Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

A perda da função das células beta acelera a deterioração do controle metabólico em pessoas com diabetes tipo 2. Além da lipo- e da glicotoxicidade, os AGEs parecem contribuir para esse processo, promovendo a apoptose das ilhotas pancreáticas. Em outros tecidos, os AGEs interagem com seu receptor específico (RAGE), produzindo espécies reativas de oxigênio (ROS) e ativando o NF-kB. Para investigar o efeito temporal dos AGEs sobre a apoptose de ilhotas, bem como o potencial de compostos antioxidantes para diminuir danos causados pelos AGEs, ilhotas pancreáticas de ratos foram tratadas durante 24, 48, 72, 96 e 120 h com AGEs gerados a partir de co-incubação de albumina de soro bovino (BSA) com Dgliceraldeído (GAD, 5 mg/mL) ou tampão fostato (controle). A apoptose foi avaliada pela quantificação do DNA fragmentado (ELISA), atividade de caspase 3 e detecção da permeabilidade da membrana mitocondrial (MitoProbe JC-1). O estresse oxidativo foi avaliado pela detecção de espécies de oxigênio (Image-iT LIVE Green) e a atividade da NADPH oxidase foi mensurada pelo método de quimioluminescência da lucigenina. A expressão dos genes Bax, Bcl2 e Nfkb1 foi avaliada por reação em cadeia da polimerase quantitativa após transcrição reversa (RT-qPCR). Em um dos tempos em que foi detectado o aumento da apoptose, o efeito de dois compostos antioxidantes foi avaliado: benfotiamina (350 M), uma vitamina B1 lipossolúvel, e Mito Q (1 M), um derivado da ubiquinona com alvo seletivo para a mitocôndria. Em 24 e 48 h, os AGES promoveram um aumento do índice de apoptose em relação ao controle, concomitantemente com o aumento na expresssão do gene Bcl2 (gene anti-apoptótico) e uma redução na expressão do gene Nfkb1. Em contraste, após 72, 96 h e 120 h, os AGEs promoveram um aumento do índice de apoptose em comparação com a condição de controle, concomitantemente com uma diminuição na expressão do gene Bcl2 e um aumento na expressão do gene Nfkb1. Em 24 h, os AGEs promoveram uma diminuição do conteúdo de ROS nas ilhotas, enquanto que nos tempos de 48 e 72 h, os AGEs promoveram um efeito oposto. A benfotiamina e o Mito Q foram capazes de diminuir o índice de apoptose e o estresse oxidativo de ilhotas expostas aos AGEs por 72 h. Em conclusão, os AGEs exerceram um duplo efeito em cultura de ilhotas pancreáticas, sendo de proteção contra a apoptose após exposição curta, mas pró-apoptótica após exposição prolongada. O Mito Q e e a benfotiamina merecem ser adicionalmente estudados como drogas com o potencial de oferecer proteção às ilhotas pancreáticas em condições de hiperglicemia crônica / Loss of beta cell function hastens the deterioration of metabolic control in people with type 2 diabetes. Besides lipo- and glucotoxicity, AGEs seem to contribute to this process by promoting islet apoptosis. In other tissues, AGEs interact with their specific receptors (RAGE) and elicit reactive oxygen species (ROS) generation and NF-kB activation. In order to investigate the temporal effect of AGEs on islet apoptosis as well as the potential of antioxidant compounds to decrease islet damage caused by AGEs, rat pancreatic islets were treated for 24, 48, 72, 96 and 120 h with either AGEs generated from co-incubation of bovine serum albumin (BSA) with D-glyceraldehyde (GAD, 5 mg/mL) or phosphate-buffered saline (PBS, control). Apoptosis was evaluated by quantification of DNA fragmentation (ELISA), caspase-3 enzyme activity and detection of mitochondrial permeability transition (MitoProbe JC-1). Oxidative stress was evaluated by oxygen species detection (Image-iT LIVE Green) and the activity of NADPH oxidase was measured by the lucigenin-enhanced chemiluminescence method. The expression of the genes Bax, Bcl2 and Nfkb1 was evaluated by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). In one of the time points at which increased apoptosis was detected, the effect of two antioxidant compounds was evaluated: benfotiamine (350 M), a liposoluble vitamin B1, and Mito Q (1 M), a derivative of ubiquinone targeted to mitochondria. In 24 and 48 h, AGEs elicited a significant decrease in the apoptosis rate in comparison to the control condition concomitantly with a significant increase in the RNA expression of the antiapoptotic gene Bcl2 and a significant decrease in the Nfkb1 RNA expression. In contrast, after 72 and 96 h, AGEs promoted a significant increase in the apoptosis rate in comparison to the control condition concomitantly with a significant decrease in Bcl2 RNA expression and a significant increase in Nfkb1 RNA expression. In 24 h, AGEs elicited a significant decrease in the islet content of ROS while after 48 and 72 h, AGEs promoted an opposite effect. Benfotiamine and Mito Q were able to decrease the apoptosis rate and the ROS content in islets exposed to AGEs for 72 h. In conclusion, AGEs exerted a dual effect in cultured pancreatic islets, being protective against apoptosis after short exposition but proapoptotic after prolonged exposition. Mito Q and benfotiamine deserve further evaluation as drugs that could offer islet protection in conditions of chronic hyperglycemia

