published_or_final_version / Physiology / Master / Master of Philosophy

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This study was undertaken as a result of the occurrence of a large number of deaths among the local Black population from the use of herbal medicines prepared from the rootstock of Callilepis laureola known to the Zulus as impila. The salient clinico-pathological features in these cases were hypoglycaemia, centrilobular zonal liver necrosis and acute renal tubular necrosis. The purpose of this study was to investigate fully the clinical, biochemical and pathological aspects of the toxicity produced by Callilepis laureola (impila). The first part of the investigation consisted of an assessment of all cases of death due to acute liver necrosis diagnosed by necropsy at King Edward VIII Hospital, Durban. A review of clinical and necropsy records of 21687 consecutive post-mortems performed on Black patients during a 20 year period showed that acute liver necrosis was the major contributing cause of death in 447 patients. In 263 cases the hepatic lesion was centri lobular zonal necrosis with associated acute tubular necrosis (Group A); while in 184 cases the I iver necrosis was of the massive or submassive type (Group B). A comparative assessment of these two groups as regards necropsy prevalence, age and sex distribution and the clinical, biochemical and pathological findings was undertaken. This study shows that the combination of hypoglycaemia, centri lobular zonal liver necrosis and acute renal tubular necrosis due to Callilepis laureola (impila) poisoning is a distinct clinico-pathological entity and differentiates this group from cases of acute massive and submassive liver necrosis resulting in most cases from fulminant viral hepatitis. In the search for the toxic components of the root of Callilepsis laureola several compounds were isolated. These were atractyloside, carboxyatractyloside, two thymol related oils and a carbohydrate. The thymol related oils as well as the carbohydrate were found to be non-toxic in laboratory rats. The crude methanol extract of the root of Callilepsis laureola, when injected intraperitoneally into laboratory rats, produced centrilobular zonal liver necrosis and acute renal tubular necrosis, the lesions identical to those seen in patients who had died after intake of impila prescribed by witchdoctors and other dispensers of herbal medicines. On the other hand intraperitoneal injections of the purified compound atractyloside caused acute renal tubular necrosis and hypoglycaemia in laboratory rats but failed to produce liver necrosis. Carboxyatractyloside also failed to cause liver necrosis. This indicated that there may be at least two toxins contained in the rootstock of Callilepsis laureola, one causing the liver lesion and the other (atractyloside) causing nephrotoxicity and hypoglycaemia. Repeated attempts at isolating the hepatotoxin have failed; the liver toxin or toxins being lost during the process of extraction and purification. Identification of the hepatotoxin awaits further investigation. It is possible that the liver necrosis may be caused by a metabolite or that it may be a synergistic effect of two or more compounds. / Thesis (MD)-University of Natal, 1983.

Entre os meses de junho e agosto 2006 dois surtos de intoxicação pelas larvas de P. flavipes ocorreram, um em ovinos (surto 1) e outro em bovinos (surto 2). O surto nos ovinos ocorreu nos meses de junho e julho no município de Encruzilhada do Sul RS e morreram 25 ovinos de um rebanho de 175 ovinos e 11 bovinos. Os ovinos eram mantidos em uma área de 40 hectares de pasto nativo. O surto nos bovinos ocorreu no mês de agosto no município de Sombrio SC e morreram 17 animais que estavam num lote de 77 bovinos, o rebanho total da propriedade era de 280 animais. O lote de bovinos estava em uma área de 90 hectares onde dois animais morreram devido à ingestão das larvas de P. flavipes. O lote foi transferido para outra área onde morreram mais 15 animais em um período de 5 dias. Seis ovinos do surto 1 e seis bovinos do surto 2 foram necropsiados. Grande quantidade de agrupamentos de larvas de P. flavipes foram encontradas no campo e no rúmen dos animais de ambos os surtos. Larvas de P. flavipes foram coletadas em ambos os surtos e foram armazenadas congeladas a -20º. Larvas frescas e larvas congeladas foram administradas para 6 ovinos, por meio de uma seringa com a ponta cortada. A menor dose letal foi de 7,5 g/kg em administração única. Animais que receberam doses sub-letais de 5 g/kg e posteriormente doses letais de 10 g/kg e 15 g/kg em intervalos de 15 e 30 dias, mostraram menor sensibilidade à intoxicação, um animal não adoeceu e outro adoeceu apenas depois de receber uma dose de 15 g/kg. Os