<p><strong>Background:</strong> Real-time quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) is recently used for characterization and expression analysis of miRNAs. The data from such experiments need effective analysis methods to produce reliable and high-quality data. For the miRNA prostate cancer qRT-PCR data used in this study, standard housekeeping normalization method fails due to non-stability of endogenous controls used. Therefore, identifying appropriate normalization method(s) for data analysis based on other data driven principles is an important aspect of this study.</p><p><strong>Results:</strong> In this study, different normalization methods were tested, which are available in the R packages <em>Affy</em> and <em>qpcrNorm</em> for normalization of the raw data. These methods reduce the technical variation and represent robust alternatives to the standard housekeeping normalization method. The performance of different normalization methods was evaluated statistically and compared against each other as well as with the standard housekeeping normalization method. The results suggest that <em>qpcrNorm</em> Quantile normalization method performs best for all methods tested.</p><p><strong>Conclusions:</strong> The <em>qpcrNorm</em> Quantile normalization method outperforms the other normalization methods and standard housekeeping normalization method, thus proving the hypothesis of the study. The data driven methods used in this study can be applied as standard procedures in cases where endogenous controls are not stable.</p>

Background: Real-time quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) is recently used for characterization and expression analysis of miRNAs. The data from such experiments need effective analysis methods to produce reliable and high-quality data. For the miRNA prostate cancer qRT-PCR data used in this study, standard housekeeping normalization method fails due to non-stability of endogenous controls used. Therefore, identifying appropriate normalization method(s) for data analysis based on other data driven principles is an important aspect of this study. Results: In this study, different normalization methods were tested, which are available in the R packages Affy and qpcrNorm for normalization of the raw data. These methods reduce the technical variation and represent robust alternatives to the standard housekeeping normalization method. The performance of different normalization methods was evaluated statistically and compared against each other as well as with the standard housekeeping normalization method. The results suggest that qpcrNorm Quantile normalization method performs best for all methods tested. Conclusions: The qpcrNorm Quantile normalization method outperforms the other normalization methods and standard housekeeping normalization method, thus proving the hypothesis of the study. The data driven methods used in this study can be applied as standard procedures in cases where endogenous controls are not stable.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20255号 / 医博第4214号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 萩原 正敏, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cyclosporine is used in veterinary medicine to treat a number of inflammatory and immune-mediated conditions, however firm oral dosing protocols have yet to be established in the dog. Traditionally a pharmacokinetic approach, through measurement of blood drug concentrations, has been the primary method of establishing if the given dose is effectively suppressing the immune system. However, there is some debate over how well blood drug concentrations correspond to immunosuppression, since individuals can vary in response to the same drug concentration. Our research group believes that a pharmacodynamic approach could alternatively be used to accurately determine cyclosporine dosages in individual patients since this will give a measurement of the immune system’s response to the drug, rather than simply how the body is processing it. This method will give a more accurate assessment of the patient’s immune system, and allow for better immunosuppressant therapy. The objective of this thesis was to develop a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that could reliably predict patient outcome during cyclosporine treatment. This assay would essentially work as a diagnostic tool that clinicians can use to help determine if they were using an appropriate cyclosporine dose for their patients. The assay measures cytokine expression of activated T cells, which are the target cell for the active metabolite of cyclosporine. Our objectives were achieved, firstly, through validation of the assay. Since this assay will be used by clinicians throughout the nation, we first established if shipping conditions affected the sample, and therefore assay results. Once the effect of sample storage time and temperature were determined, optimal sample collection timing was established. Finally, cytokine levels were measured in samples from clinical cases and healthy control dogs to examine the difference in cytokine expression between these two groups. An effective and reliable treatment method for cyclosporine has yet to be established in the dog; therefore the results of this thesis will lead to better therapeutic monitoring and more efficient use of cyclosporine therapy in canine patients.

