No description available.

No description available.

The objective of the first study was to quantify the effects of feeding Schizochytrium sp. microalgae (SP-1, Alltech, Inc., Nicholasville, KY) on milk fat and DHA content. Eight cows were fed: 0, 100, 300, or 600 g of algae per day. Fat percentage was greater (P < 0.05) for cows on treatments 0 g and 100 g than for cows on treatments 300 g and 600 g (P < 0.05). Docosahexaenoic acid in milk was greater for cows on treatment 300 and 600 than for cows on treatment 0 and 100 (P < 0.05). The objective of the second study was to assess yeast supplementation effects on high and low forage dairy cow diets. Four cows were assigned to 1 of 4 treatments: 1) low forage (LF), 2) low forage with 10 g/d yeast (Yea-Sacc®; Alltech Inc., Nicholasville, KY; LFY), 3) high forage (HF), or 4) high forage with 10 g/d yeast (HFY). Only rumination time and DMI were influenced by treatment (P < 0.01). Dry matter intake was 17.05, 13.41, 19.44, and 20.29 ± 1.40 kg/d and rumination time was 442.88, 323.09, 433.34, and 475.50 ± 21.93 min/d for cows on the LF, LFY, HF, and HFY treatments, respectively.

Hereditary myopathy with lactic acidosis (HML, OMIM#255125) is an autosomal recessive disorder which originates from Västerbotten and Ångermanland in the Northern part of Sweden. HML is characterized by severe exercise intolerance which manifests with tachycardia, dyspnea, muscle pain, cramps, elevated lactate and pyruvate levels, weakness and myoglobinuria. The symptoms arise from malfunction of the energy metabolism in skeletal muscles with defects in several important enzymes involved in the TCA cycle and the electron transport chain. All affected proteins contain iron-sulfur (Fe-S) clusters, which led to the suggestion that the disease was caused by malfunctions in either the transportation, assembly or processing of Fe-S clusters. The aim of my thesis was to identify the disease causing gene of HML and to investigate the underlying disease-mechanisms. In paper I we identified a disease-critical region on chromosome 12; a region containing 16 genes. One of the genes coded for the Fe-S cluster assembly protein ISCU and an intronic base pair substitution (g.7044G&gt;C) was identified in the last intron of this gene. The mutation gave rise to the insertion of intron sequence into the mRNA, leading to a protein containing 15 abberant amino acids and a premature stop. In paper II we investigated why a mutation in an evolutionary well conserved protein with a very important cellular role, which in addition is expressed in almost all tissues, gives rise to a muscle-restricted phenotype. Semi-quantitative RT-PCR analysis showed that the mutant transcript constituted almost 80% of total ISCU mRNA in muscle, while in both heart and liver the normal splice form was dominant. We could also show that, in mice, complete absence of Iscu protein was coupled with early embryonic death, further emphasizing the importance of the protein in all tissues. These data strongly suggested that tissue-specific splicing was the main mechanism responsible for the muscle-specific phenotype of HML. In paper III the splicing mechanisms that give rise to the mutant ISCU transcript was further investigated. We identified three proteins; PTBP1, IGF2BP1 and RBM39, that could bind to the region containing the mutation and could affect the splicing pattern of ISCU in an in vitro system. PTBP1 repressed the inclusion of the intronic sequence, while IGF2BP1 and RBM39 repressed the total ISCU mRNA level though the effect was more pronounced for the normal transcript. Moreover, IGF2BP1 and RBM39 were also able to reverse the effect of PTBP1. IGF2BP1, though not a splicing factor, had higher affinity for the mutant sequence. This suggested that the mutation enables IGF2BP1 binding, thereby preventing the PTBP1 induced repression seen in the normal case. In conclusion, we have determined the genetic cause of HML, identifying a base pair substitution in the last intron of the ISCU gene that gives rise to abnormally spliced transcript. The muscle-specific phenotype was also analyzed and tissue-specific splicing was identified as the main disease-mechanism. Furthermore, nuclear factors with ability to affect the splicing pattern of the mutant ISCU gene were identified. This work has thoroughly investigated the fundamental disease mechanisms, thus providing deeper understanding for this hereditary myopathy.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