animais intoxicados experimentalmente mostraram sinais de doença cerca de 48 horas e morreram cerca de 54 horas após a intoxicação. O exame bioquímico realizado em intervalos de 12 horas revelou alterações apenas nos animais que adoeceram. Os níveis séricos de GGT apresentavam-se elevados depois de 24 horas da intoxicação e continuaram a se elevar até a morte, AST apresentou aumento significativo cerca de 30 horas após a dosificação e posteriormente decaindo até a morte, os níveis séricos de glicose sofreram queda próximo à morte dos animais. As lesões de necropsia observadas nos casos espontâneos e experimentais foram semelhantes e mais consistentes no fígado, que se apresentava com acentuação do padrão lobular e com áreas de coloração amarelada com petéquias subcapsulares. Foram observados também edemas cavitários, edema da parede da vesícula biliar, perirrenal e na região inicial do duodeno, pâncreas e abomaso. Além das hemorragias no fígado havia hemorragias no tecido subcutâneo, coração, e mucosas e serosas da cavidade abdominal. O principal achado histopatológico era caracterizado por necrose coagulativa centrolobular ou massiva associada à hemorragia e congestão centrolobular e degeneração e tumefação hepática na região periportal. Observou-se ainda, depleção e necrose linfóide nos centros germinativos de linfonodos, nas placas de Peyer e na polpa branca do baço. A microscopia eletrônica demonstrou lesão hepática com hepatócitos necróticos, e dilatação dos espaços de Disse e endotélio vascular. Foi observada também intensa proliferação de reticulo endoplasmático liso no animal que recebeu doses graduais das larvas. As larvas descongeladas e dessecadas a 100ºC por 24 horas foram administradas a um coelho e mostraram-se tóxicas. Outros dois coelhos receberam frações sólidas e liquidas das larvas respectivamente e apenas o coelho que recebeu a fração sólida morreu. / Between June and August 2006, two outbreaks of P. flavipes larvae poisoning were observed. In outbreak 1 occurred in a farm located at the county of Encruzilhada do Sul, Rio Grande do Sul State, Brazil, 25 out of 175 sheep were affected and died. Although there were 11 cattle in the same paddock, none of them was affected. Animals were kept in a 40 hectares paddock of native pastures. In outbreak 2, occurred in a farm located at county of Sombrio, Santa Catarina State, 11 out of 77 cattle were affected and died. In total, 6 sheep and 6 cattle from respective outbreaks were necropsied. High numbers of compact masses containing up to 150 larvae were scattered on the paddocks in which animals were grazing. Larvae were collected and frozen at –20ºC. Perreyia flavipes larval body fragments and heads were found in the forestomach contents of all necropsied animals. Fresh and thawed larvae were administered to six sheep by a tip-cut plastic syringe. The lower single lethal dosis was 7,5 g/kg. Animals which received an initial sublethal doses of 5 g/kg and subsequently were dosed with lethal 10 to 15 g/kg at 15 and 30 days intervals showed lower susceptibility to the intoxication. Animals that were experimentally poisoned showed signs of disease about 48 h after dosing and died in approximately 54 h. Biochemical tests performed at 12 h intervals showed changes only in diseased animals. Serum levels of GGT started being higher 24 h after intoxication and kept enhancement until death. AST serum levels were significatively enhanced about 30 h after dosing and then, decreased to death. Glucose serum levels decreased close to the death. Necropsy lesions were similar in both, spontaneous and experimental cases and were most prominent in the livers, which had enhanced lobular pattern and yellowish areas with subcapsular pinpoint hemorrhages. Edema in body cavities, gallbladder wall, perirenal tissues, initial portion of duodenum, pancreas, and abomasum was also seen. Hemorrhages were also present in subcutaneous tissues, heart, and in mucosal and serosal membranes of the abdominal cavity. The principal histological finding was centrolobular to massive coagulative necrosis associated with centrolobular hemorrhage and congestion and periportal degeneration and tumefaction. There also were lymphoid depletion and necrosis in the germinative centres of lymph nodes, Peyer’s patches, and white pulp of the spleen. Transmission electron microscopy showed necrotic hepatocytes and dilatation in the space of Disse and vascular endothelium. Severe proliferation of smooth endoplasmic reticulum was also seen in animal receiving more than one dosis of larvae. Thawed larvae were desiccated at 100ºC for 24 h and administered to one rabbit that also became ill. Additional two rabbit were dosed with solid or liquid fractions of larvae and only which received the solid fraction died.