A busca de marcadores moleculares para o refinamento diagnóstico, classificação e estabelecimento do prognóstico dos tumores, e individualização terapêutica tem sido foco de várias pesquisas. O presente estudo teve como objetivo investigar, em carcinoma epidermóide de língua e/ou assoalho bucal, a presença de transcritos dos genes homeobox que pudessem se revelar marcadores moleculares de prognóstico e/ou agressividade tumoral. Após análise por microarray utilizando-se amostras de tumores e margens classificados como mais e menos agressivos, os genes homeobox HOXC13, HOXD10, HOXD11, IRX4, PROX1 e ZHX1 foram selecionados e sua hiper-expressão foi parcialmente validada por qRT-PCR. Observou-se aumento da expressão de HOXD10, HOXD11 e IRX4 em tumores com relação às margens correspondentes, bem como nos tumores menos agressivos em relação às suas respectivas margens. Por outro lado, os genes PROX1 e ZHX1 estavam mais expressos nas margens que nos tumores correspondentes. Esses resultados sugerem que a expressão alterada de HOXD10, HOXD11 e IRX4 pode participar no desenvolvimento do carcinoma epidermóide de língua e/ou assoalho bucal, enquanto os genes PROX1 e ZHX1 provavelmente exibem perda de função ou estão silenciados na neoplasia. Houve uma tendência de associação entre a expressão elevada de HOXD11 e presença de infiltrações linfática e perineural, e grau moderado de diferenciação da neoplasia, bem como entre a expressão elevada de HOXD10 e infiltração linfática. O gene IXR4 foi relacionado com um menor tempo de sobrevida global. Não foi possível estabelecer, dentre os genes homeobox validados por qRT-PCR, um gene ou uma combinação deles que pudesse(m) ser utilizado(s) como marcador(es) de agressividade tumoral. / The search for molecular markers to diagnosis improvement, treatment individualization and establishment of oral squamous cell carcinoma prognosis has been the focus of several studies. The present study investigated the presence of specific transcript of homeobox genes in squamous cell carcinoma of the tongue and/or floor of the mouth that might reflect relevant molecular markers of prognosis and/or tumor aggressiveness. After microarray analysis of tumor samples classified as more or less aggressive, and non tumoral margins, HOXC13, HOXD10, HOXD11, IRX4, PROX1 and ZHX1 selected and partially validated by qRT-PCR. Increased expression of HOXD10, HOXD11, IRX4 in tumors in comparison to margins as well as in less aggressive tumors related to their margins was observed. On the other hand, a decreased expression of PROX1 and ZHX1 was observed in margins compared to their respective tumors. These results suggest that the altered expression of HOXD10, HOXD11 and IRX4 may participate in the development of squamous cell carcinoma of the tongue and/or floor of the mouth, while PROX1 and ZHX1 probably present loss of function or are silenced in tumors. A tendency of association between increased expression of HOXD11 and lymphatic and perineural infiltration, as well as moderately differentiated tumors, and increased expression of HOXD10 and lymphatic infiltration was observed. Still, increased expression of IRX4 may apparently influence global survival rate. However, the results of the present study must be confirmed in a greater number of samples, and complemented with the evaluation of HOXD10, HOXD11, IRX4 protein levels. It was not possible to establish, among homeobox genes validated through qRT-PCR, a gene or a combination of genes capable of predicting tumor aggressiveness.

