IV), we investigate the functional significance of an intronic duplication in the Syntaxin 17 (STX17) gene causing hair greying and melanoma in horses. We found two Microphtalmia-associated transcription factor (MITF) binding sites within the duplication and showed that the duplicated sequence up-regulates reporter gene expression in a melanocyte-specific manner both by reporter assays in mouse melanocytes and in transgenic zebrafish. These results established that the intronic duplication acts as a melanocyte-specific enhancer that becomes much stronger when it is duplicated.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2001. / Vita. Bibliography: 108-125.

Made available in DSpace on 2014-06-11T19:33:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-24Bitstream added on 2014-06-13T18:45:17Z : No. of bitstreams: 1 ferraz_alj_dr_jabo.pdf: 613372 bytes, checksum: 0c16778d62f1e6d94f350b8efbbbd6d8 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A divergência genética entre Bos taurus taurus e Bos taurus indicus é estimada em ~ 250.000 anos mas, apesar dessa proximidade filogenética, estes dois grupos de bovinos apresentam diferenças marcantes em relação à taxa de crescimento muscular e a maciez da carne. De maneira que este estudo foi delineado para avaliar perfil da expressão gênica diferencial no musculo Longissimus dorsi das raças Nelore e Aberdeen Angus com o objetivo de identificar conjuntos de genes cuja expressão está associada à manifestação das características acima mencionadas. Vinte bezerros machos de cada raça foram criados e terminados em regime de confinamento, metade dos quais foi abatida aos 15 e a metade restante aos 19 meses de idade. Nossos resultados sugerem fortemente que a ação autócrina e/ou parácrina do IGF-1 produzido no tecido muscular deve exercer um papel crucial na modulação da taxa de crescimento muscular e na maciez da carne de bovinos. Por outro lado, nossa análise das redes de interação gênica mostrou um grande número de genes que codificam proteínas que agem na proteólise muscular, pequeno número de inibidores e reguladores de proteases, assim como diversos genes relacionados com a morte celular programada nos animais que apresentaram maior maciez da carne, reforçando a hipótese do envolvimento de um complexo multi-enzimático (incluindo as calpaínas e outras proteases) no processo de amaciamento da carne, o qual seria semelhante ao processo de apoptose. / The genetic divergence between Bos taurus taurus and Bos taurus indicus is estimated to be ~ 250.000 years but, in despite of their phylogenetic proximity, this two bovine groups show remarkable differences in regard to the muscle growth rate and meat tenderness. Thus, this study was aimed to evaluate the differential gene expression profile in Longissimus dorsi muscle of Nellore and Aberdeen Angus breeds in order to identify set of genes whose expression is associated with the manifestation of the above-mentioned traits. Twenty male calves of each breed were grown and finished in the feedlot system, half of which was slaughtered at 15 and the remaining half at 19 months of age. Our results strongly suggest that the autocrine and/or paracrine action of the IGF-1 produced in the muscle might play a crucial role in the modulation of muscle growth rate and meat tenderness in beef cattle. On the other hand, our gene interaction network analysis revealed a large number of genes encoding proteins acting in the skeletal muscle proteolysis, a small number of inhibitors and regulators of proteases, as well as several genes related to the programmed cell death in animals that had greater tenderness of meat, reinforcing the hypothesis of the involvement of a multi-enzymatic complex (including calpains and other proteases) in the meat tenderization process, which resembles the apoptosis process.