<p>In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. Additionally, cancer cells exposed to these stresses are more resistant to therapies, more likely to metastasize and often are worse for patient prognosis. While the presence of these stresses is generally negative for cancer patients, since these stresses are mostly unique to the TME, they also offer an opportunity to develop more selective therapeutics. If we achieve a better understanding of the adaptive mechanisms cancer cells employ to survive the TME stresses, then hopefully we, as a scientific community, can devise more effective cancer therapeutics specifically targeting cancer cells under stress. To systematically identify genes that modulate cancer cell survival under stresses, we performed shRNA screens under hypoxia or lactic acidosis. From these screens, we discovered that genetic depletion of acetyl-CoA carboxylase alpha (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Furthermore, the loss of ACLY or ACC1 reduced the levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate was paradoxically increased under hypoxia when ACC1 or ACLY were depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulated the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.</p> / Dissertation

Orientador: Danilo Domingues Millen / Resumo: O objetivo foi avaliar a produção de proteína microbiana, pH e dinâmica ruminal, digestibilidade e degradabilidade ruminal de nutrientes, comportamento ingestivo, seletividade da dieta e imagens termográficas em bovinos Nelore canulados que passaram por período de restrição alimentar ou que consumiram concentrado antes da entrada no confinamento na fase de terminação. O delineamento experimental foi em quadrado latino contemporâneo 3 x 3. Foram utilizados 6 bovinos inteiros, com peso vivo aproximado de 236 ± 23 kg e com 20 meses de idade. Os tratamentos diferiram somente com relação ao tipo de dieta estabelecida na fase de pré-adaptação: Controle (volumoso ad libitum + suplemento mineral); Restrição (volumoso restrito a 1,4% do peso vivo + suplemento mineral) e Concentrado (volumoso ad libitum+ 0,5% do peso vivo de ingredientes). A duração do estudo foi em 115 dias, sendo 3 períodos experimentais (33 dias/período) e dois intervalos de washout (8 dias). Cada período foi dividido em: 14 dias de pré-adaptação, 6 dias em adaptação 1, 6 dias em adaptação 2 e 7 dias na dieta de terminação (72%, 79% e 86% de concentrado, respectivamente). Os dados deste estudo foram analisados pelo PROC MIXED do SAS (2003), sendo o teste de Tukey utilizado para comparação entre médias quando necessário, considerando o nível de 10% de significância. Conclui-se que que animais submetidos à restrição nutricional ou que consumiram ingredientes concentrados antes do confinamento apresentam comportamento ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Ampliar nosso conhecimento sobre a biologia do carcinoma espinocelular bucal é fundamental para o desenvolvimento de novas estratégias terapêuticas e para melhorar a sobrevida dos pacientes acometidos por essa patologia. Para tanto, compreender as contribuições do microambiente tumoral à carcinogênese é muito importante. Um característica importante a ser avaliada do microambiente tumoral é a acidificação do meio extracelular. Considerando que o pH ácido tumoral está relacionado à maior agressividade da lesão, o objetivo deste projeto é estudar os efeitos de um microambiente ácido na biologia de células de carcinoma espinocelular bucal. Para tanto, foram realizadas duas revisões da literatura, nas quais foram avaliados os seguintes temas: influência da acidez extracelular na invasão e migração; e os mecanismos moleculares envolvidos na resistência ao tratamento quimioterápico, induzida pela acidez do microambiente tumoral. Tais revisões embasaram a construção do terceiro artigo desta tese, o qual se propôs a comparar o comportamento de células linhagens de carcinoma espinocelular bucal (SCC-4 e SCC-9) e queratinócitos (HaCat) cultivadas em meio de cultura de pH 6.8 com células mantidas em meio neutro pH 7.4. As células foram expostas de forma contínua ou intermitente ao pH 6.8 e a adaptação das células foi avaliada pelo ensaio clonogênico. Além disso, as células foram avaliadas quanto à sua capacidade migratória pelo ensaio de cicatrização de feridas e de time lapse. A expressão gênica relacionada à indiferenciação e pluripotência foi investigada por PCR em tempo real com os marcadores Bmi-1 e CD44, assim como pelo ensaio de orosferas. A resistência desses grupos de células ao tratamento anti-câncer foi avaliada pelo ensaio de viabilidade celular da sulforodamina B após o tratamento com Cisplatina. Para a análise estatística, inicialmente foi realizada a distribuição dos dados, seguido da comparação