O Semliki Forest Vírus recombinante (rSFV) vem sendo apontado como um dos vetores virais mais promissores, tanto para a expressão de proteínas de interesse biotecnológico, como para a utilização como vetor vacinal. Neste trabalho obtivemos um rSFV carregando o gene da proteína verde fluorescente GFP (rSFV-GFP), que foi utilizado para a padronização de métodos de obtenção do vetor viral e de sua titulação, duas etapas importantes para os trabalhos que utilizam rSFVs. Por meio da análise da expressão da GFP em células BHK-21 infectadas por lotes de vírus obtidos por um reagente de transfecção lipídico ou por eletroporação, estabelecemos as melhores condições de obtenção dos rSFV e de sua utilização para a infecção e produção de proteínas recombinantes de interesse. Além disso, padronizamos criteriosamente uma metodologia de RT-PCR quantitativa (qRT-PCR) absoluta, baseada em uma curva padrão, para realizar a titulação de partículas rSFV, independentemente do gene heterólogo clonado. A análise direta da quantidade de células infectadas, por citometria de fluxo e por microscopia de fluorescência, fez do rSFV-GFP a ferramenta adequada para nossos estudos de titulação viral por qRT-PCR. Foi demonstrada a associação entre o número de cópias do RNA viral utilizado para a infecção das células e a quantidade de células expressando GFP. Essa abordagem levou ao desenvolvimento de competências técnica e tecnológica (construção, avaliação e titulação) que facilitarão a obtenção de outros rSFV de interesse. Concluímos que a eletroporação é o melhor método de obtenção das partículas e que o título viral, determinado por qRT-PCR, pode ser utilizado para calcular o volume de um lote de rSFV necessário para obter a multiplicidade de infeção desejada. / The recombinant Semliki Forest Virus (rSFV) has been considered as one of the most promising viral vectors, both for the expression of proteins of biotechnological interest or as a vector for immunizations. In this study, an rSFV carrying the gene of the green fluorescent protein (GFP) was obtained (rSFV-GFP) and used for the standardization of rSFV production and of a novel titration methodology. Through the analysis of the amounts of GFP expressed in BHK-21 cells infected with virus stocks obtained by transfection with a lipid reagent or electroporation, we established the best conditions for rSFV production based on the production of this recombinant protein. In addition, a standardized method of absolute quantitative RT-PCR (qRT-PCR), based in a standard curve and applicable to virtually all rSFV particles, regardless of the heterologous gene cloned, was validated. The direct analysis of the quantity of infected cells, by flow cytometry and fluorescence microscopy, confirmed that the rSFV-GFP was an appropriated tool to support ours studies of viral titration by qRT-PCR. It was demonstrated the association between the amount of copies of viral RNA used for culture infection and the amount of GFP expression. This approach led to the development of technical and technological competence (construction, evaluation and titration) in the laboratory, that shall help the establishment of other rSFV of interest. We concluded that electroporation is the best methodology to obtain rSFV particles and the viral titre, as determined by qRT-PCR, can be used to calculate the volume of an rSFV stock necessary to obtain the desired multiplicity of infection.

A busca de marcadores moleculares para o refinamento diagnóstico, classificação e estabelecimento do prognóstico dos tumores, e individualização terapêutica tem sido foco de várias pesquisas. O presente estudo teve como objetivo investigar, em carcinoma epidermóide de língua e/ou assoalho bucal, a presença de transcritos dos genes homeobox que pudessem se revelar marcadores moleculares de prognóstico e/ou agressividade tumoral. Após análise por microarray utilizando-se amostras de tumores e margens classificados como mais e menos agressivos, os genes homeobox HOXC13, HOXD10, HOXD11, IRX4, PROX1 e ZHX1 foram selecionados e sua hiper-expressão foi parcialmente validada por qRT-PCR. Observou-se aumento da expressão de HOXD10, HOXD11 e IRX4 em tumores com relação às margens correspondentes, bem como nos tumores menos agressivos em relação às suas respectivas margens. Por outro lado, os genes PROX1 e ZHX1 estavam mais expressos nas margens que nos tumores