A influência do butirato de sódio (NaBu), do cloreto de manganês (MnCl2), combinados ou isolados, e da cicloheximida (CHX) na síntese de tireotrofina humana recombinante (r-hTSH) derivada de células CHO foi investigada pela primeira vez. Com exceção da CHX, que gerou uma redução de 1,5 vezes na produtividade volumétrica, todos os reagentes geraram um aumento (1,4 vezes para o MnCl2 e 3 vezes para o NaBu e NaBu+MnCl2) na produtividade volumétrica. Também foi observada uma diminuição no número de células viáveis com a utilização de todos os reagentes. A adição destes reagentes ao meio de cultura de células CHO produtoras de hTSH gerou também alterações na glicosilação do r-hTSH. As alterações geradas pela presença de CHX foram todas negativas, resultando em uma diminuição de 5,5% no conteúdo de ácido siálico, 10% na ocupação dos sítios de glicosilação, além de uma diminuição no conteúdo de todos os monossacarídeos neutros. Os demais reagentes, por outro lado, resultaram em alterações positivas. A presença de NaBu no meio de cultura acarretou um aumento de 12% no conteúdo de ácido siálico e de 3% na ocupação dos sítios de glicosilação. Com relação às estruturas de carboidratos presentes no hTSH, foi observado um aumento de 14% nas estruturas bi-antenárias, aumento no conteúdo de manose e fucose e uma diminuição no conteúdo de galactose e N-acetilglucosamina. A presença de MnCl2 no meio de cultura resultou em um aumento de 1,7% no conteúdo de ácido siálico e de 1,3% na ocupação dos sítios de glicosilação. Houve ainda um aumento no conteúdo de manose e N-acetilglucosamina e uma diminuição no conteúdo de fucose e galactose. Um aumento de 7% na freqüência de estruturas bi-antenárias foi também observado quando MnCl2 foi utilizado. O uso simultâneo de NaBu e MnCl2 proporcionou um aumento de 14% no conteúdo de ácido siálico e de 3% na ocupação dos sítios de glicosilação, bem como um aumento no conteúdo de todos os monossacarídeos neutros. Não houve, entretanto, alterações na freqüência das estruturas de carboidratos. Apesar de todas as alterações causadas pelos diferentes reagentes, não foram observadas diferenças na atividade biológica ou no comportamento farmacocinético das diferentes preparações de hTSH estudadas. Este estudo é extremamente relevante, tanto do ponto de vista do desenvolvimento de novos biofármacos, quanto do ponto de vista do controle de qualidade das glicoproteínas hormonais já utilizadas na clínica médica. / The influence of sodium butyrate (NaBu), manganese chloride (MnCl2), combined or isolated, and cycloheximide (CHX) on the synthesis of human thyrotropin CHO-derived (r-hTSH) was investigated for the first time. Exception made for CHX, which caused 1.5-fold reduction on volumetric productivity, all other reagents caused an increase in the volumetric productivity of 1.4-fold with MnCl2 and 3-fold with NaBu and NaBu+MnCl2. The number of viable cells decreased in presence of all these reagents. They also caused alterations in the glycosylation of r-hTSH. When CHX was utilized, a decrease in the content of sialic acid (5.5%), in the site occupancy (10%) and in the content of neutral monosaccharides was observed. In contrast, the addition of NaBu to culture medium caused an increase of about 12% in the sialic acid content and of 3% in the site occupancy. Concerning to carbohydrate structures, an increase of 14% of bi-antennary structures was achieved. In addition, an increased content of mannose and fucose and a decrease in the content of galactose and N-acetyl glucosamine was also observed. When MnCl2 was utilized, an increase of 1.7% in the sialic acid content and of 1.3% in site occupancy occurred. An increased content of mannose and N-acetyl glucosamine was also observed, while the content of fucose and galactose decreased. Concerning to carbohydrate structures, an increase of 7% on bi-antennary structures was achieved. When NaBu and MnCl2 were utilized simultaneously, we observed an increase of about 14% in the sialic acid content, of 3% in the site occupancy, as well as an increase in the content of all neutral monosaccharides. The frequency of carbohydrates structures, however, was not altered. Despite all the changes caused by these different reagents, in vivo biological activity and pharmacokinetic behavior were found not significant different in all the hTSH preparations studied in the present work.

