Rogdi was a novel gene with unknown function. According to GeneBank database, the gene is located on chromsome 10q12 and the length of coding regeion is 864 bp that encods 287 animo acids. Earlier studies in our laboratory showed that human ROGDI influenced rate of cell proliferaion in HeLa, Hep3B and NIH3T3 cells. In addition, we found Rogdi protien was up-regulated in fibrotic livers. Following various types of injury to liver, quiescent hepatic stellate cells (HSCs) transform to activated phenotype, leading to exprssion of £\-SMA, increasing rate of cell proliferation and depositing of extracellular matrix. In this study, we found that Rogdi protein was up-regulated in activated HSCs isolated and cultured from rat livers. By either overexpression or RNA interference of Rogdi, we found that Rogdi affected rate of HSCs proliferation, and expressions of £\-SMA and collagen type I. Expression of Rogdi protein was induced after PDGF treatment of rat HSCs. Additionally, we found that Rogdi was involved in MAPK and PI3K/Akt pathways. Furthermore, using wound healing assay and migration assay, Rogdi was found to regulate migration of activated HSCs.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18508号 / 医博第3928号 / 新制||医||1005(附属図書館) / 31394 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 稲垣 暢也, 教授 千葉 勉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

Doenças pulmonares intersticiais fibrosantes são doenças que afetam homens, mulheres e crianças, tem prognóstico ruim e os pacientes possuem sobrevida estimada entre 3 e 5 anos após a confirmação diagnóstica, sobretudo os portadores de Fibrose pulmonar idiopática. Estudos recentes demonstram a capacidade das células-tronco em se diferenciar em diferentes linhagens celulares e tecidos, como já comprovado em órgãos como coração, fígado, trato gastrointestinal, sistema nervoso e pulmão. Os objetivos deste trabalho foram os de estabelecer a espécie suína como modelo experimental e utilizar a terapia celular experimentalmente como possibilidade de estudo para tratamento das doenças pulmonares intersticiais fibrosantes. Para estabelecer o modelo experimental e induzir a doença nos dois grupos de animais estudados (grupos tratado e controle) foi utilizado sulfato de bleomicina pela via intratraqueal em procedimento único. Após a instalação da doença, os animais dos grupos tratado e controle foram submetidos a tomografia computadorizada de alta resolução (TCAR); um grupo tratado com terapia celular e após noventa dias os dois grupos reavaliados com TCAR antes da eutanásia, totalizando para os dois grupos, cento e oitenta dias de doença instalada. As análises tomográficas mostraram que o tempo para que a doença intersticial seja estabelecida ocorre até três meses após a infusão de bleomicina. As provas histológicas corroboram a viabilidade do modelo testado e as análises imunohistoquímicas sugerem a migração das células mononucleares de medula óssea para os pulmões, bem como a presença de populações celulares que indicam provável reestruturação do parênquima pulmonar. / Fibrotic Interstitial pulmonary illnesses affect men, women and children, and presents bad prognostic, 3-5 years depending on the diagnostic confirmation, mainly in idiopathic pulmonary fibrosis. Recent studies demonstrate the capacity of the stem cells in differentiating into different cellular lineages and different tissues, as in heart, liver, gastrointestinal, nervous system and lung. The objectives of this study were to investigate the possibility to consider the swine as experimental model of fibrotic pulmonary disease and experimental stem cell therapy. Bleomycin sulphate was injected into the trachea to induce the pulmonary disease in control and treatment groups. High resolution computed scan (TCAR) was carried out in both groups after the confirmation of the disease. The tomographic analyses showed that the interstitial illness was established after three months of the bleomicine infusion. Histologic investigation revealed the viability of the tested model and the imunohistochemical analyses suggest the migration of the mononuclear cells to the lungs, as well as the presence of new cellular populations that would indicate probable reorganization of the pulmonary parenchyma.

