<p>Phenylalanine (Phe) is a proteinogenic aromatic amino acid that also serves as a precursor for numerous primary and secondary metabolites in plants. Phe is synthesized from chorismate, the final product of the shikimate pathway. In plants, Phe is predominantly synthesized in the plastids via the arogenate pathway, while most Phe-derived compounds are produced in the cytoplasm, requiring exportation of Phe from plastids to the cytosol. Here, we provided genetic evidences that a<i> Petunia hybrida</i> plastidial cationic amino acid transporter (PhpCAT) participates in the exportation of Phe from plastids, as well as regulation of carbon flux through Phe biosynthesis.</p> <p> By using reverse genetics, we demonstrated that a petunia phenylpyruvate aminotransferase (PhPPY-AT) is able to convert phenylpyruvate to Phe in the cytosol <i>in vivo</i>, and that a cytosolic chorismate mutase (CM2), which converts chorismate to prephenate, directs carbon flux from the plastidial Phe biosynthesis pathway towards the cytosolic pathway. Downregulation of <i>PhPPY-AT</i> and <i>PhCM2</i> resulted in significant decreases in Phe levels and emission of Phe-derived volatiles in petunia flowers, respectively. Metabolic flux analysis showed that the carbon flux through the cytosolic Phe biosynthesis pathway is significantly lower in <i>PhCM2</i> RNAi petunia flowers relative to wild type control. We also demonstrated that the conversion of prephenate to phenylpyruvate in the cytosol is catalyzed by a cytosolic prephenate dehydratase (PDT) produced from an alternative transcription start site of a known plastidial arogenate dehydratase (ADT). These results suggest that a microbial-like phenylpyruvate pathway for Phe biosynthesis operates in the cytosol of plant cells and the cytosolic pathway splits from the plastidial pathway at chorismate.</p> <p> To evaluate the metabolic potential of the cytosolic phenylpyruvate pathway, <i>PhCM2 </i>overexpressing transgenic petunia plants were generated. Unexpectedly, Phe levels and emission of Phe-derived volatiles were both reduced, even though the flux through the cytosolic pathway was increased relative to wild type control. Electron microscopy, metabolic profiling and metabolic flux analysis revealed that the number of leucoplasts, starch levels and flux through the plastidial pathway were all reduced in <i>PhCM2</i> overexpression lines, while the concentrations of auxin and its biosynthetic intermediate, indole-3-pyruvic acid (IPA), were elevated. Overexpression of Arabidopsis aminotransferase VAS1, which converts IPA to Trp, in <i>PhCM2</i> overexpression petunia background recovered Phe levels and Phe-derived volatiles emission. These results indicate that there exists a metabolic crosstalk between cytosolic Phe production and Trp-dependent auxin biosynthesis .</p> <p> Our research completed the post-chorismate cytosolic Phe biosynthesis pathway in plants and revealed possible metabolic crosstalk between cytosolic Phe production and auxin biosynthesis in plant cells, providing targets for future genetic modification of metabolites in plants.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19673号 / 薬科博第61号 / 新制||薬科||7(附属図書館) / 32709 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 馬見塚 拓, 教授 掛谷 秀昭, 教授 緒方 博之 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