Research into the gut microbiome has revealed the widespread influence that microbial species have on their host. Host genetics and environmental factors influence the abundance and diversity of the bacterial species living within the gastrointestinal tract. When the normal composition of the gut microbiota is altered, a dysbiotic state incurs. Inflammatory bowel disease (IBD) is a chronic/relapsing inflammatory disorder of the intestinal mucosa, which is characterized by a state of dysbiosis. Despite the large amount of information studying the role dysbiosis has in the pathogenesis of IBD, it is not clear how the altered microbial composition of the gut in IBD patients leads to susceptibility to enteric pathogens such as Clostridium difficile. This study aims to highlight the features of the gastrointestinal tract that are modified as a result of dysbiosis in the IBD population, and how these features facilitate colonization by C. difficile and symptom development. Review of the available literature demonstrated that the depletion of Clostridial cluster XIVa in IBD-associated dysbiosis alters bile acid metabolism and butyrate fermentation in the colon, ultimately promoting germination of C. difficile spores and weakening the gut's immune response against toxin-mediated inflammation. From continued research into the gut microbiota, more will be understood of how these microbial organisms influence human health and disease.

Introdução: Gliomas são os tumores cerebrais mais frequentes em pacientes com neoplasias de Sistema Nervoso Central (SNC), sendo o Glioblastoma Multiforme (GBM) o mais agressivo e letal deles. Apesar dos esforços na melhoria dos tratamentos atuais, o prognóstico para os pacientes com GBM continua sendo incerto. Sendo necessário o uso de novas estratégias terapêuticas que visem melhorar o manejo dos gliomas malignos. A combinação de terapias que agem nas principais vias de sinalização celular envolvidas na progressão do câncer poderia potencializar o efeito antitumoral das monoterapias. Métodos: As linhagens celulares U-87 e A-172 foram tratadas com o anti-EGFR tyrphostin AG1478, o inibidor de histonas deacetilases butirato de sódio (NaB) ou a combinação de ambos, por 72 horas. Tanto a viabilidade avaliada em 72 horas quanto a proliferação celular a longo prazo foram medidas através do ensaio de exclusão com azul de tripan em câmara de Neubauer. A influência do tratamento no ciclo celular e a capacidade de formar colônias foram avaliadas através da marcação com iodeto de propídeo e ensaio clonogênico, respectivamente. Resultados: Foi possível observar que o tratamento combinado com AG1478 e NaB foi capaz de reduzir a viabilidade e a proliferação celular na linhagem U-87 de GBM. Conclusão: Nosso trabalho mostrou que a inibição da via do receptor do fator de crescimento epidérmico (EGFR) combinada com a inibição das histonas deacetilases foi mais efetiva que as monoterapias na inibição da viabilidade e a proliferação celular. Esta redução foi significativa na linhagem U-87. Futuros estudos devem ser feitos para descobrir as possíveis interações entre as duas vias de sinalização em GBM. / Introduction: Gliomas are the most frequent brain tumors, in patients with Central Nervous system (NCS) malignancies, being the Glioblastoma Multiforme the most aggressive and lethal of all. Despite current multimodality treatment efforts, the prognosis for GBM patients remains poor. New therapeutic strategies that target these pathways to improve the treatment of malignant gliomas are needed. Combination of therapies with synergistic effects in the cellular signaling pathways of cancer could potentiate the anti-tumor effect of monotherapy alone. Methods: U87 and A172 cell lines were treated with the anti-EGFR Thyrphostin AG1478, the Histone Deacetylase inhibitor (HDACi) Sodyum Butyrate (NaB), or combination of both, for 72 hours. The cellular proliferation in short and in a long time was measured through the trypan-blue assay on neubauer chamber, the influence on the cell cycle and the capability of form colonies was evaluated by nuclear staining with propidium iodide and clonogenic assay respectively. Results: We found that combined treatment with AG1478 and NaB, are able to reduce the viability and proliferation in U-87. Conclusion: Our work show that combined inhibition of both epidermal growth factor receptor and histone deacetylases was able to reduce cell proliferation in GBM cell lines. This reduction was considerably significant in U-87 cell lines when compared with individual treatments. Further studies should be performed to discover the possible crosstalk between the signaling pathways of both targets in GBM.