Introdução: A podocitopenia e a podocitúria são marcadores de injúria glomerular em podocitopatias (POD) e glomerulonefrites proliferativas (GNsP), e mesmo em fases iniciais destas doenças mecanismos pró-fibróticos indutores de glomeruloesclerose e fibrose renal progressiva estão ativados. Objetivo: Avaliar pacientes portadores de glomerulopatias biopsiados em diferentes tempos de evolução clínica, correlacionando lesões morfológicas dos compartimentos glomerular e túbulo-intersticial com a expressão dos RNAm de proteínas associadas ao podócito e de fatores pró-fibróticos no tecido renal. Materiais e Métodos: Foram incluídos no estudo oitenta e quatro pacientes adultos portadores de glomerulopatias de diferentes etiologias submetidos à biópsia renal por indicação clínica. As lesões histológicas foram individualizadas e a porcentagem de fibrose intersticial e atrofia tubular foi quantificada na coloração de Tricrômio de Masson. Foram mensurados no tecido renal o log 10 do RNAm pela reação em cadeia da polimerase em tempo real das proteínas associadas ao podócito alfa actinina-4, podocina e podocalixina e dos fatores pró-fibróticos fator de crescimento transformador ₁ (TGF₁), fator de crescimento do tecido conectivo (CTGF) e fator de crescimento derivado do endotélio A (VEGF-A). A secção livre de neoplasia de rins removidos por câncer renal foram usados como controles da expressão dos RNAm. Resultados: No grupo POD, os diagnósticos histopatológicos foram: Glomeruloesclerose segmentar e focal (n=20), GN membranosa (n=12), Nefropatia diabética (n=9) e Lesões mínimas (n=7); no grupo GNsP foram Nefropatia por IgA (n=15), GN membranoproliferativa (n=5), Nefrite lúpica (n=5) e GN proliferativa mesangial (n=4), e outros diagnósticos (n=7). O RNAm do tecido renal nos pacientes com POD e GNsP foi significativamente menor comparado ao dos controles para todos os genes estudados. A presença de crescentes, independente do estágio evolutivo, foi associada à maior expressão do RNAm de alfa actinina-4 (p=0,04), podocina (p=0,01) e podocalixina (p=0,038). O RNAm dos genes pró-fibróticos também estava inibido comparado a sua expressão no rim normal. Nas GNsP, o VEGF-A (p<0,001) e o CTGF (p<0,001) foram os genes com menor nível de expressão comparado aos controles. Em relação às biópsias com lesões crescênticas, tanto o RNAm do TGFβ1 (p=0,001) como do CTGF (p=0,041) tiveram maior expressão comparado ao RNAm das biópsias sem crescentes. Nas biópsias com fibrose intersticial superior a 30%, a expressão do RNAm de TGFβ1, (p=0,038) e do VEGF-A (p=0,040) foi maior do que nas biópsias com fibrose leve. O maior tempo entre o início da doença clínica e a realização da biópsia renal não teve influência detectável na expressão tecidual do RNAm dos biomarcadores estudados. Conclusões: Pacientes com podocitopatias ou glomerulonefrites proliferativas apresentaram inibição da expressão do RNAm de proteínas associadas ao podócito e de fatores indutores de fibrose renal, achados compatíveis com injúria podocitária e podocitopenia. Nas biópsias renais com maior grau de fibrose intersticial e atrofia tubular, assim também como naquelas com lesões crescênticas, a expressão do RNAm de fatores fibrogênicos como TGF-β1 e CTGF foi significativamente aumentada, o que pode sugerir supra-regulação de moléculas associadas a mecanismos de fibrose renal e patologia glomerular. / Introduction: Both podocitopenia and podocyturia are markers of glomerular injury in podocytopathies (POD) and proliferative glomerulonephritis (PGNs), and even in the early stages of these diseases pro-fibrotic mechanisms leading to glomerulosclerosis and progressive renal fibrosis are running. Objective: This study evaluated patients with glomerulopathies who were biopsied at different times of clinical evolution, correlating morphological lesions of the glomerular and tubulointerstitial compartments with renal messenger RNA (mRNA) expression of podocyteassociated proteins and pro-fibrotic factors. Materials and Methods: The study included eighty-four adult patients with glomerulopathies of different etiologies undergoing kidney biopsy as clinically indicated. The histological lesions were individualized and the percentage of interstitial fibrosis and tubular atrophy was quantified on Trichrome Masson staining. Tissue log 10 mRNA of the podocyte proteins alpha-actinin-4, podocin and podocalyxin and of the pro-fibrotic factors transforming