O objetivo desse experimento foi verificar diferentes planos nutricionais através da utilização de diferentes tipos de levedura na alimentação de frangos de corte no período de 1 a 42 dias. Foram utilizadas leveduras autolizada, hidrolizada e levedura íntegra e sua influência sobre parâmetros de desempenho (ganho de peso, consumo de ração e conversão alimentar) e características de carcaça (rendimento de carcaça, rendimento de peito e rendimento de pernas). Foram utilizados 1.080 pintinhos de corte, machos de um dia de idade, da linhagem Cobb, distribuídos em delineamento experimental inteiramente casualizado, em esquema fatorial 3 x 3: 3 formas de suplementação de levedura no período de 1 a 14 dias (sem levedura, levedura autolisada e levedura hidrolisada) e 3 formas de suplementação no período de 15 a 42 dias (sem suplementação, levedura autolisada e levedura íntegra) mais um tratamento controle positivo, com a adição de um antibiótico promotor de crescimento (APC), totalizando 10 tratamentos, 9 repetições e 12 aves por unidade experimental. As dietas utilizadas foram formuladas a base de milho e farelo de soja, sendo utilizado como APC a bacitracina de zinco na inclusão de 0,5 kg/ton em todas as dietas. No período de 1 a 14 dias foi utilizado o nível de inclusão de 10kg/ton das leveduras autolisada e hidrolisada e no período de 15 a 42 dias, 5kg/ton das leveduras autolisada e íntegra. As análises estatísticas dos dados foram realizadas pelo método da análise de variância com o auxílio do procedimento GLM do SAS (2002) e em caso de significância estatística, as médias foram comparadas pelo teste de Tukey, no nível de 5% de probabilidade. No período de 1 a 14 dias a levedura hidrolisada apresentou melhor viabilidade de uso na dieta. Para a utilização de um programa contínuo, a combinação do uso da levedura hidrolisada no período de 1 a 14 dias e de levedura autolizada no período de 15 a 42 dias, apresenta-se como melhor resposta pelas aves. / The objective of this experiment was to evaluate different nutritional plans using various types of yeasts in broiler chicken feed during a 42-day period. We used autolyzed, hydrolyzed, and whole yeasts and evaluated their influence on performance parameters (weight gain, feed intake, and food conversion) and carcass features (whole chicken yield, breast yield, and leg yield). A total of 1080 1-day-old male Cobb lineage chicks were used and distributed across a completely randomized 3 x 3 factorial experimental design. The treatments were comprised of 3 yeast supplement types during days 1 to 14 (i.e., without yeast, autolyzed yeast, and hydrolyzed yeast), 3 supplement types during days 15 to 42 (as above), and growth-promoting antibiotic (GPA) as positive control for a total of 10 treatments, 9 replications, and 12 birds per experimental unit. The diets were formulated based on corn and soybean meal, and 0.5 Kg/ton of zinc bacitracin was included as the GPA in the control diets. We used 10 Kg/ton of autolyzed and hydrolyzed yeast in days 1 to 14, and 5 Kg/ton of autolyzed and whole yeast during days 15 to 42. Statistical data analyses were conducted using an analysis of variance and the SAS GLM procedure (2002), and statistically significant means were compared using Tukey´s test at the 5% probability level. In days 1 - 14, the hydrolyzed yeast presented the best viability of use in the chicken diet. As an ongoing program, the birds responded best to the combination of hydrolyzed yeast in days 1 to 14 and autolyzed yeast in days 15 to 42.