Colorectal cancer (CRC) is a major problem in affluent countries worldwide. In Australia it is the second most commonly diagnosed malignancy with approximately 13,000 new cases diagnosed each year. This disease is also the leading cause of cancer related death in Australia with approximately 4,500 fatalities each year. Epidemiological studies have shown geographical variation in the incidence of disease, with diet considered to be a key contributing factor to CRC risk. In particular, diets high in fibre and low in fat have been demonstrated to reduce the risk of developing CRC. Fibre is heterogeneous in nature and can be categorised into different subtypes. Resistant starch is a component of fibre which remains largely intact throughout the gastrointestinal tract until it reaches the colon. Here it undergoes bacterial fermentation to produce the short chain fatty acids (SCFAs) acetate, propionate and butyrate (BuA). Each of the SCFAs are bioactive in the colon, with the most active being BuA. The beneficial effects of fibre have been linked to BuA’s ability to induce colon cancer cell differentiation, reduce proliferation and initiate apoptosis. Interestingly, in normal cells BuA is utilised as the preferential energy source and has been shown to promote proliferation. With an apparent “paradoxical effect” on normal and cancerous cells BuA has been the subject of much investigation as a potential anticancer agent. Despite numerous studies investigating BuA actions, the exact biological mechanisms remain largely undefined. This thesis explored a possible mechanism for BuA-induced apoptosis and inhibition of proliferation. In 2003, two publications provided evidence that SCFAs, including BuA, were ligands to two members of a previously orphan family of G-protein coupled receptors (GPCRs); GPCR41 and 43. Of the two receptors BuA had the strongest effect on GPCR43. Consequently this thesis investigated the possibility that BuA acts to decrease CRC proliferation and induce apoptosis by binding to and activating GPCR43 on CRC cells. It was hypothesised that GPCR43 acted as a “BuA sensor” on the surface of the cell to mediate the effects of BuA. This experimental work utilised PCR, Q-PCR, measures of apoptosis, proliferation and differentiation and RNAi knockdown. The key areas of investigation included: (1) Determining if GPCR43 was present on a range of CRC cell lines with a cell line to represent adenocarcinoma, carcinoma and metastatic stage of disease. (2) Investigating the expression of GPCR43 with manipulated nutrient media and different levels of cell confluence. (3) Exploring GPCR43 expression in normal and malignant human patient biopsies. (4) Determining if the inhibition of G-protein function using inhibitors influenced BuAinduced changes to apoptosis and proliferation. (5) Using RNAi, investigating the effect that GPCR43 knockdown would have on BuA-induced changes to proliferation and apoptosis. The key findings from this work included: (1) Presence of GPCR43 on some but not all CRC cell lines. (2) Modulation of GPCR43 expression with exposure to BuA and altered glucose concentrations in the media. (3) An influence of G-protein inhibition on BuA-induced apoptosis but not proliferation in some cell lines. (4) GPCR43 knockdown using RNAi indicated that GPCR43 is not exclusively required for BuA to regulate apoptosis and proliferation. The results from this work indicate that GPCR43 is not likely to exclusively mediate BuA’s effects, but opens up new areas of research into the exact role of GPCR43 on CRC cells. / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Colon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously.

Colon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously.

Thèse doctorat : Biologie-Santé : Lille 1 : 2003. / Trois articles publiés en anglais reproduits dans la thèse. N° d'ordre (Lille 1) : 3398. Résumé. Bibliogr. p. 133-156.