growth factor β₁ (TGFβ₁), connective tissue growth factor (CTGF) and vascular endothelium growth factor A (VEGF-A) was measured by real time polymerase chain reaction. The sections free of neoplasia of kidneys removed for renal cancer were used as controls for the mRNA tissue expression. Results: Results: In the POD group, the histopathological diagnoses were: focal segmental glomerulosclerosis (n=20), membranous (n=12), diabetic nephropathy (n=9) and minimal changes (n=7); in PGNs group were IgA nephropathy (n=15), membranoproliferative (n=5), lupus nephritis (n=5) and mesangial proliferative (n=4), and other diagnoses (n=7). Messenger RNA expression of POD and PGNs groups was significantly lower compared to controls for all the studied genes. The presence of crescents, regardless of their evolutive stage, was associated with higher mRNA expression of alpha-actinin-4 (p=0.04), podocin (p=0.01) and podocalyxin (p=0.038). The mRNA of pro-fibrotic genes was also inhibited compared to their expression in normal kidneys. In PGNs, VEGF-A (p<0.001) and CTGF (p<0.001) were the genes with lowest mRNA levels compared to controls. Regarding the biopsies with crescentic lesions, both the mRNA of TGFβ1 (p=0.001) and CTGF (p=0.041) were highly expressed as compared to those of biopsies without crescents. In biopsies with moderate to severe interstitial fibrosis (more than 30%), the mRNA expression of TGFβ1 (p=0.038) and VEGF-A (p=0.040) was highly expressed compared to biopsies with mild fibrosis. A longer interval between the clinical disease and the performance of kidney biopsy did not have a detectable influence on tissue mRNA expression of the studied biomarkers. Conclusions: Patients with POD or PGNs presented inhibition of the mRNA expression of podocyte-associated proteins and pro-fibrotic factors, findings that are consistent with podocyte injury and podocitopenia. In renal biopsies with a higher degree of interstitial fibrosis and tubular atrophy, as well as those with crescentic lesions, the mRNA expression of fibrogenic factors such as TGF-β1 and CTGF was significantly increased, which may suggest upregulation of molecules associated with mechanisms of renal fibrosis and glomerular pathology.

Introdução: A podocitopenia e a podocitúria são marcadores de injúria glomerular em podocitopatias (POD) e glomerulonefrites proliferativas (GNsP), e mesmo em fases iniciais destas doenças mecanismos pró-fibróticos indutores de glomeruloesclerose e fibrose renal progressiva estão ativados. Objetivo: Avaliar pacientes portadores de glomerulopatias biopsiados em diferentes tempos de evolução clínica, correlacionando lesões morfológicas dos compartimentos glomerular e túbulo-intersticial com a expressão dos RNAm de proteínas associadas ao podócito e de fatores pró-fibróticos no tecido renal. Materiais e Métodos: Foram incluídos no estudo oitenta e quatro pacientes adultos portadores de glomerulopatias de diferentes etiologias submetidos à biópsia renal por indicação clínica. As lesões histológicas foram individualizadas e a porcentagem de fibrose intersticial e atrofia tubular foi quantificada na coloração de Tricrômio de Masson. Foram mensurados no tecido renal o log 10 do RNAm pela reação em cadeia da polimerase em tempo real das proteínas associadas ao podócito alfa actinina-4, podocina e podocalixina e dos fatores pró-fibróticos fator de crescimento transformador ₁ (TGF₁), fator de crescimento do tecido conectivo (CTGF) e fator de crescimento derivado do endotélio A (VEGF-A). A secção livre de neoplasia de rins removidos por câncer renal foram usados como controles da expressão dos RNAm. Resultados: No grupo POD, os diagnósticos histopatológicos foram: Glomeruloesclerose segmentar e focal (n=20), GN membranosa (n=12), Nefropatia diabética (n=9) e Lesões mínimas (n=7); no grupo GNsP foram Nefropatia por IgA (n=15), GN membranoproliferativa (n=5), Nefrite lúpica (n=5) e GN proliferativa mesangial (n=4), e outros diagnósticos (n=7). O RNAm do tecido renal nos pacientes com POD e GNsP foi significativamente menor comparado ao dos controles para todos os genes estudados. A presença de crescentes, independente do estágio evolutivo, foi associada à maior expressão do RNAm de alfa actinina-4 (p=0,04), podocina (p=0,01) e podocalixina (p=0,038). O RNAm dos genes pró-fibróticos também estava inibido comparado a sua expressão no rim normal. Nas GNsP, o VEGF-A (p<0,001) e o CTGF (p<0,001) foram os