The thesis combines laboratory experiments and field expeditions to study production, transfer and consumption of non-enzymatic antioxidants and thiamine in an aquatic food web. In particular, I (1) documented spatial and seasonal variation of tocopherols and carotenoids in the Baltic Sea pelagic food web, and (2) examined the effects of abiotic and biotic factors on tocopherol, carotenoid and thiamine concentrations in phytoplankton, zooplankton and fish. Moderate differences in temperature and salinity affected α-tocopherol, β-carotene and thiamine production in microalgae. Furthermore, the results suggest that acute stress favors the expression of non-enzymatic antioxidants rather than enzymatic antioxidants. Because production of α-tocopherol, β-carotene and thiamine differ markedly between microalgae, the availability of non-enzymatic antioxidants and thiamine is likely to be highly variable in the Baltic Sea and is difficult to predict. The transfer of non-enzymatic antioxidants from phytoplankton to zooplankton was biomass dependent. The field expeditions revealed that phytoplankton biomass was negatively associated with α-tocopherol concentration in mesozooplankton. Thus, increased eutrophication of the Baltic Sea followed by an increase in phytoplankton biomass could decrease the transfer of essential biochemicals to higher levels in the pelagic food web. This could lead to deficiency syndromes, of the kind already observed in the Baltic Sea. Astaxanthin is synthesized from precursors provided by the phytoplankton community. Thus biomass dependent transfer of astaxanthin precursors from phytoplankton to zooplankton could be responsible for astaxanthin deficiency in zooplanktivorous herring. Astaxanthin in herring consists mostly of all-Z-isomers, which are characterized by low bioavailability. Therefore, astaxanthin deficiency in salmon could be explained by the low concentration of this substance and its isomeric composition in herring.

No description available.