correspondentes. Esses resultados sugerem que a expressão alterada de HOXD10, HOXD11 e IRX4 pode participar no desenvolvimento do carcinoma epidermóide de língua e/ou assoalho bucal, enquanto os genes PROX1 e ZHX1 provavelmente exibem perda de função ou estão silenciados na neoplasia. Houve uma tendência de associação entre a expressão elevada de HOXD11 e presença de infiltrações linfática e perineural, e grau moderado de diferenciação da neoplasia, bem como entre a expressão elevada de HOXD10 e infiltração linfática. O gene IXR4 foi relacionado com um menor tempo de sobrevida global. Não foi possível estabelecer, dentre os genes homeobox validados por qRT-PCR, um gene ou uma combinação deles que pudesse(m) ser utilizado(s) como marcador(es) de agressividade tumoral. / The search for molecular markers to diagnosis improvement, treatment individualization and establishment of oral squamous cell carcinoma prognosis has been the focus of several studies. The present study investigated the presence of specific transcript of homeobox genes in squamous cell carcinoma of the tongue and/or floor of the mouth that might reflect relevant molecular markers of prognosis and/or tumor aggressiveness. After microarray analysis of tumor samples classified as more or less aggressive, and non tumoral margins, HOXC13, HOXD10, HOXD11, IRX4, PROX1 and ZHX1 selected and partially validated by qRT-PCR. Increased expression of HOXD10, HOXD11, IRX4 in tumors in comparison to margins as well as in less aggressive tumors related to their margins was observed. On the other hand, a decreased expression of PROX1 and ZHX1 was observed in margins compared to their respective tumors. These results suggest that the altered expression of HOXD10, HOXD11 and IRX4 may participate in the development of squamous cell carcinoma of the tongue and/or floor of the mouth, while PROX1 and ZHX1 probably present loss of function or are silenced in tumors. A tendency of association between increased expression of HOXD11 and lymphatic and perineural infiltration, as well as moderately differentiated tumors, and increased expression of HOXD10 and lymphatic infiltration was observed. Still, increased expression of IRX4 may apparently influence global survival rate. However, the results of the present study must be confirmed in a greater number of samples, and complemented with the evaluation of HOXD10, HOXD11, IRX4 protein levels. It was not possible to establish, among homeobox genes validated through qRT-PCR, a gene or a combination of genes capable of predicting tumor aggressiveness.

Nitrogen is an essential macronutrient for plants and nitrogen fertilizers are indispensable for modern agriculture. Unfortunately, we know too little about how plants regulate their use of soil nitrogen, to maximize fertilizers-N use by crops and pastures. This project took a dual approach, involving forward and reverse genetics, to identify N-regulators in plants, which may prove useful in the future to improve nitrogen-use efficiency in agriculture. To identify nitrogen-regulated transcription factor genes in Arabidopsis that may control N-use efficiency we developed a unique resource for qRT-PCR measurements on all Arabidpsis transcription factor genes. Using closely spaced, gene-specific primer pairs and SYBR® Green to monitor amplification of double-stranded DNA, transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products, and 13% of TF transcripts were undetectable in these organs. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001-100 copies per cell. Real-time RT-PCR revealed 26 root-specific and 39 shoot-specific TF genes, most of which have not been identified as organ-specific previously.<br><br> An enlarged and improved version of the TF qRT-PCR platform contains now primer pairs for 2256 Arabidopsis TF genes, representing 53 gene families and sub-families arrayed on six 384-well plates. Set-up of real-time PCR reactions is now fully robotized. One researcher is able to measure expression of all 2256 TF genes in a single biological sample in a just one working day. The Arabidopsis qRT-PCT platform was successfully used to identify 37 TF genes which transcriptionaly responded at the transcriptional level to N-deprivation or to nitrate per se. Most of these genes have not been characterized previously. Further selection of TF genes based on the responses of selected candidates to other