O objetivo deste estudo foi investigar os efeitos de dois modelos experimentais de dietas hipercalóricas em comportamentos de ansiedade, processos de aprendizagem e memória e alterações metabólicas. Os animais foram divididos em seis grupos experimentais, de acordo com a condição nutricional. 1) Controle (C); 2) Dieta de Cafeteria (DC); 3) Dieta Hiperlipídica (DH); 4) Controle AIN-93 (C/AIN-93); 5) Dieta de Cafeteria AIN-93 (DC/AIN-93), e 6) Dieta Hiperlipídica AIN-93 (DH/AIN-93). Posteriormente, os grupos foram subdivididos em dois grupos independentes, conforme a tarefa à qual foram submetidos. Pesagens foram realizadas semanalmente até os 98 dias de vida; foram verificados os pesos do fígado, do coração e o peso de tecido adiposo retroperitoneal e epididimal e foram realizadas dosagens de glicose, triglicérides, TGO e TGP no soro e gordura total, colesterol total e triglicérides no fígado. Os testes utilizados: Labirinto em T Elevado (LTE), Caixa Claro/Escuro e Labirinto Aquático de Morris (LAM). Os resultados de peso corporal, os dados comportamentais do LAM, do LTE e os dados de peso dos tecidos extraídos no dia do sacrifício e as análises bioquímicas foram submetidos a uma Análise de Variância (ANOVA). Quando apropriado, foi utilizado o teste de comparações múltiplas de Newman-Keuls (p< 0,05). Os dados comportamentais do teste claro/escuro foram submetidos ao teste t-Student (p< 0,05). Animais tratados com dieta hiperlipídica apresentaram maiores medidas de peso e ganho de peso comparados aos animais controle e dieta de cafeteria, tratados com pellet e com dieta AIN-93. Animais DH1, DC1, DH1 AIN-93, DH2 AIN-93 e DH2 apresentaram maior peso no dia do sacrifício. Animais DH1, DH1 AIN-93, DH2 e DH2 AIN-93 apresentaram maior acúmulo dos tecidos adiposos retroperitoneal e epididimal. Animais DH1 AIN-93 e DC2 AIN-93 apresentaram maiores níveis de glicose. Animais C2, DH2 e DC2 apresentaram maiores níveis de triglicérides. Animais DH1 e C1 apresentaram menores valores de TGO. Animais C2 e C2 AIN-93 apresentaram maiores níveis de TGO. Animais C1, DH1, C2 e DH2 apresentaram maiores níveis de TGP. Animais DH1 AIN-93, DH1, DH2 e DH2 AIN-93 apresentaram maiores valores de gordura total no fígado. Animais DH1 AIN-93 e DH2 apresentaram maiores níveis de colesterol no fígado. Animais DH1, DC1, DH2 e DH2 AIN-93 apresentaram maiores níveis de triglicérides no fígado. Com relação ao consumo alimentar, animais DH apresentaram maior consumo calórico e maior consumo lipídico quando comparados aos animais C e DC, com ração em pellet ou dieta AIN-93. Com relação ao LTE, não foram verificadas diferenças nas esquivas e na fuga. Animais DC1, DH1 e DH1 AIN-93 apresentaram menores níveis de ansiedade verificados a partir dos dados do teste da caixa claro-escuro. Animais DC2 AIN-93 apresentaram pior desempenho em tarefa de memória. Os dados obtidos a partir deste estudo demonstraram que as dietas utilizadas foram capazes de acarretar ganho de peso, acúmulo de tecido adiposo, alterações metabólicas, diminuição da ansiedade nos animais e pior desempenho em uma tarefa de memória em um dos grupos nutricionais. / The aim of this study was to investigate the effects of two experimental hypercaloric diets on anxiety behaviors, learning and memory processes and metabolic changes. The animals were divided into six experimental groups, according to the nutritional condition. 1) Control (C); 2) Cafeteria Diet (CD); 3) Fat Diet (FD); 4) Control AIN-93 (C/AIN-93); 5) Cafeteria Diet AIN-93 (CD/AIN-93), and 6) Fat Diet AIN-93 (FD/AIN-93). Posteriorly, the groups were subdivided into two independent groups, according to the task that they were submitted. Weighing were carried out weekly until 98 days of life; weights of liver, heart, retroperitoneal and epididymal adipose tissue were checked and glucose, triglycerides, AST, ALT levels in serum and total fat liver, total cholesterol and triglycerides levels in liver were measured. Elevated T Maze (ETM), Light/Dark Box and Morris Water Maze (MWM) tests were used. The body weight data, the behavioral data of MWM and ETM, the tissues weight data extracted on the sacrifice day and the biochemical analysis were subjected to analysis of variance (ANOVA). When appropriate, the multiple comparisons of Newman-Keuls test were used (p<0.05). The behavioral data at the