estatística dos grupos utilizando, para distribuição normal, os testes ANOVA e ANOVA de duas vias. Toda a análise foi realizada no programa GraphPad Prism 5.0 e o nível de significância considerado foi de p< 0.05.Observamos que ambas as linhagens mudaram sua morfologia para um aspecto mesenquimal. Ao avaliar o perfil migratório observou-se que as células SCC-9 apresentaram maior capacidade de migração após a exposição ao pH6.8. O aumento da migração celular pode ser causado pela indução da transição epitélio-mesênquima, visto que observamos o aumento da expressão de N-caderina (SCC-4:p<0.05) concomitante à diminuição de E-caderina (SCC-4: p<0.05). A exposição à acidez também provocou, em ambas as linhagens, aumento da capacidade de formar orosferas em placa de baixa aderência, denotando um fenótipo pluripotente (SCC-4: p=0.007/ SCC-9: p= 0.1202). Tal resultado foi reforçado com aumento da expressão gênica do marcador de célula-tronco tumoral CD44 (p= 0.0325). na linhagem SCC-4. No entanto, observamos diminuição da expressão de Bmi-1(p=0.0572) em relação ao controle. A resistência à Cisplatina aumentou nos casos de exposição contínua à acidez (SCC-4: p<0,05). O recondicionamento em meio neutro reverteu a sensibilidade celular (SCC-4: p>0,05). Concluímos que a acidez extracelular no carcinoma espinocelular bucal aumenta a capacidade de migração, induz o fenótipo de células tronco-tumorais e aumenta a resistência a quimioterápicos. / To expand our knowledge about the biology of oral squamous cell carcinoma is crucial for the development of new therapeutic strategies and to improve the survival of the patients affected by this pathology. Therefore, understanding the contributions of the tumor microenvironment to carcinogenesis is very important. An important feature to be evaluated of the tumor microenvironment is the acidification of the extracellular medium. Considering that acidic pH is related to the greater aggressiveness of the tumor, the aim of this study is to analyze the effects of an acidic microenvironment on the biology of oral squamous cell carcinoma cells. We realized two reviews of the literature, in which the following themes were evaluated: the influence of extracellular acidity on the invasion and migration; and the molecular mechanisms involved in the resistance to chemotherapeutic treatment, induced by the acidity of the tumor microenvironment. These revisions helped to construct the third article of this thesis, which proposed to compare the behavior of squamous cell carcinoma lines (SCC-4 and SCC-9) and keratinocytes (HaCat), cultured under pH 6.8 was compared to cells maintained at pH 7.4. For the statistical analysis, the data distribution was initially performed, followed by the statistical comparison of the groups using, for normal distribution, ANOVA and ANOVA two-way tests. All analysis was performed in the GraphPad Prism 5.0 program and the level of significance considered was p <0.05.After continuous or intermittent exposure to pH 6.8, cell adaptation was assessed by the clonogenic assay. In addition, the migratory capacity of the cells was evaluated by the wound healing and time-lapse assays. The gene expression related to undifferentiation and pluripotency was assessed by real-time PCR analysis of the Bmi-1 and CD44 markers, as well as by the orosphere assay. The resistance of these cell groups to anti-cancer treatment was assessed by the sulforhodamine B cell viability assay after treatment with Cisplatin. We observed that both cell lines changed their morphology to a mesenchymal aspect. When assessed the migratory profile, it was observed that SCC-9 cells showed higher migration capacity after exposure to pH6.8. Increased cell migration may be caused by the induction of the epithelial-mesenchymal transition, as we observed increased N-cadherin (SCC-4:p<0.05) expression concomitant with decreased E-cadherin (SCC-4: p<0.05). Exposure to acidity also led to increased ability to form orospheres on low-attachment dishes, denoting a pluripotent phenotype in both strains (SCC-4: p=0.007/ SCC-9: p= 0.1202). This result was reinforced by increased expression of the CD44 (p= 0.0325) tumor cell marker in the SCC- 4 cells. However, we observed a decrease in the expression of Bmi-1(p=0.0572) in relation to the control. Resistance to Cisplatin increased when cells were continuously exposed to acidity(SCC-4: p<0,05). Neutral reconditioning reversed cell sensitivity (SCC-4: p>0,05). We conclude that extracellular acidity in oral squamous cell carcinoma increases the migration capacity, induces the cancer stem cell phenotype and increases resistance to chemotherapy.