Colorectal cancer (CRC) is a major problem in affluent countries worldwide. In Australia it is the second most commonly diagnosed malignancy with approximately 13,000 new cases diagnosed each year. This disease is also the leading cause of cancer related death in Australia with approximately 4,500 fatalities each year. Epidemiological studies have shown geographical variation in the incidence of disease, with diet considered to be a key contributing factor to CRC risk. In particular, diets high in fibre and low in fat have been demonstrated to reduce the risk of developing CRC. Fibre is heterogeneous in nature and can be categorised into different subtypes. Resistant starch is a component of fibre which remains largely intact throughout the gastrointestinal tract until it reaches the colon. Here it undergoes bacterial fermentation to produce the short chain fatty acids (SCFAs) acetate, propionate and butyrate (BuA). Each of the SCFAs are bioactive in the colon, with the most active being BuA. The beneficial effects of fibre have been linked to BuA’s ability to induce colon cancer cell differentiation, reduce proliferation and initiate apoptosis. Interestingly, in normal cells BuA is utilised as the preferential energy source and has been shown to promote proliferation. With an apparent “paradoxical effect” on normal and cancerous cells BuA has been the subject of much investigation as a potential anticancer agent. Despite numerous studies investigating BuA actions, the exact biological mechanisms remain largely undefined. This thesis explored a possible mechanism for BuA-induced apoptosis and inhibition of proliferation. In 2003, two publications provided evidence that SCFAs, including BuA, were ligands to two members of a previously orphan family of G-protein coupled receptors (GPCRs); GPCR41 and 43. Of the two receptors BuA had the strongest effect on GPCR43. Consequently this thesis investigated the possibility that BuA acts to decrease CRC proliferation and induce apoptosis by binding to and activating GPCR43 on CRC cells. It was hypothesised that GPCR43 acted as a “BuA sensor” on the surface of the cell to mediate the effects of BuA. This experimental work utilised PCR, Q-PCR, measures of apoptosis, proliferation and differentiation and RNAi knockdown. The key areas of investigation included: (1) Determining if GPCR43 was present on a range of CRC cell lines with a cell line to represent adenocarcinoma, carcinoma and metastatic stage of disease. (2) Investigating the expression of GPCR43 with manipulated nutrient media and different levels of cell confluence. (3) Exploring GPCR43 expression in normal and malignant human patient biopsies. (4) Determining if the inhibition of G-protein function using inhibitors influenced BuAinduced changes to apoptosis and proliferation. (5) Using RNAi, investigating the effect that GPCR43 knockdown would have on BuA-induced changes to proliferation and apoptosis. The key findings from this work included: (1) Presence of GPCR43 on some but not all CRC cell lines. (2) Modulation of GPCR43 expression with exposure to BuA and altered glucose concentrations in the media. (3) An influence of G-protein inhibition on BuA-induced apoptosis but not proliferation in some cell lines. (4) GPCR43 knockdown using RNAi indicated that GPCR43 is not exclusively required for BuA to regulate apoptosis and proliferation. The results from this work indicate that GPCR43 is not likely to exclusively mediate BuA’s effects, but opens up new areas of research into the exact role of GPCR43 on CRC cells. / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Introdução: Gliomas são os tumores cerebrais mais frequentes em pacientes com neoplasias de Sistema Nervoso Central (SNC), sendo o Glioblastoma Multiforme (GBM) o mais agressivo e letal deles. Apesar dos esforços na melhoria dos tratamentos atuais, o prognóstico para os pacientes com GBM continua sendo incerto. Sendo necessário o uso de novas estratégias terapêuticas que visem melhorar o manejo dos gliomas malignos. A combinação de terapias que agem nas principais vias de sinalização celular envolvidas na progressão do câncer poderia potencializar o efeito antitumoral das monoterapias. Métodos: As linhagens celulares U-87 e A-172 foram tratadas com o anti-EGFR tyrphostin AG1478, o inibidor de histonas deacetilases butirato de sódio (NaB) ou a combinação de ambos, por 72 horas. Tanto a viabilidade avaliada em 72 horas quanto a proliferação celular a longo prazo foram medidas através do ensaio de exclusão com azul de tripan em câmara de Neubauer. A influência do tratamento no ciclo celular e a capacidade de formar colônias foram avaliadas através da marcação com iodeto de propídeo e ensaio clonogênico, respectivamente. Resultados: Foi possível observar que o tratamento combinado com AG1478 e NaB foi capaz de reduzir a viabilidade e a proliferação celular na linhagem U-87 de GBM. Conclusão: Nosso trabalho mostrou que a inibição da via do receptor do fator de crescimento epidérmico (EGFR) combinada com a inibição das histonas deacetilases foi mais efetiva que as monoterapias na inibição da viabilidade e a proliferação celular. Esta redução foi significativa na linhagem U-87. Futuros estudos devem ser feitos para descobrir as possíveis interações entre as duas vias de sinalização em GBM. / Introduction: Gliomas are the most frequent brain tumors, in patients with Central Nervous system (NCS) malignancies, being the Glioblastoma Multiforme the most aggressive and lethal of all. Despite current multimodality treatment efforts, the prognosis for GBM patients remains poor. New therapeutic strategies that target these pathways to improve the treatment of malignant gliomas are needed. Combination of therapies with synergistic effects in the cellular signaling pathways of cancer could potentiate the anti-tumor effect of monotherapy alone. Methods: U87 and A172 cell lines were treated with the anti-EGFR Thyrphostin AG1478, the Histone Deacetylase inhibitor (HDACi) Sodyum Butyrate (NaB), or combination of both, for 72 hours. The cellular proliferation in short and in a long time was measured through the trypan-blue assay on neubauer chamber, the influence on the cell cycle and the capability of form colonies was evaluated by nuclear staining with propidium iodide and clonogenic assay respectively. Results: We found that combined treatment with AG1478 and NaB, are able to reduce the viability and proliferation in U-87. Conclusion: Our work show that combined inhibition of both epidermal growth factor receptor and histone deacetylases was able to reduce cell proliferation in GBM cell lines. This reduction was considerably significant in U-87 cell lines when compared with individual treatments. Further studies should be performed to discover the possible crosstalk between the signaling pathways of both targets in GBM.