light/dark box were subjected to the t-Student test (p<0.05). Animals treated with fat diet presented higher body weight and weight gain compared to the control and cafeteria diet animals, treated with lab chow and AIN-93 diet. FD1, CD1, FD1 AIN-93, FD2 AIN-93 and FD2 animals presented higher weight at the sacrifice day. FD1, FD1 AIN-93, FD2 and FD2 AIN-93 animals presented higher retroperitoneal and epididymal adipose tissue accumulation. FD1 AIN-93 and CD2 AIN-93 animals presented higher glucose levels. C2, FD2 and CD2 animals presented higher triglycerides levels. FD1 and C1 animals presented lower AST levels. C2 and C2 AIN-93 animals presented higher AST levels. C1, FD1, C2 and FD2 animals presented higher ALT levels. FD1 AIN-93, FD1, FD2 and FD2 AIN-93 animals presented higher total liver fat. FD1 AIN-93 and FD2 animals presented higher total cholesterol levels at liver. FD1, CD1, FD2 and FD2 AIN-93 animals presented higher triglycerides levels at liver. Relative to food consumption, FD animals presented higher caloric intake and higher lipid intake compared to C and CD animals, whether they have received lab chow or AIN-93 diet. Regarding the ETM, it has not been verified differences regarding the avoidances and escape. CD1, FD1 and FD1 AIN-93 animals presented lower anxiety levels checked at the light/dark box. CD2 AIN-93 animals presented worse performance at the memory retention test. The data obtained from this study suggest that the diets were capable of causing weight gain, adipose tissue accumulation, metabolic changes, decreased anxiety levels and lead to worse performance memory at a memory retention test in one of the nutritional groups.

O objetivo deste estudo foi investigar os efeitos de dois modelos experimentais de dietas hipercalóricas em comportamentos de ansiedade, processos de aprendizagem e memória e alterações metabólicas. Os animais foram divididos em seis grupos experimentais, de acordo com a condição nutricional. 1) Controle (C); 2) Dieta de Cafeteria (DC); 3) Dieta Hiperlipídica (DH); 4) Controle AIN-93 (C/AIN-93); 5) Dieta de Cafeteria AIN-93 (DC/AIN-93), e 6) Dieta Hiperlipídica AIN-93 (DH/AIN-93). Posteriormente, os grupos foram subdivididos em dois grupos independentes, conforme a tarefa à qual foram submetidos. Pesagens foram realizadas semanalmente até os 98 dias de vida; foram verificados os pesos do fígado, do coração e o peso de tecido adiposo retroperitoneal e epididimal e foram realizadas dosagens de glicose, triglicérides, TGO e TGP no soro e gordura total, colesterol total e triglicérides no fígado. Os testes utilizados: Labirinto em T Elevado (LTE), Caixa Claro/Escuro e Labirinto Aquático de Morris (LAM). Os resultados de peso corporal, os dados comportamentais do LAM, do LTE e os dados de peso dos tecidos extraídos no dia do sacrifício e as análises bioquímicas foram submetidos a uma Análise de Variância (ANOVA). Quando apropriado, foi utilizado o teste de comparações múltiplas de Newman-Keuls (p< 0,05). Os dados comportamentais do teste claro/escuro foram submetidos ao teste t-Student (p< 0,05). Animais tratados com dieta hiperlipídica apresentaram maiores medidas de peso e ganho de peso comparados aos animais controle e dieta de cafeteria, tratados com pellet e com dieta AIN-93. Animais DH1, DC1, DH1 AIN-93, DH2 AIN-93 e DH2 apresentaram maior peso no dia do sacrifício. Animais DH1, DH1 AIN-93, DH2 e DH2 AIN-93 apresentaram maior acúmulo dos tecidos adiposos retroperitoneal e epididimal. Animais DH1 AIN-93 e DC2 AIN-93 apresentaram maiores níveis de glicose. Animais C2, DH2 e DC2 apresentaram maiores níveis de triglicérides. Animais DH1 e C1 apresentaram menores valores de TGO. Animais C2 e C2 AIN-93 apresentaram maiores níveis de TGO. Animais C1, DH1, C2 e DH2 apresentaram maiores níveis de TGP. Animais DH1 AIN-93, DH1, DH2 e DH2 AIN-93 