Orientador: Lucia Elvira Alvares / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T18:28:57Z (GMT). No. of bitstreams: 1 Grade_CarlaVermeulenCarvalho_D.pdf: 3007033 bytes, checksum: 522d9d385305f8744861d38f7dabf5aa (MD5) Previous issue date: 2013 / Resumo: A Miostatina é um regulador negativo da deposição de musculatura esquelética e mutações no gene que codifica esta proteína têm sido associadas a um aumento marcante na massa muscular de organismos vertebrados, resultado de hiperplasia e hipertrofia das fibras musculares. Nosso grupo identificou previamente o promotor basal do gene da Miostatina e análises de bioinformática revelaram a presença de sítios evolutivamente conservados para a ligação de CREB, Meis, FXR e NFY, além de um sítio TATA. No presente trabalho nós utilizamos mutagênese sítio-dirigida para gerar diversas construções delecionais que possuem um ou mais sítios mutados, e testamos sua atividade in vitro usando mioblastos C2C12 de camundongo sob condições de proliferação e diferenciação, para analisar o papel destes sítios de ligação sobre a atividade do promotor. Os resultados mostraram que FXR aparentemente não confere efeito na atividade transcricional do promotor da Miostatina em ambos os momentos analisados, indicando que o papel regulador desta proteína pode estar relacionado ao controle da expressão da Miostatina em outro tipo celular, que não o mioblasto. O NFY apresentou um papel de ativador transcricional, enquanto CREB e Meis atuaram inicialmente como repressores durante a proliferação, passando a relaxar esta repressão durante a diferenciação dos mioblastos, permitindo que a atividade do promotor aumentasse significativamente. Trabalhando juntos, estes fatores de transcrição são capazes de manter a atividade do promotor em níveis mais baixos durante a proliferação dos mioblastos e, com o início da diferenciação, a repressão é liberada, e os níveis de atividade podem aumentar. Este padrão está de acordo com o padrão de expressão dinâmico observado para a proteína da Miostatina durante o desenvolvimento da musculatura esquelética em vertebrados / Abstract: Myostatin is a negative regulator of skeletal muscle deposition and mutations in the gene that encodes this protein have been associated to a remarkable increase in skeletal muscle mass, attributable to both hyperplasia and hypertrophy. We have previously identified Myostatin's basal promoter and bioinformatic analyses revealed the presence of evolutionarily conserved binding sites for CREB, Meis, FXR and NFY, besides a TATA box. In the present study we used site-directed mutagenesis to generate several expression constructs possessing one or more mutated sites, and tested their activity in vitro using mouse C2C12 myoblasts in proliferation and differentiation conditions, to analyze the role of these sites on the activity of the promoter. The results show that FXR appears not to confer any effect on the transcriptional activity of the promoter in both conditions, indicating that the regulatory role of this protein might be involved in the control of Myostatin expression in another cell type. NFY presents a role as transcriptional activator, while CREB and Meis act initially as repressors during proliferation, releasing this repression upon differentiation, which allows the activity of the promoter to significantly increase. Working together, these transcription factors are capable of maintaining the promoter activity at lower levels during the proliferation of myoblasts and, upon differentiation, the repression is released, and activity levels can be increased. This pattern is in agreement with the dynamic expression pattern observed for Myostatin during the skeletal muscle development in vertebrates / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Abstract Replicative DNA polymerases (pols) synthesize chromosomal DNA with high accuracy and speed during cell division. In eukaryotes the process involves three family B pols (α, δ, ε), whereas in Archaea, two types of pols, families B and D, are involved. In this study the B-subunits of replicative pols were analysed at the DNA, RNA and protein levels. By cloning the cDNAs for the B-subunits of human and mouse pol ε we were able to show that the encoded proteins are not only homologous to budding yeast pol ε, but also to the second largest subunit of pol α. Later studies have revealed that the B-subunits are conserved from Archaea to human, and also that they belong to the large calcineurin-like phosphoesterase superfamily consisting of a wide variety of hydrolases. At the mRNA level, the expression of the human pol ε B-subunit was strongly dependent on cell proliferation as has been observed for the A-subunit of pol ε and also for other eukaryotic replicative pols. By analysing the promoter of the POLE2 gene encoding the human pol ε B-subunit we show that the gene is regulated by two E2F-pocket protein complexes associated with the Sp1 and NF-1 transcription factors. Comparison of the promoters of the human pol ε and the pol α B-subunit indicates that the genes for the B-subunits may be generally regulated through E2F-complexes whereas adjustment of the basal activity may be achieved by distinct transcription factors. To clarify the function of the B-subunits, we screened through the expression of 13 different recombinant B-subunits. Although they were mainly expressed as insoluble proteins in E. coli, we were able to optimize the expression and purification for the B-subunit (DP1) of Methanococcus jannaschii pol D (MjaDP1). We show that MjaDP1 alone was a manganese dependent 3'-5' exonuclease with a preference for mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreader of archaeal pol D. So far, pol D is the only pol family utilising an enzyme of the calcineurin-like phosphoesterase superfamily as a proofreader.