macronutrients and abiotic stresses allowed to distinguish between TFs regulated (i) specifically by nitrogen (29 genes) (ii) regulated by general macronutrient or by salt and osmotic stress (6 genes), and (iii) responding to all major macronutrients and to abiotic stresses. Most of the N-regulated TF genes were also regulated by carbon. Further characterization of sixteen selected TF genes, revealed: (i) lack of transcriptional response to organic nitrogen, (ii) two major types of kinetics of induction by nitrate, (iii) specific responses for the majority of the genes to nitrate but not downstream products of nitrate assimilation. All sixteen TF genes were cloned into binary vectors for constitutive and ethanol inducible over expression, and the first generation of transgenic plants were obtained for almost all of them. Some of the plants constitutively over expressing TF genes under control of the 35S promoter revealed visible phenotypes in T1 generation. Homozygous T-DNA knock out lines were also obtained for many of the candidate TF genes. So far, one knock out line revealed a visible phenotype: retardation of flowering time.<br><br> A forward genetic approach using an Arabidopsis ATNRT2.1 promoter : Luciferase reporter line, resulted in identification of eleven EMS mutant reporter lines affected in induction of ATNRT2.1 expression by nitrate. These lines could by divided in the following classes according to expression of other genes involved in primary nitrogen and carbon metabolism: (i) lines affected exclusively in nitrate transport, (ii) those affected in nitrate transport, acquisition, but also in glycolysis and oxidative pentose pathway, (iii) mutants affected moderately in nitrate transport, oxidative pentose pathway and glycolysis but not in primary nitrate assimilation. Thus, several different N-regulatory genes may have been mutated in this set of mutants. Map-based cloning has begun to identify the genes affected in these mutants. / Stickstoff ist einer der wichtigsten Makroelemente in der Natur und sein eingeschränktes Vorkommen ist häufig ein limitierender Faktor für pflanzliches Wachstum. In der Landwirtschaft eingesetzte Stickstoff-Dünger werden häufig nicht vollständig von Getreide- oder anderen kultivierten Pflanzen genutzt, sondern in die umliegenden Gewässer oder das Grundwasser ausgewaschen. Das Verständnis von pflanzlichen Signalprozessen kann helfen, Stickstoffaufnahme und -assimilation zu kontrollieren und somit den Einsatz von stickstoffhaltigen Düngemitteln in der Landwirtschaft zu reduzieren.<br><br> Die meisten der in den pflanzlichen Stickstoffstoffwechsel involvierten Gene werden auf Transkriptionsebene reguliert. Dies geschieht durch sogenannte Transkriptionsfaktoren (TFs), Proteine, die von Genen anderer Genfamilien kodiert werden. Im Rahmen dieser Promotion wurde eine einzigartige und neue Ressource zur Quantifizierung der Expressionsniveaus solcher Transkriptionsfaktoren der Modellpflanze <i>Arabidopsis thaliana</i> entwickelt und getestet. Dabei konnte die beispiellose Robustheit, Genauigkeit und Präzision dieser PCR-basierten Methode gezeigt werden. Mit Hilfe dieses experimentellen Aufbaus wurden Transkriptionsfaktoren, potentielle Regulatoren von Genen, die in Stickstoffmetabolismus involviert sind, identifiziert und charakterisiert. Um die Funktionsweise dieser Gene besser zu verstehen, wurden transgene Pflanzen erzeugt und identifiziert, die entweder erhöhte oder chemisch induzierbare Transkription und/oder einen partiellen oder vollständigen Verlust der Aktivität dieser Gene aufweisen. Die Analyse der Transkriptionsfaktoren, die unter die Kontrolle eines induzierbaren Promoters gestellt wurden, soll helfen, die genauen Zielgene dieser TFs zu identifizieren und ihre Rolle im Stickstoffmetabolismus zu erklären. Darüber hinaus bieten sie die Chance, Hierarchieebenen innerhalb der verschiedenen TFs zu erkennen. Überexpression von Transkriptionsfaktoren kann zur Generierung von Phänotypen führen, die von direktem biotechnologischen Interesse sind, wie z.B. Pflanzen mit erhöhtem Stickstoffgehalt (Aminosäuregehalt), die besser an Situationen mit Stickstoffmangel angepasst sind. Neben diesen Transkriptionsfaktoren wurden allerdings auch Mutanten mit einem genetischen Defekt in einem der wichtigsten Gene, das für den Nitrattransport in Wurzeln von <i>Arabidopsis</i> verantwortlich ist, identifiziert.