Submitted by DANIELA DUTRA ESTEVAM null (daniela_estevam@yahoo.com.br) on 2016-02-01T14:08:30Z No. of bitstreams: 1 DISSERTAÇÃO DANIELA.pdf: 1615348 bytes, checksum: 18226c859c846688ff9fa236460a54de (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-02-01T18:01:26Z (GMT) No. of bitstreams: 1 estevam_dd_me_bot.pdf: 1615348 bytes, checksum: 18226c859c846688ff9fa236460a54de (MD5) / Made available in DSpace on 2016-02-01T18:01:26Z (GMT). No. of bitstreams: 1 estevam_dd_me_bot.pdf: 1615348 bytes, checksum: 18226c859c846688ff9fa236460a54de (MD5) Previous issue date: 2016-01-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste estudo foi avaliar os efeitos dos períodos de 6, 9, 14 e 21 dias de adaptação de bovinos da raça Nelore confinados a dietas de alto teor de concentrado em relação ao desempenho, variação na ingestão de massa seca, energia líquida, perfil metabólico sanguíneo, comportamento ingestivo, saúde ruminal e características de carcaça. Os animais foram alimentados durante 88 dias, independentemente do período de adaptação adotado e o delineamento utilizado foi em blocos casualizados, com seis repetições cada, em que 96 bovinos da raça Nelore não castrados (391,1 ± 30,9 kg) foram alimentados em 24 baias (4 animais/baia), de acordo com os diferentes períodos de adaptação adotados: 6, 9, 14 e 21 dias. O programa de adaptação utilizado foi em escadas, com três dietas ao longo de períodos de adaptação com seguintes níveis de concentrado: 70, 75, 80,5 e 86% da massa seca na dieta. Contrastes ortogonais foram utilizados para avaliar a relação linear, quadrática e cúbica entre os dias de adaptação e a variável dependente, além da interação de tratamento e fase (P≤0,10). Os animais adaptados por 14 dias apresentaram resultados superiores em relação ao ganho de peso diário, peso vivo final e eficiência alimentar, bem como peso de carcaça quente (P=0,04) e área de olho de lombo final (P=0,01). A adaptação por 14 dias também proporcionou aos animais melhor desenvolvimento do epitélio ruminal, pois apresentaram maior área de superfície absortiva (P=0,02) e largura de papilas (P=0,06). Além disso, esses animais demonstraram que estavam adequadamente adaptados, pois a renovação celular do epitélio ruminal demonstrou-se estabilizada no índice de proliferação celular (P=0,003) e nos núcleos em morte celular (P=0,0004). Com base nesses resultados, recomenda-se adaptar bovinos da raça Nelore a dietas de alto concentrado com o protocolo em escadas por 14 dias, pois este proporcionou maior desempenho produtivo e desenvolvimento do epitélio ruminal. / This study was designed to determine the effects of adaptation periods of 6, 9, 14 and 21 days on feedlot performance, dry matter intake fluctuations, energy gain, blood metabolic profile, feeding behavior, rumen health and carcass traits of Nellore cattle. Cattle were fed for 88-d regardless of adaptation period adopted and the experiment was designed as a completely randomized block, replicated 6 times, in which ninety-six 20-mo-old yearling Nellore bulls (391.1 ± 30.9 kg) were fed in 24 pens (4 animals/pen) according to the different adaptation periods adopted: 6, 9, 14, and 21 days. The adaptation program consisted of ad libitum feeding of 3 diets over adaptation periods with concentrate level increasing from 70, 75, 80.5 and 86% of diet dry matter. Orthogonal contrasts were used to evaluate linear, quadratic, and cubic relationship between adaptation periods and the dependent variable, moreover the interaction the treatment and phase (P≤0.10). Cattle adapted for 14 days had significantly greater for average daily gain, final body weight, G:F ratio, hot carcass weight (P=0,04) and rib-eye area (P=0,01). The adaptation for 14 days the animals also provided better development of the rumen epithelium for a greater absorptive surface area (P=0.02) and width papillae (P=0.06). Moreover, these cattle showed that were properly adapted because the cell epithelium renovation proved to be stable in cell proliferation index (P=0.003) and cell death (P=0.0004). Thus, based on the results of this study, yearling Nellore bulls should be adapted for the step protocol for 14 days, because these provided greater performance and development of the rumen epithelium. / FAPESP: 2013/25403-0