apresentaram maiores valores de gordura total no fígado. Animais DH1 AIN-93 e DH2 apresentaram maiores níveis de colesterol no fígado. Animais DH1, DC1, DH2 e DH2 AIN-93 apresentaram maiores níveis de triglicérides no fígado. Com relação ao consumo alimentar, animais DH apresentaram maior consumo calórico e maior consumo lipídico quando comparados aos animais C e DC, com ração em pellet ou dieta AIN-93. Com relação ao LTE, não foram verificadas diferenças nas esquivas e na fuga. Animais DC1, DH1 e DH1 AIN-93 apresentaram menores níveis de ansiedade verificados a partir dos dados do teste da caixa claro-escuro. Animais DC2 AIN-93 apresentaram pior desempenho em tarefa de memória. Os dados obtidos a partir deste estudo demonstraram que as dietas utilizadas foram capazes de acarretar ganho de peso, acúmulo de tecido adiposo, alterações metabólicas, diminuição da ansiedade nos animais e pior desempenho em uma tarefa de memória em um dos grupos nutricionais. / The aim of this study was to investigate the effects of two experimental hypercaloric diets on anxiety behaviors, learning and memory processes and metabolic changes. The animals were divided into six experimental groups, according to the nutritional condition. 1) Control (C); 2) Cafeteria Diet (CD); 3) Fat Diet (FD); 4) Control AIN-93 (C/AIN-93); 5) Cafeteria Diet AIN-93 (CD/AIN-93), and 6) Fat Diet AIN-93 (FD/AIN-93). Posteriorly, the groups were subdivided into two independent groups, according to the task that they were submitted. Weighing were carried out weekly until 98 days of life; weights of liver, heart, retroperitoneal and epididymal adipose tissue were checked and glucose, triglycerides, AST, ALT levels in serum and total fat liver, total cholesterol and triglycerides levels in liver were measured. Elevated T Maze (ETM), Light/Dark Box and Morris Water Maze (MWM) tests were used. The body weight data, the behavioral data of MWM and ETM, the tissues weight data extracted on the sacrifice day and the biochemical analysis were subjected to analysis of variance (ANOVA). When appropriate, the multiple comparisons of Newman-Keuls test were used (p<0.05). The behavioral data at the light/dark box were subjected to the t-Student test (p<0.05). Animals treated with fat diet presented higher body weight and weight gain compared to the control and cafeteria diet animals, treated with lab chow and AIN-93 diet. FD1, CD1, FD1 AIN-93, FD2 AIN-93 and FD2 animals presented higher weight at the sacrifice day. FD1, FD1 AIN-93, FD2 and FD2 AIN-93 animals presented higher retroperitoneal and epididymal adipose tissue accumulation. FD1 AIN-93 and CD2 AIN-93 animals presented higher glucose levels. C2, FD2 and CD2 animals presented higher triglycerides levels. FD1 and C1 animals presented lower AST levels. C2 and C2 AIN-93 animals presented higher AST levels. C1, FD1, C2 and FD2 animals presented higher ALT levels. FD1 AIN-93, FD1, FD2 and FD2 AIN-93 animals presented higher total liver fat. FD1 AIN-93 and FD2 animals presented higher total cholesterol levels at liver. FD1, CD1, FD2 and FD2 AIN-93 animals presented higher triglycerides levels at liver. Relative to food consumption, FD animals presented higher caloric intake and higher lipid intake compared to C and CD animals, whether they have received lab chow or AIN-93 diet. Regarding the ETM, it has not been verified differences regarding the avoidances and escape. CD1, FD1 and FD1 AIN-93 animals presented lower anxiety levels checked at the light/dark box. CD2 AIN-93 animals presented worse performance at the memory retention test. The data obtained from this study suggest that the diets were capable of causing weight gain, adipose tissue accumulation, metabolic changes, decreased anxiety levels and lead to worse performance memory at a memory retention test in one of the nutritional groups.