genes com menor nível de expressão comparado aos controles. Em relação às biópsias com lesões crescênticas, tanto o RNAm do TGFβ1 (p=0,001) como do CTGF (p=0,041) tiveram maior expressão comparado ao RNAm das biópsias sem crescentes. Nas biópsias com fibrose intersticial superior a 30%, a expressão do RNAm de TGFβ1, (p=0,038) e do VEGF-A (p=0,040) foi maior do que nas biópsias com fibrose leve. O maior tempo entre o início da doença clínica e a realização da biópsia renal não teve influência detectável na expressão tecidual do RNAm dos biomarcadores estudados. Conclusões: Pacientes com podocitopatias ou glomerulonefrites proliferativas apresentaram inibição da expressão do RNAm de proteínas associadas ao podócito e de fatores indutores de fibrose renal, achados compatíveis com injúria podocitária e podocitopenia. Nas biópsias renais com maior grau de fibrose intersticial e atrofia tubular, assim também como naquelas com lesões crescênticas, a expressão do RNAm de fatores fibrogênicos como TGF-β1 e CTGF foi significativamente aumentada, o que pode sugerir supra-regulação de moléculas associadas a mecanismos de fibrose renal e patologia glomerular. / Introduction: Both podocitopenia and podocyturia are markers of glomerular injury in podocytopathies (POD) and proliferative glomerulonephritis (PGNs), and even in the early stages of these diseases pro-fibrotic mechanisms leading to glomerulosclerosis and progressive renal fibrosis are running. Objective: This study evaluated patients with glomerulopathies who were biopsied at different times of clinical evolution, correlating morphological lesions of the glomerular and tubulointerstitial compartments with renal messenger RNA (mRNA) expression of podocyteassociated proteins and pro-fibrotic factors. Materials and Methods: The study included eighty-four adult patients with glomerulopathies of different etiologies undergoing kidney biopsy as clinically indicated. The histological lesions were individualized and the percentage of interstitial fibrosis and tubular atrophy was quantified on Trichrome Masson staining. Tissue log 10 mRNA of the podocyte proteins alpha-actinin-4, podocin and podocalyxin and of the pro-fibrotic factors transforming growth factor β₁ (TGFβ₁), connective tissue growth factor (CTGF) and vascular endothelium growth factor A (VEGF-A) was measured by real time polymerase chain reaction. The sections free of neoplasia of kidneys removed for renal cancer were used as controls for the mRNA tissue expression. Results: Results: In the POD group, the histopathological diagnoses were: focal segmental glomerulosclerosis (n=20), membranous (n=12), diabetic nephropathy (n=9) and minimal changes (n=7); in PGNs group were IgA nephropathy (n=15), membranoproliferative (n=5), lupus nephritis (n=5) and mesangial proliferative (n=4), and other diagnoses (n=7). Messenger RNA expression of POD and PGNs groups was significantly lower compared to controls for all the studied genes. The presence of crescents, regardless of their evolutive stage, was associated with higher mRNA expression of alpha-actinin-4 (p=0.04), podocin (p=0.01) and podocalyxin (p=0.038). The mRNA of pro-fibrotic genes was also inhibited compared to their expression in normal kidneys. In PGNs, VEGF-A (p<0.001) and CTGF (p<0.001) were the genes with lowest mRNA levels compared to controls. Regarding the biopsies with crescentic lesions, both the mRNA of TGFβ1 (p=0.001) and CTGF (p=0.041) were highly expressed as compared to those of biopsies without crescents. In biopsies with moderate to severe interstitial fibrosis (more than 30%), the mRNA expression of TGFβ1 (p=0.038) and VEGF-A (p=0.040) was highly expressed compared to biopsies with mild fibrosis. A longer interval between the clinical disease and the performance of kidney biopsy did not have a detectable influence on tissue mRNA expression of the studied biomarkers. Conclusions: Patients with POD or PGNs presented inhibition of the mRNA expression of podocyte-associated proteins and pro-fibrotic factors, findings that are consistent with podocyte injury and podocitopenia. In renal biopsies with a higher degree of interstitial fibrosis and tubular atrophy, as well as those with crescentic lesions, the mRNA expression of fibrogenic factors such as TGF-β1 and CTGF was significantly increased, which may suggest upregulation of molecules associated with mechanisms of renal fibrosis and glomerular pathology.