Entre os meses de junho e agosto 2006 dois surtos de intoxicação pelas larvas de P. flavipes ocorreram, um em ovinos (surto 1) e outro em bovinos (surto 2). O surto nos ovinos ocorreu nos meses de junho e julho no município de Encruzilhada do Sul RS e morreram 25 ovinos de um rebanho de 175 ovinos e 11 bovinos. Os ovinos eram mantidos em uma área de 40 hectares de pasto nativo. O surto nos bovinos ocorreu no mês de agosto no município de Sombrio SC e morreram 17 animais que estavam num lote de 77 bovinos, o rebanho total da propriedade era de 280 animais. O lote de bovinos estava em uma área de 90 hectares onde dois animais morreram devido à ingestão das larvas de P. flavipes. O lote foi transferido para outra área onde morreram mais 15 animais em um período de 5 dias. Seis ovinos do surto 1 e seis bovinos do surto 2 foram necropsiados. Grande quantidade de agrupamentos de larvas de P. flavipes foram encontradas no campo e no rúmen dos animais de ambos os surtos. Larvas de P. flavipes foram coletadas em ambos os surtos e foram armazenadas congeladas a -20º. Larvas frescas e larvas congeladas foram administradas para 6 ovinos, por meio de uma seringa com a ponta cortada. A menor dose letal foi de 7,5 g/kg em administração única. Animais que receberam doses sub-letais de 5 g/kg e posteriormente doses letais de 10 g/kg e 15 g/kg em intervalos de 15 e 30 dias, mostraram menor sensibilidade à intoxicação, um animal não adoeceu e outro adoeceu apenas depois de receber uma dose de 15 g/kg. Os animais intoxicados experimentalmente mostraram sinais de doença cerca de 48 horas e morreram cerca de 54 horas após a intoxicação. O exame bioquímico realizado em intervalos de 12 horas revelou alterações apenas nos animais que adoeceram. Os níveis séricos de GGT apresentavam-se elevados depois de 24 horas da intoxicação e continuaram a se elevar até a morte, AST apresentou aumento significativo cerca de 30 horas após a dosificação e posteriormente decaindo até a morte, os níveis séricos de glicose sofreram queda próximo à morte dos animais. As lesões de necropsia observadas nos casos espontâneos e experimentais foram semelhantes e mais consistentes no fígado, que se apresentava com acentuação do padrão lobular e com áreas de coloração amarelada com petéquias subcapsulares. Foram observados também edemas cavitários, edema da parede da vesícula biliar, perirrenal e na região inicial do duodeno, pâncreas e abomaso. Além das hemorragias no fígado havia hemorragias no tecido subcutâneo, coração, e mucosas e serosas da cavidade abdominal. O principal achado histopatológico era caracterizado por necrose coagulativa centrolobular ou massiva associada à hemorragia e congestão centrolobular e degeneração e tumefação hepática na região periportal. Observou-se ainda, depleção e necrose linfóide nos centros germinativos de linfonodos, nas placas de Peyer e na polpa branca do baço. A microscopia eletrônica demonstrou lesão hepática com hepatócitos necróticos, e dilatação dos espaços de Disse e endotélio vascular. Foi observada também intensa proliferação de reticulo endoplasmático liso no animal que recebeu doses graduais das larvas. As larvas descongeladas e dessecadas a 100ºC por 24 horas foram administradas a um coelho e mostraram-se tóxicas. Outros dois coelhos receberam frações sólidas e liquidas das larvas respectivamente e apenas o coelho que recebeu a fração sólida morreu. / Between June and August 2006, two outbreaks of P. flavipes larvae poisoning were observed. In outbreak 1 occurred in a farm located at the county of Encruzilhada do Sul, Rio Grande do Sul State, Brazil, 25 out of 175 sheep were affected and died. Although there were 11 cattle in the same paddock, none of them was affected. Animals were kept in a 40 hectares paddock of native pastures. In outbreak 2, occurred in a farm located at county of Sombrio, Santa Catarina State, 11 out of 77 cattle were affected and died. In total, 6 sheep and 6 cattle from respective outbreaks were necropsied. High numbers of compact masses containing up to 150 larvae were scattered on the paddocks in which animals were grazing. Larvae were collected and frozen at –20ºC. Perreyia flavipes larval body fragments