Introdução: Gliomas são os tumores cerebrais mais frequentes em pacientes com neoplasias de Sistema Nervoso Central (SNC), sendo o Glioblastoma Multiforme (GBM) o mais agressivo e letal deles. Apesar dos esforços na melhoria dos tratamentos atuais, o prognóstico para os pacientes com GBM continua sendo incerto. Sendo necessário o uso de novas estratégias terapêuticas que visem melhorar o manejo dos gliomas malignos. A combinação de terapias que agem nas principais vias de sinalização celular envolvidas na progressão do câncer poderia potencializar o efeito antitumoral das monoterapias. Métodos: As linhagens celulares U-87 e A-172 foram tratadas com o anti-EGFR tyrphostin AG1478, o inibidor de histonas deacetilases butirato de sódio (NaB) ou a combinação de ambos, por 72 horas. Tanto a viabilidade avaliada em 72 horas quanto a proliferação celular a longo prazo foram medidas através do ensaio de exclusão com azul de tripan em câmara de Neubauer. A influência do tratamento no ciclo celular e a capacidade de formar colônias foram avaliadas através da marcação com iodeto de propídeo e ensaio clonogênico, respectivamente. Resultados: Foi possível observar que o tratamento combinado com AG1478 e NaB foi capaz de reduzir a viabilidade e a proliferação celular na linhagem U-87 de GBM. Conclusão: Nosso trabalho mostrou que a inibição da via do receptor do fator de crescimento epidérmico (EGFR) combinada com a inibição das histonas deacetilases foi mais efetiva que as monoterapias na inibição da viabilidade e a proliferação celular. Esta redução foi significativa na linhagem U-87. Futuros estudos devem ser feitos para descobrir as possíveis interações entre as duas vias de sinalização em GBM. / Introduction: Gliomas are the most frequent brain tumors, in patients with Central Nervous system (NCS) malignancies, being the Glioblastoma Multiforme the most aggressive and lethal of all. Despite current multimodality treatment efforts, the prognosis for GBM patients remains poor. New therapeutic strategies that target these pathways to improve the treatment of malignant gliomas are needed. Combination of therapies with synergistic effects in the cellular signaling pathways of cancer could potentiate the anti-tumor effect of monotherapy alone. Methods: U87 and A172 cell lines were treated with the anti-EGFR Thyrphostin AG1478, the Histone Deacetylase inhibitor (HDACi) Sodyum Butyrate (NaB), or combination of both, for 72 hours. The cellular proliferation in short and in a long time was measured through the trypan-blue assay on neubauer chamber, the influence on the cell cycle and the capability of form colonies was evaluated by nuclear staining with propidium iodide and clonogenic assay respectively. Results: We found that combined treatment with AG1478 and NaB, are able to reduce the viability and proliferation in U-87. Conclusion: Our work show that combined inhibition of both epidermal growth factor receptor and histone deacetylases was able to reduce cell proliferation in GBM cell lines. This reduction was considerably significant in U-87 cell lines when compared with individual treatments. Further studies should be performed to discover the possible crosstalk between the signaling pathways of both targets in GBM.