Abstract Clarification of the mechanisms of cardiac-specific gene expression provides not only basic knowledge about how the gene expression is regulated in the heart, but also about the changes in the gene expression during the development of cardiovascular diseases. The purpose of this study was to analyze the mechanisms of cardiac chamber-specific gene expression and cardiac gene activation induced by mechanical load. In the present study, the experiments were carried out by using two cardiac genes, salmon cardiac peptide (sCP) and rat B-type natriuretic peptide (BNP) genes as models. sCP was discovered previously in our laboratory and turned out to be extremely cardiac-specific, representing A-type natriuretic peptide characters in an exaggerated way. In neonatal rat cardiomyocytes, the sCP promoter activity was shown to be strictly restricted to atrial cells and the promoter to be inert to cardiac hypertrophy-inducing factors. In order to find out the mechanisms of earlier proved BNP gene activation by mechanical load, BNP promoter activity was studied in vivo in adult rat hearts. The tandem GATA transcription factor binding site at position -80/-91 was shown to be essential for the BNP gene induction by angiotensin II. To clarify the possiblity to transfer the characters of the BNP gene into the sCP gene, short BNP fragments were inserted to the sCP gene promoter. The otherwise atrial-restricted sCP promoter was shown to be switched on in rat ventricular cardiomyocytes by adding a short BNP proximal promoter element to the sCP promoter, preferably near to the transcription start site. This activity was partly dependent on the -80/-91 GATA sites in the BNP promoter. Thus, A-type natriuretic peptide regulation can be switched to B-type regulation by a short proximal BNP promoter element. In conclusion, these studies reveal certain basic differences in cardiac atrial and ventricular gene expression.

Previously studies used correlation of DNase I hypersensitivity sites sequencing (DNase-seq) experiments to predict interactions between enhancers and its target promoter gene. We investigate the correlation methods Pearson’s correlation and Mutual Information, using DNase-seq data for 100 cell-types in regions on chromosome one. To assess the performances, we compared our results of correlation scores to Hi-C data from Jin et al. 2013. We showed that the performances are low when comparing it to the Hi-C data, and there is a need of improved correlation metrics. We also demonstrate that the use of Hi-C data as a gold standard is limited, because of its low resolution, and we suggest using another gold standard in further studies.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of the present document was carried an experiment and a metaanalysis, involving the use of vegetal extract for sows in gestation, lactation and piglets at nursery phase. The study with sows was carried with thirty genetically homogeneous sows were distributed in two treatments: one control diet (CD) and a CD plus 160ppm of saponins. The experimental design was a completely randomized block. The studied variables in sows were feed supply, fecal characteristics (color and consistency) and corporal score. In the piglets the studied variables were mummified fetuses, stillborn, born alive and total born, birth and weaning weights. Were not found differences in color for sows feces. The fecal consistency for sows that received diets with saponins was more solid. In the end nursing the sows fed with saponins presented a corporal score higher compared to the control females. The piglets of sows that received diets with saponins presented higher weights on birth (1.2 x 1.4kg) and at weaning (5.5 x 5.9kg) weights, both at P<0.05. Sows fed in the last 15 days of gestation and during the nursing period with diets containing 160ppm of saponins, had better corporal score in the end of nursing and their piglets had higher birth and weaning weights. In meta-analyses study were utilized eleven publications containing 48 treatments and 2752 piglets. The treatments were separated in two groups: synthesis antimicrobial (AS) and vegetal extracts (EV). Were analyzed the experimental variables and piglet s performance. For experimental variables no differences (P>0.05) were found. The average piglets number for each treatment was 57, the average experimental period was 35 days and average initial age was 25 days. The average initial weight (7.4kg) was 16% greater (P<0.05) for piglets that received vegetal extracts. The average final weight was 25kg. The average nutritional values (P<0.05) were: 3345 kcal of EM/kg, 0.39% of methionine, 1.42% of 9 lysine and 21.8% of crude protein. Were no differences (P>0.05) among piglet groups for average feed intake (818g/d), average daily gain (480g/d) and gain ratio (1.70) (P>0.05) Post weaning piglets feeding with diets containing vegetal extracts and synthesis antimicrobials don t change: feed intake, daily gain, gain ratio, ingestion of metabolizable energy, crude protein, lysine, methionine, calcium and phosphorus. The live weight is not the best predictor to estimate performance variables for post weaning piglets feed with diets containing vegetal extracts and synthesis antimicrobials, being the meta-analysis better to analyses these variables. / O