Introdução: A podocitopenia e a podocitúria são marcadores de injúria glomerular em podocitopatias (POD) e glomerulonefrites proliferativas (GNsP), e mesmo em fases iniciais destas doenças mecanismos pró-fibróticos indutores de glomeruloesclerose e fibrose renal progressiva estão ativados. Objetivo: Avaliar pacientes portadores de glomerulopatias biopsiados em diferentes tempos de evolução clínica, correlacionando lesões morfológicas dos compartimentos glomerular e túbulo-intersticial com a expressão dos RNAm de proteínas associadas ao podócito e de fatores pró-fibróticos no tecido renal. Materiais e Métodos: Foram incluídos no estudo oitenta e quatro pacientes adultos portadores de glomerulopatias de diferentes etiologias submetidos à biópsia renal por indicação clínica. As lesões histológicas foram individualizadas e a porcentagem de fibrose intersticial e atrofia tubular foi quantificada na coloração de Tricrômio de Masson. Foram mensurados no tecido renal o log 10 do RNAm pela reação em cadeia da polimerase em tempo real das proteínas associadas ao podócito alfa actinina-4, podocina e podocalixina e dos fatores pró-fibróticos fator de crescimento transformador ₁ (TGF₁), fator de crescimento do tecido conectivo (CTGF) e fator de crescimento derivado do endotélio A (VEGF-A). A secção livre de neoplasia de rins removidos por câncer renal foram usados como controles da expressão dos RNAm. Resultados: No grupo POD, os diagnósticos histopatológicos foram: Glomeruloesclerose segmentar e focal (n=20), GN membranosa (n=12), Nefropatia diabética (n=9) e Lesões mínimas (n=7); no grupo GNsP foram Nefropatia por IgA (n=15), GN membranoproliferativa (n=5), Nefrite lúpica (n=5) e GN proliferativa mesangial (n=4), e outros diagnósticos (n=7). O RNAm do tecido renal nos pacientes com POD e GNsP foi significativamente menor comparado ao dos controles para todos os genes estudados. A presença de crescentes, independente do estágio evolutivo, foi associada à maior expressão do RNAm de alfa actinina-4 (p=0,04), podocina (p=0,01) e podocalixina (p=0,038). O RNAm dos genes pró-fibróticos também estava inibido comparado a sua expressão no rim normal. Nas GNsP, o VEGF-A (p<0,001) e o CTGF (p<0,001) foram os genes com menor nível de expressão comparado aos controles. Em relação às biópsias com lesões crescênticas, tanto o RNAm do TGFβ1 (p=0,001) como do CTGF (p=0,041) tiveram maior expressão comparado ao RNAm das biópsias sem crescentes. Nas biópsias com fibrose intersticial superior a 30%, a expressão do RNAm de TGFβ1, (p=0,038) e do VEGF-A (p=0,040) foi maior do que nas biópsias com fibrose leve. O maior tempo entre o início da doença clínica e a realização da biópsia renal não teve influência detectável na expressão tecidual do RNAm dos biomarcadores estudados. Conclusões: Pacientes com podocitopatias ou glomerulonefrites proliferativas apresentaram inibição da expressão do RNAm de proteínas associadas ao podócito e de fatores indutores de fibrose renal, achados compatíveis com injúria podocitária e podocitopenia. Nas biópsias renais com maior grau de fibrose intersticial e atrofia tubular, assim também como naquelas com lesões crescênticas, a expressão do RNAm de fatores fibrogênicos como TGF-β1 e CTGF foi significativamente aumentada, o que pode sugerir supra-regulação de moléculas associadas a