A perda da função das células beta acelera a deterioração do controle metabólico em pessoas com diabetes tipo 2. Além da lipo- e da glicotoxicidade, os AGEs parecem contribuir para esse processo, promovendo a apoptose das ilhotas pancreáticas. Em outros tecidos, os AGEs interagem com seu receptor específico (RAGE), produzindo espécies reativas de oxigênio (ROS) e ativando o NF-kB. Para investigar o efeito temporal dos AGEs sobre a apoptose de ilhotas, bem como o potencial de compostos antioxidantes para diminuir danos causados pelos AGEs, ilhotas pancreáticas de ratos foram tratadas durante 24, 48, 72, 96 e 120 h com AGEs gerados a partir de co-incubação de albumina de soro bovino (BSA) com Dgliceraldeído (GAD, 5 mg/mL) ou tampão fostato (controle). A apoptose foi avaliada pela quantificação do DNA fragmentado (ELISA), atividade de caspase 3 e detecção da permeabilidade da membrana mitocondrial (MitoProbe JC-1). O estresse oxidativo foi avaliado pela detecção de espécies de oxigênio (Image-iT LIVE Green) e a atividade da NADPH oxidase foi mensurada pelo método de quimioluminescência da lucigenina. A expressão dos genes Bax, Bcl2 e Nfkb1 foi avaliada por reação em cadeia da polimerase quantitativa após transcrição reversa (RT-qPCR). Em um dos tempos em que foi detectado o aumento da apoptose, o efeito de dois compostos antioxidantes foi avaliado: benfotiamina (350 M), uma vitamina B1 lipossolúvel, e Mito Q (1 M), um derivado da ubiquinona com alvo seletivo para a mitocôndria. Em 24 e 48 h, os AGES promoveram um aumento do índice de apoptose em relação ao controle, concomitantemente com o aumento na expresssão do gene Bcl2 (gene anti-apoptótico) e uma redução na expressão do gene Nfkb1. Em contraste, após 72, 96 h e 120 h, os AGEs promoveram um aumento do índice de apoptose em comparação com a condição de controle, concomitantemente com uma diminuição na expressão do gene Bcl2 e um aumento na expressão do gene Nfkb1. Em 24 h, os AGEs promoveram uma diminuição do conteúdo de ROS nas ilhotas, enquanto que nos tempos de 48 e 72 h, os AGEs promoveram um efeito oposto. A benfotiamina e o Mito Q foram capazes de diminuir o índice de apoptose e o estresse oxidativo de ilhotas expostas aos AGEs por 72 h. Em conclusão, os AGEs exerceram um duplo efeito em cultura de ilhotas pancreáticas, sendo de proteção contra a apoptose após exposição curta, mas pró-apoptótica após exposição prolongada. O Mito Q e e a benfotiamina merecem ser adicionalmente estudados como drogas com o potencial de oferecer proteção às ilhotas pancreáticas em condições de hiperglicemia crônica / Loss of beta cell function hastens the deterioration of metabolic control in people with type 2 diabetes. Besides lipo- and glucotoxicity, AGEs seem to contribute to this process by promoting islet apoptosis. In other tissues, AGEs interact with their specific receptors (RAGE) and elicit reactive oxygen species (ROS) generation and NF-kB activation. In order to investigate the temporal effect of AGEs on islet apoptosis as well as the potential of antioxidant compounds to decrease islet damage caused by AGEs, rat pancreatic islets were treated for 24, 48, 72, 96 and 120 h with either AGEs generated from co-incubation of bovine serum albumin (BSA) with D-glyceraldehyde (GAD, 5 mg/mL) or phosphate-buffered saline (PBS, control). Apoptosis was evaluated by quantification of DNA fragmentation (ELISA), caspase-3 enzyme activity and detection of mitochondrial permeability transition (MitoProbe JC-1). Oxidative stress was evaluated by oxygen species detection (Image-iT LIVE Green) and the activity of NADPH oxidase was measured by the lucigenin-enhanced chemiluminescence method. The expression of the genes Bax, Bcl2 and Nfkb1 was evaluated by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR). In one of the time points at which increased apoptosis was detected, the effect of two antioxidant compounds was evaluated: benfotiamine (350 M), a liposoluble vitamin B1, and Mito Q (1 M), a derivative of ubiquinone targeted to mitochondria. In 24 and 48 h, AGEs elicited a significant decrease in the apoptosis rate in comparison to the control condition concomitantly with a significant increase in the RNA expression of the antiapoptotic gene Bcl2 and a significant decrease in the Nfkb1 RNA expression. In contrast, after 72 and 96 h, AGEs promoted a significant increase in the apoptosis rate in comparison to the control condition concomitantly with a significant decrease in Bcl2 RNA expression and a significant increase in Nfkb1 RNA expression. In 24 h, AGEs elicited a significant decrease in the islet content of ROS while after 48 and 72 h, AGEs promoted an opposite effect. Benfotiamine and Mito Q were able to decrease the apoptosis rate and the ROS content in islets exposed to AGEs for 72 h. In conclusion, AGEs exerted a dual effect in cultured pancreatic islets, being protective against apoptosis after short exposition but proapoptotic after prolonged exposition. Mito Q and benfotiamine deserve further evaluation as drugs that could offer islet protection in conditions of chronic hyperglycemia

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)