A redução no desenvolvimento muscular de filhotes cujas mães foram submetidas ao consumo de dietas baseadas no padrão ocidental pode ser, ao menos em parte, explicada pela resistência periférica à insulina, condição na qual a atividade de proteínas relacionadas à via de sinalização intracelular sensível a esse hormônio encontra-se reduzida. A regulação positiva dessa via resulta no aumento da atividade do Alvo da Rapamicina em Mamíferos (mTOR) que atua como efetor positivo da taxa de tradução de RNAm e, consequentemente, da síntese proteica. Estudos que avaliam a atividade dessa proteína frente ao consumo crônico de dietas hiperlipídicas são escassos e controversos e, até o momento, não são conhecidos trabalhos que avaliaram esses marcadores em animais neonatos ou desmamados, provenientes de mães alimentadas com dieta hiperlipídica gestacional e pós-gestacional. O presente estudo objetiva avaliar o efeito do consumo de uma dieta hiperlipídica por ratas adultas sobre a morfologia e sobre a expressão e a fosforilação das proteínas que compõem a via de sinalização intracelular do mTOR no músculo esquelético da prole em dois momentos: nascimento e desmame. Para isso, inicialmente, 39 ratas foram distribuídas em dois grupos, de acordo com a dieta oferecida: controle (n=19) e hiperlipídica (n=20). Após o nascimento, cerca de seis filhotes por mãe foram eutanasiados para coleta de amostras e análise dos marcadores investigados. Os filhotes selecionados para dar continuidade ao experimento foram dispostos junto às mães que, por sua vez, foram distribuídas em outros quatro grupos, segundo a dieta gestacional e pós-gestacional: CON/CON (n=8); CON/HL (n=9); HL/HL (n=8); HL/CON (n=7). Ao final da lactação, os filhotes foram eutanasiados e amostras foram coletadas para análise. Os resultados obtidos indicam que, em relação aos animais neonatos, há redução das concentrações séricas de leptina e de IGF-I e aumento da fosforilação da Akt e do mTOR musculares, em resposta ao consumo materno da dieta hiperlipídica. Por sua vez, nos animais lactentes, observamos influência da dieta hiperlipídica materna pós-gestacional sobre a promoção de fenótipo obesogênico, com concomitante redução do desenvolvimento muscular e da fosforilação de proteínas alvo do mTOR em estado pós-prandial. Com base nos resultados obtidos, concluímos que a dieta hiperlipídica materna afeta a atividade do mTOR, sendo, esse efeito, dependente da idade e da condição fisiológica dos animais. / The decrease in muscle development of offspring whose mothers consume a typical Western diet can be partly explained by the progression of peripheral insulin resistance, a condition in which the activity of proteins related to the intracellular signaling pathway sensitive to this hormone is reduced. The positive regulation of this pathway results in increased activity of the Mammalian Target of Rapamycin (mTOR) that acts as a positive regulator of the rate of mRNA translation and protein synthesis. Studies that assess the activity of this protein in response to chronic consumption of high fat diets are scarce and controversial and, to date, studies that evaluated these markers in the offspring of mothers fed a high fat diet during gestational and lactation are not known. This study aims to evaluate the effect of consuming a high fat diet for female adult rats in morphology and expression and phosphorylation of proteins that comprise the intracellular signaling pathway of mTOR in skeletal muscle of offspring in two stages: birth and weaning. Therefore, initially, 39 rats were divided into two groups, according to the available diet: control (n = 19) and diet (n = 20). After birth, around six pups per mother were killed for sample collection and analysis of the markers investigated. The pups selected to continue the experiment were placed with the mothers who, in turn, were divided into four groups according to gestational and post-gestational diets: CON/CON (n = 8), CON/HL (n = 9), HL/HL (n = 8), HL/CON (n = 7). At the end of lactation, the pups were euthanized and samples were collected for analysis. The results indicate that, for the newborn animals, there is a reduction of serum leptin and IGF-I concentrations and increased phosphorylation of Akt and mTOR in muscle in response to maternal consumption of high fat diet. In turn, we found that maternal high-fat diet during lactation promoted an obese phenotype in weaned animals, with concomitant reduction of muscle development and mTOR target proteins phosphorylation in the postprandial state. Based on these results, we conclude that maternal high-fat diet affects the activity of mTOR, depending on age and physiological condition of the animals.

The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).