mecanismos de fibrose renal e patologia glomerular. / Introduction: Both podocitopenia and podocyturia are markers of glomerular injury in podocytopathies (POD) and proliferative glomerulonephritis (PGNs), and even in the early stages of these diseases pro-fibrotic mechanisms leading to glomerulosclerosis and progressive renal fibrosis are running. Objective: This study evaluated patients with glomerulopathies who were biopsied at different times of clinical evolution, correlating morphological lesions of the glomerular and tubulointerstitial compartments with renal messenger RNA (mRNA) expression of podocyteassociated proteins and pro-fibrotic factors. Materials and Methods: The study included eighty-four adult patients with glomerulopathies of different etiologies undergoing kidney biopsy as clinically indicated. The histological lesions were individualized and the percentage of interstitial fibrosis and tubular atrophy was quantified on Trichrome Masson staining. Tissue log 10 mRNA of the podocyte proteins alpha-actinin-4, podocin and podocalyxin and of the pro-fibrotic factors transforming growth factor β₁ (TGFβ₁), connective tissue growth factor (CTGF) and vascular endothelium growth factor A (VEGF-A) was measured by real time polymerase chain reaction. The sections free of neoplasia of kidneys removed for renal cancer were used as controls for the mRNA tissue expression. Results: Results: In the POD group, the histopathological diagnoses were: focal segmental glomerulosclerosis (n=20), membranous (n=12), diabetic nephropathy (n=9) and minimal changes (n=7); in PGNs group were IgA nephropathy (n=15), membranoproliferative (n=5), lupus nephritis (n=5) and mesangial proliferative (n=4), and other diagnoses (n=7). Messenger RNA expression of POD and PGNs groups was significantly lower compared to controls for all the studied genes. The presence of crescents, regardless of their evolutive stage, was associated with higher mRNA expression of alpha-actinin-4 (p=0.04), podocin (p=0.01) and podocalyxin (p=0.038). The mRNA of pro-fibrotic genes was also inhibited compared to their expression in normal kidneys. In PGNs, VEGF-A (p<0.001) and CTGF (p<0.001) were the genes with lowest mRNA levels compared to controls. Regarding the biopsies with crescentic lesions, both the mRNA of TGFβ1 (p=0.001) and CTGF (p=0.041) were highly expressed as compared to those of biopsies without crescents. In biopsies with moderate to severe interstitial fibrosis (more than 30%), the mRNA expression of TGFβ1 (p=0.038) and VEGF-A (p=0.040) was highly expressed compared to biopsies with mild fibrosis. A longer interval between the clinical disease and the performance of kidney biopsy did not have a detectable influence on tissue mRNA expression of the studied biomarkers. Conclusions: Patients with POD or PGNs presented inhibition of the mRNA expression of podocyte-associated proteins and pro-fibrotic factors, findings that are consistent with podocyte injury and podocitopenia. In renal biopsies with a higher degree of interstitial fibrosis and tubular atrophy, as well as those with crescentic lesions, the mRNA expression of fibrogenic factors such as TGF-β1 and CTGF was significantly increased, which may suggest upregulation of molecules associated with mechanisms of renal fibrosis and glomerular pathology.