objetivo do presente documento é apresentar um experimento e uma meta análise sobre a utilização de extratos vegetais para porcas em gestação/lactação e leitões na creche. O estudo com porcas envolveu trinta fêmeas geneticamente homogêneas que distribuídas em dois tratamentos (dieta controle e dieta com adição de 160ppm de saponinas). O delineamento foi de blocos ao acaso, tendo como fator de bloqueamento a ordem de parto. Foram avaliados o consumo de ração, a cor e consistência das fezes e o escore corporal. Nos leitões lactentes foram avaliados os nativivos, natimortos, mumificados, pesos (ao nascer e ao desmame) e mortalidade. Não foram encontradas diferenças na cor das fezes das fêmeas, porém as fezes das fêmeas que receberam dieta com saponinas foram mais duras. Na última semana de lactação as fêmeas alimentadas com saponinas apresentaram um escore corporal superior as fêmeas que receberam dieta controle. Não foram encontradas diferenças (P>0,05) nos leitões para as variáveis de nascimento. Os leitões das fêmeas que receberam dietas com saponinas foram mais pesados (P<0,05) ao nascer (1,2 x 1,4kg) e ao desmame (5,5 x 5,9kg). Porcas alimentadas na fase final de gestação e na lactação com dietas com 160ppm de saponinas têm melhor escore corporal no final da lactação e suas leitegadas são mais pesadas ao nascer e ao desmame. No estudo de meta-análise foram utilizadas 11 publicações contendo 48 tratamentos e 2752 animais. Os tratamentos foram agrupados antimicrobianos de síntese (AS) e extratos vegetais (EV). Não houve diferença (P>0,05) para as variáveis experimentais. O número médio de animais por tratamento foi de 57, o período experimental médio foi 35 dias e a idade inicial de 25 dias. O peso vivo médio inicial foi de 7,4kg, sendo 16% superior (P<0,05) para leitões que receberam extratos vegetais. O peso vivo médio final foi de 25kg. Os valores nutricionais médios (P<0,05) foram de 3345 kcal EM/kg, 0,39% de metionina, 7 1,42% de lisina e 21,8% de proteína bruta. O consumo médio de ração de 818g/d, o ganho de peso de 480g/d e conversão alimentar de 1,70 não diferiram (P>0,05) entre os dois aditivos. A alimentação de leitões na creche com dietas contendo determinados antimicrobianos de síntese e extratos vegetais não altera o consumo de ração, ganho de peso, conversão alimentar, ingestão de energia metabolizável, proteína bruta, lisina, metionina, cálcio e fósforo. O peso vivo não é um estimador adequado para as variáveis de desempenho de leitões na creche alimentados com aditivos antimicrobianos de síntese e extratos vegetais, sendo a meta-análise mais adequada para estimar essas variáveis.

Abstract Gene products that are only expressed in one tissue or cell type are useful models for investigating the biochemical and molecular mechanisms of tissue/cell-specific gene regulation. The regulatory regions of such genes are also practical tools in gene therapy. In this work, prostate-specific and androgen-dependent gene regulation was investigated by using human prostatic acid phosphatase (hPAP) and rat probasin (rPB) as models. In DNase I footprinting, a protected 12 bp region was found in the PB gene between the nucleotides -251 and -240 only with nuclear extracts of prostatic origin. The sequence of this area was GAAAATATGATA. Weak interaction could be detected between the DNA-binding domain of AR and the prostatic transcription factor. The results also suggested that the prostatic regulatory protein makes AR binding to its response element more effective and concomitantly magnifies the effect of androgen. A hPAP construct containing the sequence between the nucleotides -734 and +467 in front of the CAT reporter gene was highly expressed in the prostate of transgenic mice. Five homologues (A-E) for our previously identified prostate-specific GAAAATATGATA DNA-binding site were found in the area where the sites C and E could bind the regulatory protein in EMSA. The prostatic transcription factor complex bound to the GAAAATATGATA site was purified and characterized from a suspension-adapted mass culture of PC-3 prostate cancer cells by using sequence-specific DNA affinity chromatography, mass spectrometry and supershifts. Several potential transcription factors were identified, but only USF2 was confirmed to be part of the transcription factor complex. Two PC-3 cell line variants (anchorage-dependent and suspension-adapted, anchorage-independent variants) were used as a model for advanced, androgen-independent prostate cancer. Genes that were overexpressed in a suspension-adapted PC-3 cell line were further investigated, since they can be considered as putative markers of metastatic activity. The macrophage inhibitory cytokine-1 (MIC-1) gene, which was overexpressed in the suspension-adapted PC-3 cell line, was further investigated in order to clarify the mechanism behind aggressive cell growth and androgen-independent gene regulation. In patient specimens, MIC-1 had no or low expression in benign prostatic hyperplasia and normal prostate but high in prostatic cancer and therefore it could be a useful marker for aggressive prostate cancer. Indomethacin increased the expression of MIC-1 in PC-3 cells, and apoptosis was also induced in this cell line but not in saPC-3 cell line suggesting a block in MIC-1 inducible apoptosis pathway.