Made available in DSpace on 2015-03-26T13:42:24Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1800419 bytes, checksum: 3fadb87455bb2fa082c397f8824173aa (MD5) Previous issue date: 2012-02-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / O objetivo do estudo foi identificar genes codificantes para os complexos enzimáticos da cadeia tranpostadora de elétrons (CTE) que apresentam-se diferencialmente expressos no músculo Longissimus dorsi (LD) de suínos bem como seus padrões de expressãos de acordo níveis de fósforo disponíveis (Pd) na dieta e ambientes térmicos em duas fases de crescimento. Dois experimentos foram realizados utilizando-se 48 suínos de alto potencial genético para deposição de carne em duas diferentes fases de crescimento: a fase 1 de 15 a 30 kg e fase 2 de 30 a 60 kg. Estas experimentos foram construidos em modelo fatorial 2 X 3 (2 diferentes temperaturas ambientais X 3 níveis de Pd na dieta) constituindo 6 tratamentos em cada fase de crescimento. Vinte e quatro suínos da fase da 1 (15 a 30 kg) com peso inicial de 15 ± 0,41 kg, foram distribuídos em delineamento inteiramente casualizado, com 6 tratamentos e 6 repetições. Estes animais foram alojados ou no ambiente termoneutro (24,5 ± 1,2 º C e umidade relativa em 76,3 ± 8,5%) ou no ambiente de calor (34,1 ± 0,8 º C e umidade relativa em 70,1 ± 8,1%) e, em seguida, distribuídos para um dos 3 níveis de Pd na dieta (0.107, 0.321 ou 0.535%). Vinte e quatro suínos da fase de 2 (30 a 60 kg) com peso inicial de 30,19 ± 0,30 kg, foram distribuídos em delineamento inteiramente casualizado com 6 tratamentos e 6 repetições. Estes animais foram alojados ou ambiente termoneutro (21,9 ± 1,4 º C e umidade relativa em 76,9 ± 5,7%) ou ambiente de calor (31,6 ± 0,7 º C e umidade relativa em 72,8 ± 5,9%) e, em seguida, distribuídos para um dos 3 níveis de Pd na dieta (0.116 , 0.306 ou 0.496%). Os perfis de expressão dos genes ND1, ND2, SDHD, CYTB, COX1, COX2, COX3, ATP5J2 e ATP6 foram analisados pelo PCR quantitativo em tempo real (qRT-PCR) a partir de amostras de LD. Os genes ND1, ND2, CYTB, COX1, ATP5J2 e ATP6 foram menos expressos (P< 0.05) nos suínos da fase 1 mantindo em estresse por calor em relação aqueles mantidos no ambiente termoneutro. Nos suíno da fase 2 de crescimento que estavam mantidos em estresse por calor apresentaram redução (P < 0.05) na expressão dos genes ND2, SDHD, CYTB, COX1, COX2, COX3, ATP5J2 e ATP6, em relação aqueles submetido ao ambiente termoneutro. A menor expressão desses genes nos suínos estressados por calor, prove evidências dos efeitos das altas temperaturas sob o metabolismo oxidativo a fim de reduzir a produção de calor o que refletiu na redução da exigência de consumo diário de fósforo. Parte desse efeito pode estar associado a concomitante redução do consumo vonlutário e outra parte é sugerido ser devido ao aumento da produção de espécies reativas de oxigênio (ROS) nas mitocôndrias induzido pelo estresse por calor. Quanto a mudança nos padrões de expressão desses genes entre os níveis de Pd, verificou-se que nos suínos da fase 1, os genes ND1, ND2, CYTB, COX1, ATP5J2 e ATP6 mostraram-se diferencialmente expressos (P<0.05) entre os níveis de Pd no ambiente termoneutro e o gene ND2 no ambiente de calor (P<0.05). Nos suínos da fase 2, os genes COX3 e ND2 foram diferencialmente expressos (P<0.05), respectivamente, entre os níveis de Pd no ambiente termoneutro e ambiente de calor. Os dados apresentados revelam uma forte evidência de que o nutriente fósforo é um dos fatores chave que regulam a fosforilação oxidativa com implicação direta sobre a performance animal. Para a confirmação dessa hipótese de que suínos estressados por calor apresentam aumento na produção de ROS, foi realizado um segundo experimento que objetivou avaliar o perfil de expressão dos genes associados sistema antioxidante (manganes- superóxido dismutase, glutathiona peroxidase e catalase) em suínos de 2 diferentes fases de crescimeto submetido à ambientes de termoneutralidade e estresse por calor. Baseado em análises de qRTPCR, estes genes foram significativamente mais expressos em suínos expostos ao estresse de calor. Em geral, os dados deste estudo são os primeiros a revelar os efeitos de diferentes níveis de fósforo disponível na expressão de genes relacionados com a fosforilação oxidativa e suas implicações sobre o desempenho de suínos. Além disso, é demonstrado que elevadas temperaturas não só induzem a redução da capacidade metabólica oxidativa, como também, estimulam a produção de ROS em mitocôndrias de músculo LD o que leva a danos oxidativos mitocondriais, tais como, repressão da transcrição e provavelmente redução na atividade dos complexos da cadeia transportadora