Doenças pulmonares intersticiais fibrosantes são doenças que afetam homens, mulheres e crianças, tem prognóstico ruim e os pacientes possuem sobrevida estimada entre 3 e 5 anos após a confirmação diagnóstica, sobretudo os portadores de Fibrose pulmonar idiopática. Estudos recentes demonstram a capacidade das células-tronco em se diferenciar em diferentes linhagens celulares e tecidos, como já comprovado em órgãos como coração, fígado, trato gastrointestinal, sistema nervoso e pulmão. Os objetivos deste trabalho foram os de estabelecer a espécie suína como modelo experimental e utilizar a terapia celular experimentalmente como possibilidade de estudo para tratamento das doenças pulmonares intersticiais fibrosantes. Para estabelecer o modelo experimental e induzir a doença nos dois grupos de animais estudados (grupos tratado e controle) foi utilizado sulfato de bleomicina pela via intratraqueal em procedimento único. Após a instalação da doença, os animais dos grupos tratado e controle foram submetidos a tomografia computadorizada de alta resolução (TCAR); um grupo tratado com terapia celular e após noventa dias os dois grupos reavaliados com TCAR antes da eutanásia, totalizando para os dois grupos, cento e oitenta dias de doença instalada. As análises tomográficas mostraram que o tempo para que a doença intersticial seja estabelecida ocorre até três meses após a infusão de bleomicina. As provas histológicas corroboram a viabilidade do modelo testado e as análises imunohistoquímicas sugerem a migração das células mononucleares de medula óssea para os pulmões, bem como a presença de populações celulares que indicam provável reestruturação do parênquima pulmonar. / Fibrotic Interstitial pulmonary illnesses affect men, women and children, and presents bad prognostic, 3-5 years depending on the diagnostic confirmation, mainly in idiopathic pulmonary fibrosis. Recent studies demonstrate the capacity of the stem cells in differentiating into different cellular lineages and different tissues, as in heart, liver, gastrointestinal, nervous system and lung. The objectives of this study were to investigate the possibility to consider the swine as experimental model of fibrotic pulmonary disease and experimental stem cell therapy. Bleomycin sulphate was injected into the trachea to induce the pulmonary disease in control and treatment groups. High resolution computed scan (TCAR) was carried out in both groups after the confirmation of the disease. The tomographic analyses showed that the interstitial illness was established after three months of the bleomicine infusion. Histologic investigation revealed the viability of the tested model and the imunohistochemical analyses suggest the migration of the mononuclear cells to the lungs, as well as the presence of new cellular populations that would indicate probable reorganization of the pulmonary parenchyma.

This thesis describes the electrical and physiological characterization of cardiac tissue with myocardial infarction (MI) responsible for abnormal cardiac rhythms such as ventricular tachycardia (VT), using a newly-developed magnetic resonance imaging (MRI) electrophysiology system. In electrophysiology (EP), radiofrequency (RF) catheter ablation combined with cardioverter-defibrillator implantation is a first-line action to manage ventricular VT. Unfortunately, this therapy is known to have sub-optimal success rates in a large number of patients because of difficulties to accurately identifying the arrhythmic target regions. Currently, characterization of post-MI scars is performed by using catheters to measure electrical signals of the endocardial tissue (electroanatomical mapping), under x-ray fluoroscopy guidance. Prolonged radiation exposure to both the cardiologist and the patient have made the use of MRI extremely attractive; further, unlike x-ray imaging, MRI provides post-MI scars with direct visualization, characterization in three dimensions and the ability to visualize ablation lesions. Although recent research has focused on registration between pre-acquired MR images and electroanatomical maps, a potentially more useful approach is to use real-time MRI to directly locate and characterize potential arrhythmogenic regions during the EP procedure. A real-time MR-guided EP system was developed and validated to perform EP diagnostic procedures, such as mapping and pacing. In a series of animal studies, the system demonstrated the ability to use active catheter tracking and intra-procedural MR imaging to navigate to specific regions in the left ventricle and record intracardiac electrical signals. A study correlating myocardial fibrotic scar detected by multicontrast late enhancement (MCLE) MRI and electroanatomical voltage mapping demonstrated that MRI information (transmurality, tissue classification, and relaxation rate) can accurately predict areas of myocardial fibrosis identified with bipolar voltage mapping. Finally, MCLE-derived gray zone was shown to have a high correspondence to regions with a high proportion of abnormal intracardiac signals. The methods described in this thesis help advance the understanding of infarcted tissue responsible for ventricular tachycardia. Further studies are proposed to perform RF ablation lesions and correlate pre- and post-ablation tissue electrophysiological properties with MRI.