Submitted by Michelle Catarina Pires (catarinamcatarinap@gmail.com) on 2016-04-01T20:02:30Z No. of bitstreams: 1 dissertação mestrado 29022016.pdf: 3459720 bytes, checksum: d104cdf26cd66a64c36361ef64f249d8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-05T19:37:59Z (GMT) No. of bitstreams: 1 pires_mc_me_bot.pdf: 3459720 bytes, checksum: d104cdf26cd66a64c36361ef64f249d8 (MD5) / Made available in DSpace on 2016-04-05T19:37:59Z (GMT). No. of bitstreams: 1 pires_mc_me_bot.pdf: 3459720 bytes, checksum: d104cdf26cd66a64c36361ef64f249d8 (MD5) Previous issue date: 2016-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Há vinte e cinco anos, o primeiro caso de síndrome da infusão do propofol foi descrito e, até hoje, são ainda desconhecida às verdadeiras razões para seu desenvolvimento. O objetivo desta revisão sistemática e meta-análise proporcional é avaliar os fatores contribuintes, sinais clínicos e taxa de mortalidade relacionada à ocorrência da síndrome da infusão do propofol. Métodos: Revisão sistemática da literatura e meta-análise proporcional de relatos e série de casos de pacientes com síndrome da infusão do propofol. A meta-análise foi realizada avaliando o desfecho morte associada com dose (< 240mg/Kg versus ≥ 240mg/Kg) ou tempo de infusão (< 48h contra ≥ 48h) usando um modelo de efeito randômico. Os intervalos de confiança de 95% também foram calculados. Resultados: Foram incluídos 59 relatos e série de casos com um total de 75 pacientes. Acidose metabólica e doenças cardíacas foram associadas com aumento das taxas de mortalidade. Houve uma diferença estatisticamente significante favorecendo a dose total propofol inferior a 240mg/Kg 31% (IC 95% 16% a 49%) em comparação com ≥ 240mg/Kg 66% (lC 95% 52% a 79%) na redução das taxas de mortalidade. Houve também uma proporção estatisticamente maior de morte no período de administração de propofol ≥ 48h 64% (IC de 95% 51% a 76%) em comparação com < 48h 20% (IC 95% 14% a 48%). Conclusão: Estes dados sugerem que tanto a acidose metabólica, quanto os distúrbios cardíacos podem ser preditores da mortalidade em pacientes com síndrome da infusão do propofol. Além disso, os dados mostraram que período de administração e dose maiores de propofol resultam nas taxas de mortalidade mais elevadas entre os pacientes com síndrome da infusão do propofol. / Twenty-five years ago the first case of propofol infusion syndrome was described and, until today, it is yet unknown the true reasons for it’s develop. The aim of this systematic review and proportional meta-analysis is to evaluate the contributing factors, clinical signs and mortality rate related to the occurrence of the propofol infusion syndrome. Methods: Literature review and proporcional meta-analysis of both, reports and case series of all patients with propofol infusion syndrome undergoing either general surgey or ICU. The meta-analysis was performed on death associated with either dose (< 240mg/Kg versus ≥ 240 mg/Kg) or infusion time (< 48h versus ≥ 48h) using a radom-effect model. The 95% confidential intervals were also calculed. Results: 59 studies were included with a total of 75 patients. Metabolic acidosis and cardiac disorders were associated with increase rates of mortality. There was a statistically significance difference favoring propofol total dose less than 240mg/Kg 31% (CI 95% 16% to 49%) compared to ≥ 240mg/Kg 66% (CI 95% 52% to 79%) in the reduction of mortality rates. There was also a statistically higher proportion of death in the infusion propofol period ≥ 48h 64% (CI 95% 51% to 76%) compared to < 48h 29% (CI 95% 14% to 48%). Conclusion: These data suggests that both metabolic acidosis and cardiac disorders might be predictors of the development of mortality in patients with propofol infusion syndrome. Furthermore, our data showed that higher infusion period and dose of propofol results in higher mortality rates among those patients with propofol infusion syndrome.