The COVID-19 pandemic forced many countries to put forth severe lockdown restrictions in order to stop the spread of the virus. Spain was one the most affected countries in Europe, and its non-essential socio-economic activities faced strict restrictions in the free movement of people. Within the Spanish territories the island of Menorca was more vulnerable to these movement restrictions, since it is mostly dependent on external resources and tourism in order to perform its socio-economic activities. Island territories like Menorca are already vulnerable to climate change threats such as sea-level rise and extreme weather patterns, but other non-climate stressors such as pandemics also threaten to render island territories under partial or total isolation. Learning how Menorca’s socio-economic activities operate resource-wise during these periods of isolation can help the island’s government to make decisions to improve their resource security and sustainability of the island. This work studies how the societal metabolism – i.e., the use of resources through the different levels and structures that conform the social system of the island – was like in Menorca under the lockdown restrictions during 2020 using the Multi-Scale Integrated Analysis of Societal and Ecosystem Metabolism (MuSIASEM) approach for metabolic analysis; and assesses Menorca’s potential for self-sufficiency. The results of the metabolic analysis show that during 2020 Menorca had a lower energy and water demand as consequence of a great loss of activity in the economic sectors, in particular that of tourism. Lesser energy consumption has translated to lower carbon emissions, and the loss of activity of the economic sectors has meant that the total gross value added generated by the island has also been lower than what the island generates under regular circumstances. This metabolic analysis show how Menorca works under conditions of isolation, reorganizing it hours of activity and use of resources mainly from the touristic sector and giving more emphasis on internal agricultural production. Should Menorca in the future become partly or totally isolated due to other circumstances, an organized rearrangement of its metabolism away from a high dependency on tourism and imports and towards internal production would aid the island to be more self-sufficient.

Les bactéries magnétotactiques (MTB) appartiennent à différents phyla procaryotes et ont la capacité de synthétiser des magnetosomes (cristaux de magnétite entourés par une membrane). Durant la thèse, nous avons procédé à l’analyse génomique de 2 bactéries magnétotactiques: Magnetospira sp. QH-2 et Magnetococcus MO-1. La synthénie et la correlation génique des gènes impliqués dans la formation des magnétosomes montrent que l'insertion de cet îlot chez QH-2 a eu lieu après la divergence entre les Magnetospirillum sp et Magnetospira sp. L'analyse comparative a mis en évidence trois groupes distincts de MTB : Groupe I, comprenant les souches Magnetospirillum spp. et Magnetospira; Groupe II avec MO-1 et M. marinus MC-1 et le Groupe III, avec D. magneticus RS-1. QH-2 montre aussi une évolution adaptative distincte par comparaison aux souches marines ou d'eau douce. L'analyse comparative des réseaux métaboliques révèle une très grande similitude intra-Groupe et une importante variabilité inter-Groupe. Cela est probablement dû aux enzymes impliqués dans les voies métaboliques anoxiques, qui représentent ainsi la contrainte à une distribution taxonomique large des MTB. Ces enzymes permettent ainsi de prédire le phénotype métabolique nécessaire à la production des magnétosomes. Différentes analyses (des protéines ribosomales au genome entier) indiquent une composition taxonomique chimérique des gènes de MO-1 et MC-1, et peut représenter une nouvelle lignée taxonomique chez les Protéobactéries. J’ai aussi participé à l'analyse des génomes de deux bactéries piezophiles, d’une bactérie photosynthétique pourpre et l’analyse phylogénomique des tyrosine-Kinases bactériennes. / Magnetotactic bacteria (MTB) are a diverse group of aquatic prokaryotes, which synthesize membrane-Enclosed magnetic crystals known as magnetosomes. In this thesis, the genome sequences of two marine MTB strains, Magnetospira sp. QH-2 and magneto-Ovoid strain MO-1 were analyzed. The magnetosome gene cluster synteny and mam gene correlation indicate that the insertion of the magnetosome island into QH-2 chromosome occurred after divergence between freshwater and marine magnetospirilla. Comparative genomic analysis revealed three distinct groups of sequenced MTB strains: Group I with Magnetospirillum spp. strains and Magnetospira strain, Group II with MO-1 strain and M. marinus MC-1, and Group III including Desulfovibrio magneticus RS-1. In addition, it shows an adaptive evolution of two marine MTB strains to marine sediments in comparison with closely related freshwater species. Moreover, comparative metabolic network analysis reveals high level of intra-Group similarity and inter-Group variety in MTB. With anoxic network enzymes, potential “MTB” strains are predicted, and are consistent with recently isolated MTB strains. It suggested that the anoxic metabolic network might be one restricted constraint for MTB distribution in bacterial lineages. Interestingly, analyses from ribosomal proteins to the whole MTB genome strongly support a taxonomic chimeric nature of MO-1 and MC-1 genes, and may represent a novel Proteobacteria lineage. Additionally, I also participate to genome analyses of piezophilic Desulfovibrio and Phaeospirillum molischianum strains as well as genome-Wide analysis of bacterial tyrosine kinases.