This thesis describes the electrical and physiological characterization of cardiac tissue with myocardial infarction (MI) responsible for abnormal cardiac rhythms such as ventricular tachycardia (VT), using a newly-developed magnetic resonance imaging (MRI) electrophysiology system. In electrophysiology (EP), radiofrequency (RF) catheter ablation combined with cardioverter-defibrillator implantation is a first-line action to manage ventricular VT. Unfortunately, this therapy is known to have sub-optimal success rates in a large number of patients because of difficulties to accurately identifying the arrhythmic target regions. Currently, characterization of post-MI scars is performed by using catheters to measure electrical signals of the endocardial tissue (electroanatomical mapping), under x-ray fluoroscopy guidance. Prolonged radiation exposure to both the cardiologist and the patient have made the use of MRI extremely attractive; further, unlike x-ray imaging, MRI provides post-MI scars with direct visualization, characterization in three dimensions and the ability to visualize ablation lesions. Although recent research has focused on registration between pre-acquired MR images and electroanatomical maps, a potentially more useful approach is to use real-time MRI to directly locate and characterize potential arrhythmogenic regions during the EP procedure. A real-time MR-guided EP system was developed and validated to perform EP diagnostic procedures, such as mapping and pacing. In a series of animal studies, the system demonstrated the ability to use active catheter tracking and intra-procedural MR imaging to navigate to specific regions in the left ventricle and record intracardiac electrical signals. A study correlating myocardial fibrotic scar detected by multicontrast late enhancement (MCLE) MRI and electroanatomical voltage mapping demonstrated that MRI information (transmurality, tissue classification, and relaxation rate) can accurately predict areas of myocardial fibrosis identified with bipolar voltage mapping. Finally, MCLE-derived gray zone was shown to have a high correspondence to regions with a high proportion of abnormal intracardiac signals. The methods described in this thesis help advance the understanding of infarcted tissue responsible for ventricular tachycardia. Further studies are proposed to perform RF ablation lesions and correlate pre- and post-ablation tissue electrophysiological properties with MRI.

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating respiratory disorder that is characterized by a progressive decline in lung function. Originating through unknown etiology, it is essentially an unchecked wound healing response that causes the build-up of excessive scar tissue in the lung interstitial tissue with a heavy toll on the patient’s respiratory capacity. Pro-fibrotic alternatively activated macrophages (M2) have been linked as an important contributor to the fibrotic remodeling of the lung. Previous Ask research indicates that targeting M2 macrophages is possible through the use of the Dectin-1 receptor, a transmembrane cell surface receptor found in high abundance on M2 macrophages. Activating the Dectin-1 receptor through the use of beta-glucan, a ligand the receptor has a high affinity for, initiates a pro-inflammatory response within the naturally immunosuppressive macrophage and can alter its activity to be less fibrogenic. Our data suggest that M2 polarization of naïve macrophages can be inhibited in vitro by beta-glucan microparticles. Additionally, we have found that polarized M2 macrophages adopt M1-like characteristics when treated with beta-glucan microparticles, in a process that is largely Dectin-1 dependent. M2 cell surface marker CD206, increased levels of which are associated with rapidly progressing IPF, shows significantly decreased frequency of expression in M2 macrophages treated with beta-glucan microparticles. Our assessment for cell-specific uptake of beta-glucan microparticles suggests an important role of the Dectin-1 receptor for significantly increased uptake in murine wild-type M2 macrophages relative to their Dectin-1 knockout counterpart. The use of beta-glucan microparticles as a potential anti-fibrotic therapeutic was assessed in the bleomycin model of fibrotic lung disease. Mice given bleomycin and treated with beta-glucan displayed decreased soluble collagen content and TGFB expression within lung homogenate relative to fibrotic bleomycin control mice. Overall, these results provide insight into the use of beta-glucan as a potential activity modulator of macrophage function in IPF and the possibility of its use as a therapeutic. / Thesis / Master of Science (MSc)