and heads were found in the forestomach contents of all necropsied animals. Fresh and thawed larvae were administered to six sheep by a tip-cut plastic syringe. The lower single lethal dosis was 7,5 g/kg. Animals which received an initial sublethal doses of 5 g/kg and subsequently were dosed with lethal 10 to 15 g/kg at 15 and 30 days intervals showed lower susceptibility to the intoxication. Animals that were experimentally poisoned showed signs of disease about 48 h after dosing and died in approximately 54 h. Biochemical tests performed at 12 h intervals showed changes only in diseased animals. Serum levels of GGT started being higher 24 h after intoxication and kept enhancement until death. AST serum levels were significatively enhanced about 30 h after dosing and then, decreased to death. Glucose serum levels decreased close to the death. Necropsy lesions were similar in both, spontaneous and experimental cases and were most prominent in the livers, which had enhanced lobular pattern and yellowish areas with subcapsular pinpoint hemorrhages. Edema in body cavities, gallbladder wall, perirenal tissues, initial portion of duodenum, pancreas, and abomasum was also seen. Hemorrhages were also present in subcutaneous tissues, heart, and in mucosal and serosal membranes of the abdominal cavity. The principal histological finding was centrolobular to massive coagulative necrosis associated with centrolobular hemorrhage and congestion and periportal degeneration and tumefaction. There also were lymphoid depletion and necrosis in the germinative centres of lymph nodes, Peyer’s patches, and white pulp of the spleen. Transmission electron microscopy showed necrotic hepatocytes and dilatation in the space of Disse and vascular endothelium. Severe proliferation of smooth endoplasmic reticulum was also seen in animal receiving more than one dosis of larvae. Thawed larvae were desiccated at 100ºC for 24 h and administered to one rabbit that also became ill. Additional two rabbit were dosed with solid or liquid fractions of larvae and only which received the solid fraction died.

Entre os meses de junho e agosto 2006 dois surtos de intoxicação pelas larvas de P. flavipes ocorreram, um em ovinos (surto 1) e outro em bovinos (surto 2). O surto nos ovinos ocorreu nos meses de junho e julho no município de Encruzilhada do Sul RS e morreram 25 ovinos de um rebanho de 175 ovinos e 11 bovinos. Os ovinos eram mantidos em uma área de 40 hectares de pasto nativo. O surto nos bovinos ocorreu no mês de agosto no município de Sombrio SC e morreram 17 animais que estavam num lote de 77 bovinos, o rebanho total da propriedade era de 280 animais. O lote de bovinos estava em uma área de 90 hectares onde dois animais morreram devido à ingestão das larvas de P. flavipes. O lote foi transferido para outra área onde morreram mais 15 animais em um período de 5 dias. Seis ovinos do surto 1 e seis bovinos do surto 2 foram necropsiados. Grande quantidade de agrupamentos de larvas de P. flavipes foram encontradas no campo e no rúmen dos animais de ambos os surtos. Larvas de P. flavipes foram coletadas em ambos os surtos e foram armazenadas congeladas a -20º. Larvas frescas e larvas congeladas foram administradas para 6 ovinos, por meio de uma seringa com a ponta cortada. A menor dose letal foi de 7,5 g/kg em administração única. Animais que receberam doses sub-letais de 5 g/kg e posteriormente doses letais de 10 g/kg e 15 g/kg em intervalos de 15 e 30 dias, mostraram menor sensibilidade à intoxicação, um animal não adoeceu e outro adoeceu apenas depois de receber uma dose de 15 g/kg. Os animais intoxicados experimentalmente mostraram sinais de doença cerca de 48 horas e morreram cerca de 54 horas após a intoxicação. O exame bioquímico realizado em intervalos de 12 horas revelou alterações apenas nos animais que adoeceram. Os níveis séricos de GGT apresentavam-se elevados depois de 24 horas da intoxicação e continuaram a se elevar até a morte, AST apresentou aumento significativo cerca de 30 horas após a dosificação e posteriormente decaindo até a morte, os níveis séricos de glicose sofreram queda próximo à morte dos animais. As lesões de necropsia observadas nos casos