A calorimetria indireta para análise do metabolismo humano tem se mostrado um método não invasivo de crescente importância no estudo mais preciso das respostas metabólicas e cardio-respiratórias. Entretanto, o alto custo dos equipamentos aliado à falta de conhecimento das técnicas tem impedido a disseminação do uso de analisadores metabólicos no Brasil. Este trabalho foi desenvolvido com o intuito de oferecer uma visão global dos procedimentos de calorimetria indireta aplicados à avaliação do metabolismo humano sob a abordagem de Engenharia Biomédica. Foram avaliados três transdutores tipicamente utilizados em equipamentos de calorimetria indireta para análise metabólica humana: um transdutor eletroquímico de oxigênio, um transdutor de dióxido de carbono por radiação infravermelha e um transdutor de fluxo em volume tipo turbina. O transdutor de oxigênio mostrou-se bastante confiável, mas com um tempo de resposta bastante elevado para aplicações em análises de respiração a respiração. A principal deficiência do transdutor de dióxido de carbono está no fato de sua configuração proporcionar um aumento significativo na resistência à respiração do indivíduo. Verificou-se que o transdutor de fluxo em volume apresenta um melhor comportamento para um fluxo em torno de 20 litros/minuto. Apesar das limitações apresentadas pelos transdutores, os resultados dos ensaios foram úteis e representativos para a compreensão do funcionamento de um analisador metabólico humano porcalorimetria indireta, evidenciando quais deficiências devem ser sanadas em pesquisas futuras e os cuidados a serem considerados na implantação do laboratório para análise metabólica/respiratória sendo instalado no Laboratório de Engenharia Biomédica da Escola Politécnica da Universidade de São Paulo. / The indirect calorimetry for analysis of the human metabolism has been a no invasive method of increasing importance in the study of metabolic and cardiorespiratory responses. However, the high cost of the equipment ally to the lack of knowledge of the techniques have been obstacles to the use of metabolic analysers in Brazil. This research was developed with the intention to offer a global vision of the applied procedures of indirect calorimetry to the evaluation of the human metabolism under the prism of the Biomedical Engineering. Three transducers typically used in equipment of indirect calorimetry for metabolic analysis had been evaluated: an electrochemical transducer of oxygen, a carbon dioxide transducer by infrared absorption and a turbine flowmeter. The oxygen transducer revealed sufficiently trustworthy, but a too high response time for applications in breath by breath analyses. The main deficiency of the transducer of carbon dioxide is to provide a significant increase in the flow resistance. It was verified that the flowmeter presents one better behavior for a stream around 20 litres/minute. Despite the limitations presented for the transducers, the results of the assays had been useful and representative for the understanding of the functioning of a human metabolic analyser for indirect calorimetry, evidencing which points have to be improved in future researches and to be considered in the implantation of the laboratory for metabolic and respiratory analysis being installed in the Laboratório de Engenharia Biomédica da Escola Politécnica da Universidade de São Paulo.