de elétrons, o que refletiu na piora da performance animal. Tais informações irão contribuir para melhor compreensão do papel do fósforo no metabolismo energético e maior entendimento sobre as mudanças no metabolismo e funcionamento mitocondrial em resposta à altas temperaturas. / The purpose of this study was to identify genes encoding the electron transport chain (ETC) complexes that are present differentially expressed in the Longissimus dorsi (LD) of pigs and their expression patterns according to levels available of phosphorus (aP) in the diet and thermal environment in the two different growth phase. Two experiments were carried out using 48 higher-lean growth pigs from two different growth phases: phase 1 from 15 to 30 kg and phase 2 from 30 to 60 kg. These experiments were designed as a 2 x 3 (2 different environment temperatures X 3 levels of dietary aP) factorial treatments comprising of 6 treatments and each growth phase. Twenty -four pigs from growth phase 1 (15 to 30 kg) with initial weight of 15 ± 0.41kg, distributed in a completely randomized design with 6 treatments and 6 replications. These pigs were allotted to either thermoneutral (24.5 ± 1.2ºC and RH at 76.3 ± 8.5% or heat stress (34.1 ± 0.8ºC and RH at 70.1 ± 8.1%) and then allotted to one of the 3 levels of dietary aP (0.107, 0.321 or 0.535%). Twenty-four pigs from growth phase 2 (30 to 60 kg) with initial weight of 30.19 ± 0.30 kg, distributed in a completely randomized design with 6 treatments and 6 replications. These pigs were allotted to either thermoneutral ( 21.9 ± 1.4ºC and RH at 76.9 ± 5.7%) or heat stress (31.6 ± 0.7ºC and RH at 72.8 ± 5.9%) and then allotted to one of the 3 levels of dietary aP (0.116, 0.306 or 0.496%). The expression profiles of ND1, ND2, SDHD, CYTB, COX1, COX2, COX3, ATP5J2 and ATP6 genes were analyzed by quantitative real-time PCR from samples of LD. The ND1, ND2, CYTB, COX1, ATP5J2 and ATP6 genes showed reduce expression (P<0.05) in phase 1 pigs maintained under heat stress compared to those kept in thermoneutral environment. In pigs from growth phase 2 maintained under heat stress, the ND2, SDHD, CYTB, COX1, COX2, COX3, ATP5J2 and ATP6 genes showed reduce expression (P<0.05) compared to those kept in thermoneutral environment. The lower expression of these genes in pigs exposed to high temperatures provides evidence of the effect of heat stress by decreasing oxidative metabolism (oxidative phosphorylation) in order to reduce heat production, which reflecting on reduction of requirement for daily aP intake. Part of this effect is associated with concomitant reduction in voluntary feed intake and another part is suggested to be due to an increased production of reactive oxygen species in the mitochondria induced by heat stress resulting in downregulation of these genes. Significant different levels of expression were observed across aP levels. In pigs from growth phase 1, the ND1, ND2, CYTB, COX1, ATP5J2 and ATP6 genes were differentially expressed across aP levels (P<0.05) in the thermoneutral and ND2 gene in the hot environments. In pigs from growth phase 2, COX3 and ND2 genes were respectively differentially expressed across aP levels (P<0.05) in the thermoneutral and hot environments. These data revealed strong evidence that phosphorus is one of the key factors that regulates oxidative phosphorylation with direct implications on animal performance. To confirm the hypothesis that heat-stressed pigs have enhanced production of ROS, we performed a second experiment focused to identify changes in expression of genes encoding antioxidant systems (manganese superoxide dismutase, glutathione peroxidase e catalase) in pigs from high lean meat deposition at 2 different growth phases (15 to 30kg and 30 to 60kg) submitted to hot and thermoneutral environments. Based on qRT-PCR analyses, these genes were significantly upregulated in heat-exposed pigs. In general, data from this study are the first to reveal the effects of dietary phosphorus levels on gene expression related to oxidative phosphorylation and its implications on animal performance. Moreover, it is shown that high temperatures not only causes reduction in oxidative metabolic capacity, but also stimulate the ROS production in LD mitochondria which leading to oxidative damages, such as, repression of transcription of ETC complexes and likely reducing their activity in the mitochondria, which resulted in worse pig performance. The results obtained with this study will contribute to better understand the role of phosphorus in energy metabolism and will bring new insights into the comprehension of the changes metabolism and mitochondrial functioning in response to high temperature.