espontâneos e experimentais foram semelhantes e mais consistentes no fígado, que se apresentava com acentuação do padrão lobular e com áreas de coloração amarelada com petéquias subcapsulares. Foram observados também edemas cavitários, edema da parede da vesícula biliar, perirrenal e na região inicial do duodeno, pâncreas e abomaso. Além das hemorragias no fígado havia hemorragias no tecido subcutâneo, coração, e mucosas e serosas da cavidade abdominal. O principal achado histopatológico era caracterizado por necrose coagulativa centrolobular ou massiva associada à hemorragia e congestão centrolobular e degeneração e tumefação hepática na região periportal. Observou-se ainda, depleção e necrose linfóide nos centros germinativos de linfonodos, nas placas de Peyer e na polpa branca do baço. A microscopia eletrônica demonstrou lesão hepática com hepatócitos necróticos, e dilatação dos espaços de Disse e endotélio vascular. Foi observada também intensa proliferação de reticulo endoplasmático liso no animal que recebeu doses graduais das larvas. As larvas descongeladas e dessecadas a 100ºC por 24 horas foram administradas a um coelho e mostraram-se tóxicas. Outros dois coelhos receberam frações sólidas e liquidas das larvas respectivamente e apenas o coelho que recebeu a fração sólida morreu. / Between June and August 2006, two outbreaks of P. flavipes larvae poisoning were observed. In outbreak 1 occurred in a farm located at the county of Encruzilhada do Sul, Rio Grande do Sul State, Brazil, 25 out of 175 sheep were affected and died. Although there were 11 cattle in the same paddock, none of them was affected. Animals were kept in a 40 hectares paddock of native pastures. In outbreak 2, occurred in a farm located at county of Sombrio, Santa Catarina State, 11 out of 77 cattle were affected and died. In total, 6 sheep and 6 cattle from respective outbreaks were necropsied. High numbers of compact masses containing up to 150 larvae were scattered on the paddocks in which animals were grazing. Larvae were collected and frozen at –20ºC. Perreyia flavipes larval body fragments and heads were found in the forestomach contents of all necropsied animals. Fresh and thawed larvae were administered to six sheep by a tip-cut plastic syringe. The lower single lethal dosis was 7,5 g/kg. Animals which received an initial sublethal doses of 5 g/kg and subsequently were dosed with lethal 10 to 15 g/kg at 15 and 30 days intervals showed lower susceptibility to the intoxication. Animals that were experimentally poisoned showed signs of disease about 48 h after dosing and died in approximately 54 h. Biochemical tests performed at 12 h intervals showed changes only in diseased animals. Serum levels of GGT started being higher 24 h after intoxication and kept enhancement until death. AST serum levels were significatively enhanced about 30 h after dosing and then, decreased to death. Glucose serum levels decreased close to the death. Necropsy lesions were similar in both, spontaneous and experimental cases and were most prominent in the livers, which had enhanced lobular pattern and yellowish areas with subcapsular pinpoint hemorrhages. Edema in body cavities, gallbladder wall, perirenal tissues, initial portion of duodenum, pancreas, and abomasum was also seen. Hemorrhages were also present in subcutaneous tissues, heart, and in mucosal and serosal membranes of the abdominal cavity. The principal histological finding was centrolobular to massive coagulative necrosis associated with centrolobular hemorrhage and congestion and periportal degeneration and tumefaction. There also were lymphoid depletion and necrosis in the germinative centres of lymph nodes, Peyer’s patches, and white pulp of the spleen. Transmission electron microscopy showed necrotic hepatocytes and dilatation in the space of Disse and vascular endothelium. Severe proliferation of smooth endoplasmic reticulum was also seen in animal receiving more than one dosis of larvae. Thawed larvae were desiccated at 100ºC for 24 h and administered to one rabbit that also became ill. Additional two rabbit were dosed with solid or liquid fractions of larvae and only which received the solid fraction died.