A calorimetria indireta para análise do metabolismo humano tem se mostrado um método não invasivo de crescente importância no estudo mais preciso das respostas metabólicas e cardio-respiratórias. Entretanto, o alto custo dos equipamentos aliado à falta de conhecimento das técnicas tem impedido a disseminação do uso de analisadores metabólicos no Brasil. Este trabalho foi desenvolvido com o intuito de oferecer uma visão global dos procedimentos de calorimetria indireta aplicados à avaliação do metabolismo humano sob a abordagem de Engenharia Biomédica. Foram avaliados três transdutores tipicamente utilizados em equipamentos de calorimetria indireta para análise metabólica humana: um transdutor eletroquímico de oxigênio, um transdutor de dióxido de carbono por radiação infravermelha e um transdutor de fluxo em volume tipo turbina. O transdutor de oxigênio mostrou-se bastante confiável, mas com um tempo de resposta bastante elevado para aplicações em análises de respiração a respiração. A principal deficiência do transdutor de dióxido de carbono está no fato de sua configuração proporcionar um aumento significativo na resistência à respiração do indivíduo. Verificou-se que o transdutor de fluxo em volume apresenta um melhor comportamento para um fluxo em torno de 20 litros/minuto. Apesar das limitações apresentadas pelos transdutores, os resultados dos ensaios foram úteis e representativos para a compreensão do funcionamento de um analisador metabólico humano porcalorimetria indireta, evidenciando quais deficiências devem ser sanadas em pesquisas futuras e os cuidados a serem considerados na implantação do laboratório para análise metabólica/respiratória sendo instalado no Laboratório de Engenharia Biomédica da Escola Politécnica da Universidade de São Paulo. / The indirect calorimetry for analysis of the human metabolism has been a no invasive method of increasing importance in the study of metabolic and cardiorespiratory responses. However, the high cost of the equipment ally to the lack of knowledge of the techniques have been obstacles to the use of metabolic analysers in Brazil. This research was developed with the intention to offer a global vision of the applied procedures of indirect calorimetry to the evaluation of the human metabolism under the prism of the Biomedical Engineering. Three transducers typically used in equipment of indirect calorimetry for metabolic analysis had been evaluated: an electrochemical transducer of oxygen, a carbon dioxide transducer by infrared absorption and a turbine flowmeter. The oxygen transducer revealed sufficiently trustworthy, but a too high response time for applications in breath by breath analyses. The main deficiency of the transducer of carbon dioxide is to provide a significant increase in the flow resistance. It was verified that the flowmeter presents one better behavior for a stream around 20 litres/minute. Despite the limitations presented for the transducers, the results of the assays had been useful and representative for the understanding of the functioning of a human metabolic analyser for indirect calorimetry, evidencing which points have to be improved in future researches and to be considered in the implantation of the laboratory for metabolic and respiratory analysis being installed in the Laboratório de Engenharia Biomédica da Escola Politécnica da Universidade de São Paulo.

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Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8,6x106 e 1,9x106 células/mL, respectivamente) e apresentou crescimento celular durante 18 dias em cultura. A velocidade específica de crescimento máxima (?max) foi semelhante nas duas células (0,0159 h-1 para a HKB-11 e 0,0186 h-1 para SK-Hep-1). A limitação do crescimento das células adaptadas não parece estar associada à exaustão de glicose e glutamina, tampouco à formação de lactato em concentrações inibitórias. Todavia, para ambos os casos, foi observada produção de amônia em concentrações consideradas inibitórias (2 - 5 mM). De maneira geral, foi possível estabelecer culturas celulares em condições compatíveis com o desenvolvimento de um bioprocesso reprodutível, seguro e em concordância com as boas práticas de fabricação. / Human cell lines have attracted great interest since they are capable of producing glycosylated proteins in a more similar way to native human proteins, reducing the potential for immune responses against non-human epitopes. However, these human cell lines have not been extensively characterized and cultured in large scale and in serum-free suspension conditions. As a result, the main objective of this work was to adapt three human cell lines: SK-Hep-1, HepG2 and HKB-11 to serum-free suspension cultures, since they are promising systems of recombinant protein expression. For this task, four commercial serum-free media were tested. Adapted cell lines in T-flasks were further adapted to suspension cultures. Results showed that both HKB-11 and SK-Hep-1 were adapted to serum-free suspension cultures in FreeStyle and SFMII, respectively and were cryopreservated in serum-free formulations. Kinetic characterization showed that HKB-11 cell concentration was four times higher than SK-Hep-1 cell (8,6x106 and 1,9x106 cells/ml, respectively) and showed cell growth in culture over 18 days. The maximum specific growth rate (?max) was similar for both cell lines (0,0159 h-1 to HKB-11 and 0,0186h-1 to SK-Hep-1). Growth limitation of adapted human cell lines does not seem to be associated with depletion of glucose and glutamine, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production achieved inhibitory concentrations (2 - 5 mM). In general, it was possible to establish human cell cultures that are compatible with reproducible and safe bioprocess conditions and in compliance with good manufacturing practices.