O Semliki Forest Vírus recombinante (rSFV) vem sendo apontado como um dos vetores virais mais promissores, tanto para a expressão de proteínas de interesse biotecnológico, como para a utilização como vetor vacinal. Neste trabalho obtivemos um rSFV carregando o gene da proteína verde fluorescente GFP (rSFV-GFP), que foi utilizado para a padronização de métodos de obtenção do vetor viral e de sua titulação, duas etapas importantes para os trabalhos que utilizam rSFVs. Por meio da análise da expressão da GFP em células BHK-21 infectadas por lotes de vírus obtidos por um reagente de transfecção lipídico ou por eletroporação, estabelecemos as melhores condições de obtenção dos rSFV e de sua utilização para a infecção e produção de proteínas recombinantes de interesse. Além disso, padronizamos criteriosamente uma metodologia de RT-PCR quantitativa (qRT-PCR) absoluta, baseada em uma curva padrão, para realizar a titulação de partículas rSFV, independentemente do gene heterólogo clonado. A análise direta da quantidade de células infectadas, por citometria de fluxo e por microscopia de fluorescência, fez do rSFV-GFP a ferramenta adequada para nossos estudos de titulação viral por qRT-PCR. Foi demonstrada a associação entre o número de cópias do RNA viral utilizado para a infecção das células e a quantidade de células expressando GFP. Essa abordagem levou ao desenvolvimento de competências técnica e tecnológica (construção, avaliação e titulação) que facilitarão a obtenção de outros rSFV de interesse. Concluímos que a eletroporação é o melhor método de obtenção das partículas e que o título viral, determinado por qRT-PCR, pode ser utilizado para calcular o volume de um lote de rSFV necessário para obter a multiplicidade de infeção desejada. / The recombinant Semliki Forest Virus (rSFV) has been considered as one of the most promising viral vectors, both for the expression of proteins of biotechnological interest or as a vector for immunizations. In this study, an rSFV carrying the gene of the green fluorescent protein (GFP) was obtained (rSFV-GFP) and used for the standardization of rSFV production and of a novel titration methodology. Through the analysis of the amounts of GFP expressed in BHK-21 cells infected with virus stocks obtained by transfection with a lipid reagent or electroporation, we established the best conditions for rSFV production based on the production of this recombinant protein. In addition, a standardized method of absolute quantitative RT-PCR (qRT-PCR), based in a standard curve and applicable to virtually all rSFV particles, regardless of the heterologous gene cloned, was validated. The direct analysis of the quantity of infected cells, by flow cytometry and fluorescence microscopy, confirmed that the rSFV-GFP was an appropriated tool to support ours studies of viral titration by qRT-PCR. It was demonstrated the association between the amount of copies of viral RNA used for culture infection and the amount of GFP expression. This approach led to the development of technical and technological competence (construction, evaluation and titration) in the laboratory, that shall help the establishment of other rSFV of interest. We concluded that electroporation is the best methodology to obtain rSFV particles and the viral titre, as determined by qRT-PCR, can be used to calculate the volume of an rSFV stock necessary to obtain the desired multiplicity of infection.