Wong, Chi Bun. / Thesis submitted in: Nov 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 116-127). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.v / Abbreviations --- p.vii / List of Table and Figures --- p.ix / Table of Contents --- p.x / Chapter Chapter 1 --- p.1 / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Identification of the proteins regulated by BRE when BRE was over-expressed or silencedin C2C12 and D122 --- p.1 / Chapter 1.1.1 --- What is BRE? --- p.1 / Chapter 1.1.2 --- BRE gene is Highly Conserved --- p.2 / Chapter 1.1.3 --- BRE binds to the Intracellular Domain of TNFR1 and Fas --- p.3 / Chapter 1.1.4 --- BRE Suppresses Apoptosis --- p.4 / Chapter 1.1.5 --- "BRE forms a Holoenzyme Complex with BRCA1, BARD1 and BRCC36" --- p.4 / Chapter 1.16 --- Roles of the Differentially Expressed Proteins Identified in the siRNA knockdown Experiments --- p.5 / Chapter 1.1.6.1 --- Akt3 --- p.5 / Chapter 1.1.6.2 --- Mdm2/4 --- p.6 / Chapter 1.1.6.3 --- Prohibitin --- p.7 / Chapter 1.1.6.4 --- Carbonic Anhydrase III --- p.8 / Chapter 1.1.6.5 --- 26S Proteasome --- p.8 / Chapter 1.2 --- The Role of BRE in Liver: a morphological approach --- p.9 / Chapter 1.2.1 --- The General Structure of the Liver. --- p.9 / Chapter 1.2.2 --- The Essential Functions of the Liver --- p.11 / Chapter 1.2.3 --- Inflammation of the Liver --- p.11 / Chapter 1.2.3.1 --- Hepatitis --- p.11 / Chapter 1.2.3.2 --- Acute Hepatitis --- p.12 / Chapter 1.2.3.3 --- Chronic Hepatitis --- p.12 / Chapter 1.2.4 --- Necrosis and Apoptosis --- p.13 / Chapter 1.2.5 --- The Apoptotic Pathway --- p.14 / Chapter 1.2.6 --- Hepatic Necrosis is Divided into Different Zones --- p.16 / Chapter 1.2.6.1 --- Hepatitis Necrosis is Categorized into 3 Zones --- p.16 / Chapter 1.2.7 --- Carbon Tetrachloride (CCL4) --- p.16 / Chapter 1.2.8 --- TNFa is a Pleiotropic Cytokine --- p.17 / Chapter 1.3 --- The Objectives of This Project --- p.20 / Chapter Chapter 2 --- p.21 / Chapter 2. --- Materials and Methods --- p.21 / Chapter 2.1 --- Animals --- p.21 / Chapter 2.2 --- Adminstration of Carbon Tetrachloride and Corn Oil --- p.21 / Chapter 2.3 --- Cell Cultures --- p.22 / Chapter 2.4 --- Cell Culturing --- p.22 / Chapter 2.5 --- Gene Silencing with Small Interfering RNA (siRNA) --- p.23 / Chapter 2.5.1 --- Transfection with BRE siRNA --- p.24 / Chapter 2.6 --- Cell Proliferation Assays --- p.24 / Chapter 2.7 --- In-Situ Hybridization of BRE Sense and Antisense Probes --- p.25 / Chapter 2.8 --- Immunohistological Staining --- p.26 / Chapter 2.9 --- Semi-Quantitative RT-PCR --- p.28 / Chapter 2.10 --- Comparative Proteomics --- p.29 / Chapter 2.10.1 --- Sample Preparation for Two Dimensional Gel Electrophoresis --- p.29 / Chapter 2.10.2 --- Two Dimensional Polyacrylamide Gel Electrophoresis --- p.30 / Chapter 2.10.3 --- In-Gel Digestion and MALDI-TOF Analysis --- p.31 / Chapter 2.11 --- Western Blotting --- p.32 / Chapter 2.12 --- Flow Cytometry --- p.34 / Chapter 2.13 --- Haematoxylin and Eosin Staining (H&E) --- p.34 / Chapter Chapter 3 --- p.36 / Chapter 3. --- Results --- p.36 / Chapter 3.1 --- BRE expression in C2C12 cells --- p.36 / Chapter 3.2 --- Comparative Proteomic Profile of BRE silenced C2C12 cells --- p.41 / Chapter 3.3 --- Effect of Silencing BRE on C2C12 cell Proliferation --- p.49 / Chapter 3.4 --- Effects of BRE over-expression in D122 cells --- p.54 / Chapter 3.5 --- BRE Expression in the Liver --- p.62 / Chapter 3.5.1 --- Histological Analysis of Liver Sections after 24 hours of CCL4 Insult --- p.62 / Chapter 3.5.2 --- BRE Expression in the Liver --- p.62 / Chapter 3.6 --- Histological Study of Liver Treated with CCL4 --- p.67 / Chapter 3.7 --- BRE Expression in Experimental Liver --- p.76 / Chapter Chapter 4 --- p.92 / Chapter 4. --- Discussion --- p.92 / Chapter 4.1 --- Expression of BRE in C2C12 --- p.92 / Chapter 4.2 --- The Regulatory Function of BRE --- p.96 / Chapter 4.3 --- The Relationship Between BRE and p53 --- p.98 / Chapter 4.4 --- The Relationship Between BRE and NFkB --- p.104 / Chapter 4.5 --- BRE Expression in Normal Control and CCL4 Treated Livers --- p.105 / Chapter 4.6 --- A Possible Explanation for the Necrosis Pattern Observed --- p.107 / Chapter 4.7 --- The Relationship Between BRE and the TNF Receptors --- p.109 / Chapter Chapter 5 --- p.112 / Chapter 5. --- Conclusion and Future Prospects --- p.112 / References --- p.116