Abstract Mammalian extracellular matrix metalloproteinases, MMPs, are a family of enzymes capable of degrading components of the connective tissue. The in vivo regulation of the cell lineage-specific expression of MMPs, however, is not well known. This study used transgenic mice to identify cell-specific elements in the upstream regulatory regions of MMP-2 and MMP-9. Transgenic mice were generated by pronuclear microinjections into fertilised oocytes using lacZ as a reporter gene. The reporter gene constructs containing varying lengths of the MMP-9 5'-upstream region revealed an area that allowed for expression in osteoclasts and migrating keratinocytes, the cells that also express MMP-9 in vivo. The sequence driving the cell specific expression included the nucleotides from -2722 to -7745. When the same upstream regulatory fragment of MMP-9 was used to drive the expression of the human tissue specific inhibitor of MMPs, TIMP-1, instead of lacZ, the transgenic mice developed normally and the animals were fertile with normal post-embryonic growth. However, cutaneous wound healing was remarkably retarded, but not totally prevented, and the migration of keratinocytes over the wound was slow. The mice expressed the human TIMP-1 in keratinocytes during wound healing and in situ zymography revealed a total blockage of the gelatinolytic activity of MMP-2 and MMP-9, the main gelatinases active in the healing wound tissues. By using a sequence of 6500 base pairs from the 5'-upstream regulatory region of the MMP-2 gene it was possible to drive the expression of lacZ in mesenchymal cells of the developing transgenic mouse embryo. The expression pattern was similar to that found in previous in situ hybridization studies, following the different stages of tissue morphogenesis and being present in the areas of basement membrane degradation and epithelial cell invasion. Computer analyses of the sequence revealed three regulatory upstream regions conserved between human, mouse, and rat, and possibly responsible for the cell-and tissue specificity. New transgene constructs containing fragments of the conserved regions will provide a more detailed profile of the in vivo MMP-2 regulation in the future. This study defined a fragment in the upstream regulatory region of MMP-9 that is essential for expression in osteoclasts and migrating keratinocytes. Furthermore, the keratinocyte derived MMPs, including MMP-9, were found to play important role in epithelial cell migration in the area of the healing wound.

Abstract Overlapping genomic clones coding for the α1 chain of mouse type XV collagen (Col15a1) were isolated. The gene was found to be 110 kb in length and to contain 40 exons. Analysis of the proximal 5'-flanking region showed properties characteristic of a housekeeping gene promoter, and functional analysis identified cis-acting elements for both positive and negative regulation of Col15a1 gene expression. The general exon-intron pattern of the mouse Col15a1 gene was found to be highly similar to that of its human homologue, and comparison of 5'-flanking sequences indicated four conserved domains. The genomic area encoding the end of the N-terminal non-collagenous domain nevertheless showed marked divergence from the human form. Due to the lack of two exons coding for the N-terminal collagenous domain and a codon divergence in one exon, the mouse β1(XV) chain contains seven collagenous domains whereas the human equivalent contains nine. In order to understand the biological role of this protein, a null mutation in the Col15a1 gene was introduced into the germ line of mice. Despite the wide tissue distribution of type XV collagen, the null mice developed and reproduced normally and were indistinguishable from their wild-type littermates. After three months of age, however, microscopic analysis revealed progressive histological changes characteristic of myopathic disorder, and treadmill exercise resulted in greater skeletal muscle injury than in the wild-type mice. Irrespective of potential anti-angiogenic properties of type XV collagen-derived endostatin, the number of vessels appeared normal. Nevertheless, ultrastructural analyses revealed markedly abnormal capillaries and endothelial cell degeneration in the heart and skeletal muscle. Perfused hearts showed a diminished inotropic response, and exercise resulted in cardiac injury, changes that mimic early or mild heart disease. Thus type XV collagen appears to function as a necessary structural component for stabilizing cells with surrounding connective tissue in skeletal muscle cells and microvessels. Mice lacking the type XV collagen homologue, type XVIII collagen, showed delayed regression of blood vessels in the vitreous body of the eye and abnormal outgrowth of the retinal vessels. This suggests that collagen XVIII plays a role in regulating vascular development in the eye. Moreover, type XVIII collagen was found to be important at the surface between the inner limiting membrane and the collagen fibrils of the vitreous body. Col18a1 deficient mice serve as an animal model for the recessively inherited Knobloch syndrome, characterized by various eye abnormalities and occipital encephalocele. The results presented in this thesis indicate diverse biological roles for the closely related collagen types XV and XVIII.