Este estudo caracteriza a intoxicação por Trema micrantha em equinos, até então desconhecida nesta espécie. No primeiro artigo (artigo 1), é descrito um surto espontâneo de intoxicação por T. micrantha em equinos que ocorreu em junho de 2007, no Município de São José do Herval, na região da encosta da serra do Rio Grande do Sul. Dois equinos morreram na propriedade, após uma árvore de T. micrantha ter sido derrubada por um temporal e suas folhas terem sido consumidas pelos animais. O quadro clínico patológico apresentado foi característico de insuficiência hepática aguda, com desenvolvimento de encefalopatia hepática. Subsequentemente, para melhor caracterizar a intoxicação por Trema micrantha em equinos, desenvolveu-se um experimento (artigo 2). Quatro pôneis receberam e consumiram espontaneamente folhas de T. micrantha, em doses únicas de 30, 25 e 20g/kg. Um equino recebeu uma dose de 15 e outra de 25g/kg, 30 dias após. Três animais adoeceram e evoluíram para morte. Os principais sinais clínicos apresentados foram apatia, desequilíbrios, dificuldade de deglutição, decúbito esternal, decúbito lateral, movimentos de pedalagem coma e morte. Os três equinos afetados apresentaram elevação na atividade sérica de gama glutamil transferase (GGT), nos níveis séricos de amônia, além de diminuição da glicemia. Os principais achados patológicos foram encontrados no fígado e no encéfalo dos três animais. O fígado apresentava macroscopicamente acentuação do padrão lobular, enquanto que no encéfalo havia áreas amareladas na superfície de corte, mais evidentes na substância branca do cerebelo. Microscopicamente, o fígado apresentava necrose, predominantemente centrolobular e hemorragia. No encéfalo, havia edema perivascular generalizado e astrócitos Alzheimer tipo II na substância cinzenta. Esses astrócitos apresentaram marcação fraca ou negativa na imuno-histoquímica anti-GFAP e marcação positiva do antígeno S-100. A dose letal mínima de folhas de T. micrantha estabelecida nesse experimento foi de 20g/kg. A maior sensibilidade da espécie equina constatada nesse estudo, a ampla distribuição de T. micrantha, bem como sua palatabilidade reforçam a importância da planta em casos acidentais de intoxicação nessa espécie. / This study characterizes Trema micrantha poisoning in horses, previously unknown in this species. The first article (Article 1) describes an outbreak of T. micrantha poisoning in horses. The disease occurred in June 2007, in the Municipality of São José do Herval, Rio Grande do Sul State. Two horses died after consuming the leaves of the branches from a T. micrantha tree, which had been felled by a storm. Clinical pathology presented by the animals was characteristic of an acute liver failure with development of hepatic encephalopathy. To further characterize the Trema micrantha poisoning in horses, an experiment was carried out (Article 2). Four ponies received and spontaneously consumed green leaves of T. micrantha, at the doses of 30, 25, and 20 g/kg. One horse received two doses, 15 and 25 g/kg, 30 days apart. Three animals were affected and died. The main clinical signs were apathy, equilibrium deficit, deglutition difficulty, sternal or lateral recumbency, paddling, coma and death. These tree diseased ponies had also enhanced seric activity of gamma-glutamyl transferase (GGT), seric ammonia apart of diminished glycemia. The main pathological findings were observed in the liver and encephalon. There were enhanced lobular pattern of the livers and yellowish areas in the cut surface of the encephalon, especially visualized in the cerebral white matter. Microscopically, there was hepatic necrosis predominantly centrilobular apart of hemorrhages. Generalized perivascular edema and Alzheimer type II astrocytes were observed in the encephalon. The Alzheimer type II astrocytes showed weak or absent anti-glial fibrillar acid protein immunostaining associated with positive immunostaining for S-100 protein. The minimal lethal dose of Trema micrantha leaves was established at 20 g/kg. The high sensibility of this species to this plant, its wide distribution, and the high palatability of the plant reinforce the importance of Trema micrantha in accidental episodes of intoxication in horses.

Este estudo caracteriza a intoxicação por Trema micrantha em equinos, até então desconhecida nesta espécie. No primeiro artigo (artigo 1), é descrito um surto espontâneo de intoxicação por T. micrantha em equinos que ocorreu em junho de 2007, no Município de São José do Herval, na região da encosta da serra do Rio Grande do Sul. Dois equinos morreram na propriedade, após uma árvore de T. micrantha ter sido derrubada por um temporal e suas folhas terem sido consumidas pelos animais. O quadro clínico patológico apresentado foi característico de insuficiência hepática aguda, com desenvolvimento de encefalopatia hepática. Subsequentemente, para melhor caracterizar a intoxicação por Trema micrantha em equinos, desenvolveu-se um experimento (artigo 2). Quatro pôneis receberam e consumiram espontaneamente folhas de T. micrantha, em doses únicas de 30, 25 e 20g/kg. Um equino recebeu uma dose de 15 e outra de 25g/kg, 30 dias após. Três animais adoeceram e evoluíram para morte. Os principais sinais clínicos apresentados foram apatia, desequilíbrios, dificuldade de deglutição, decúbito esternal, decúbito lateral, movimentos de pedalagem coma e morte. Os três equinos afetados apresentaram elevação na atividade sérica de gama glutamil transferase (GGT), nos níveis séricos de amônia, além de diminuição da glicemia. Os principais achados patológicos foram encontrados no fígado e no encéfalo dos três animais. O fígado apresentava macroscopicamente acentuação do padrão lobular, enquanto que no encéfalo havia áreas amareladas na superfície de corte, mais evidentes na substância branca do cerebelo. Microscopicamente, o fígado apresentava necrose, predominantemente centrolobular e hemorragia. No encéfalo, havia edema perivascular generalizado e astrócitos Alzheimer tipo II na substância cinzenta. Esses astrócitos apresentaram marcação fraca ou negativa na imuno-histoquímica anti-GFAP e marcação positiva do antígeno S-100. A dose letal mínima de folhas de T. micrantha estabelecida nesse experimento foi de 20g/kg. A maior sensibilidade da espécie equina constatada nesse estudo, a ampla distribuição de T. micrantha, bem como sua palatabilidade reforçam a importância da planta em casos acidentais de intoxicação nessa espécie. / This study characterizes Trema micrantha poisoning in horses, previously unknown in this species. The first article (Article 1) describes an outbreak of T. micrantha poisoning in horses. The disease occurred in June 2007, in the Municipality of São José do Herval, Rio Grande do Sul State. Two horses died after consuming the leaves of the branches from a T. micrantha tree, which had been felled by a storm. Clinical pathology presented by the animals was characteristic of an acute liver failure with development of hepatic encephalopathy. To further characterize the Trema micrantha poisoning in horses, an experiment was carried out (Article 2). Four ponies received and spontaneously consumed green leaves of T. micrantha, at the doses of 30, 25, and 20 g/kg. One horse received two doses, 15 and 25 g/kg, 30 days apart. Three animals were affected and died. The main clinical signs were apathy, equilibrium deficit, deglutition difficulty, sternal or lateral recumbency, paddling, coma and death. These tree diseased ponies had also enhanced seric activity of gamma-glutamyl transferase (GGT), seric ammonia apart of diminished glycemia. The main pathological findings were observed in the liver and encephalon. There were enhanced lobular pattern of the livers and yellowish areas in the cut surface of the encephalon, especially visualized in the cerebral white matter. Microscopically, there was hepatic necrosis predominantly centrilobular apart of hemorrhages. Generalized perivascular edema and Alzheimer type II astrocytes were observed in the encephalon. The Alzheimer type II astrocytes showed weak or absent anti-glial fibrillar acid protein immunostaining associated with positive immunostaining for S-100 protein. The minimal lethal dose of Trema micrantha leaves was established at 20 g/kg. The high sensibility of this species to this plant, its wide distribution, and the high palatability of the plant reinforce the importance of Trema micrantha in accidental episodes of intoxication in horses.