Exposure of cells to hydrogen peroxide can cause oxidative damage to cellular constituents including lipids, protein, and DNA. At elevated concentrations, hydrogen peroxide can trigger cell death by apoptosis or necrosis. Apoptotic cell death can be prevented by overexpression of the oncoprotein Bcl-2. The exact mechanism by which Bcl-2 blocks cell death is controversial. Some researchers believe that Bcl-2 possesses antioxidant properties that protect cells from apoptosis. The purpose of this thesis was to assess oxidative stress and apoptosis following hydrogen peroxide exposure in Jurkat T cells overexpressing Bcl-2. One of the major objectives was to ascertain whether or not Bcl-2 overexpression elevated the antioxidant capacity of Jurkat T cells to provide protection from oxidant-induced cell death. Hydrogen peroxide treated Jurkat cells became apoptotic at moderate levels of oxidant (25-100 uM H2O2), and necrotic at higher doses (greater than 200 uM H2O2). Bcl-2 overexpression prevented caspase activation and cell death at the apoptotic doses of H2O2, but not the necrotic doses. Caspase inhibition studies demonstrated that Bcl-2 overexpression provided a greater level of resistance from H2O2-induced cell death than the broad-spectrum caspase inhibitor z-VAD.fmk. A systematic study was carried out examining the antioxidant status of Jurkat cells overexpressing Bcl-2. Several Bcl-2 transfectants were utilised for the study, so that any differences seen could be correlated to the level of Bcl-2 expression. Surprisingly, there were no statistically significant differences among the Bcl-2 transfectants for any of the antioxidant enzymes. Jurkat cells overexpressing Bcl-2 exhibited the same level of oxidative damage to lipids and protein in response to H2O2 exposure as the parental Jurkat cells. Interestingly, Jurkat cells overexpressing Bcl-2 continued to grow in culture after H2O2 exposure, despite harboring damage to cellular constituents. Consistent with these results, H2O2 treated Jurkat cells overexpressing Bcl-2, which failed to undergo apoptosis, were more prone to genomic instability. Together, these findings suggest that Bcl-2 overexpression protects Jurkat cells from H2O2-induced cell death by blocking apoptosis. Jurkat cells overexpressing Bcl-2 were no better at detoxifying oxidants and showed the same level of oxidative damage following H2O2 exposure. As a result, the overexpression of Bcl-2 considerably enhanced the mutagenicity of H2O2.

Thesis (Ph.D.). / Written for the Dept. of Medicine. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Neurology & Neurosurgery. Title from title page of PDF (viewed 2008/05/14). Includes bibliographical references.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 2003. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/11). Includes bibliographical references.

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 66-78. Publié aussi en version électronique.

Submitted by Hiolanda Rêgo (hiolandar@gmail.com) on 2013-08-14T20:47:20Z No. of bitstreams: 1 Dissertação_ICS_Paulo Cirino de Carvalho Filho.pdf: 1601148 bytes, checksum: 994736a47abe1ce0bf2aece42ff07568 (MD5) / Made available in DSpace on 2013-08-14T20:47:20Z (GMT). No. of bitstreams: 1 Dissertação_ICS_Paulo Cirino de Carvalho Filho.pdf: 1601148 bytes, checksum: 994736a47abe1ce0bf2aece42ff07568 (MD5) / A periodontite é uma doença multifatorial causada pela resposta imuno-inflamatória do hospedeiro sob estímulos bacterianos que destroem o periodonto. Este estudo objetivou investigar a expressão in vitro das proteínas Fas e Bcl-2 em Células Mononucleares de Sangue Periférico (CMSP) estimuladas pela proteína rHmuY de Porphyromonas gingivalis (Pg). Os 39 voluntários (18-periodontite crônica-PC e 21-sem periodontite- SP) foram avaliados seguindo descritores clínicos periodontais. As CMSP foram cultivadas sob o estímulo de P. gingivalis e os ensaios para a expressão de Bcl-2 e Fas (CD95) foram realizados após 48 horas. A fluorescência para CMSP com os marcadores para CD3, CD4, CD8, Bcl-2 e CD95 foi determinada usando Citometria de Fluxo. A expressão de Bcl-2 em células T CD3+ de indivíduos com PC estimuladas com a proteína rHmuY foi maior do que nos SP (P=0,043) e também maior do que nas CMSP estimuladas com o extrato total de Pg ATCC 33277 e sem estimulo. Não foram observadas diferenças estatisticamente significantes na co-expressão dos marcadores para CD3/CD4, CD3/CD8, Fas/Bcl-2 e CD3/Fas nas CMSP. A proteína rHmuY pode representar um importante estímulo de P. gingivalis, induzindo expressão elevada de Bcl-2, a qual possibilita a inibição de apoptose em CMSP de indivíduos com periodontite crônica. / Salvador

Adenomas hipofisários são tumores benignos, de crescimento lento, originados no interior da sela túrcica e constituem de 10% a 15% dos tumores intracranianos, Os adenomas clinicamente não funcionantes (ACNF), correspondem aproximadamente um terço dos adenomas em geral. Por não apresentarem síndrome clínica hormonal são geralmente diagnosticados devido a sintomas neurológicos ou oftalmológicos, como macroadenomas, com grandes dimensões, invasão de estruturas circunvizinhas e hipopituitarismo. A cirurgia é o tratamento de escolha para estes tumores e apesar de ser eficaz na resolução do quadro compressivo, a possibilidade de cura cirúrgica é reduzida principalmente em tumores invasivos. Seu acompanhamento pós-operatório é efetuado por exame de imagem, preferencialmente ressonância magnética, devido à indisponibilidade de marcadores séricos. Nesta pesquisa avaliou-se a relação da expressão dos genes relacionados à apoptose, Bcl-2, Bax e Caspase-3 e sua relação com o comportamento dos ACNF. Na Divisão de Neurocirurgia Funcional do Instituto de Psiquiatria do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo foram operados 119 doentes com tumores hipofisários, de 28/05/08 à 07/04/09, 50 deles com ACNF, 30 deles foram estudados. A ressonância magnética da região selar pré-operatória possibilitou a medida dos três maiores diâmetros do tumor, ou seja, antero-posterior (AP), crânio-caudal (CC), látero-lateral (LL) e avaliar a invasão do seios cavernoso e esfenoidal. O tamanho dos tumores foi avaliado pela soma dos três diâmetros, pelo maior diâmetro e pelo cálculo do volume, efetuado pela fórmula AP x CC x LL x 0,5. No intraoperatório foram avaliados, a consistência e invasão tumoral. A análise histológica por hematoxilina-eosina, foi efetuada em todos os tumores, assim como a análise imuno-histoquímica (IHQ) dos hormônios hipofisários, Ki-67, p53 e Bcl-2. Foi realizada a análise molecular dos genes Bcl-2, Bax e Caspase-3 por RT-PCR. Dados demográficos: 17 do sexo masculino, 13 do sexo feminino, mediana da idade foi de 54,5 anos e mediana da duração dos sintomas de 31 meses. Todos apresentavam macroadenoma, 87% deles com perda visual, 53% com cefaléia, 17% com outras alterações neurológicas e um assintomático diagnosticado incidentalmente. Avaliação hormonal, disponível em 26 doentes, confirmou deficiência em 92% deles, com mais de dois eixos acometidos em 50% dos casos. A mediana do volume dos tumores foi de 11,6 cm3, do maior diâmetro de 3,8cm e da soma dos três diâmetros de 8,6cm, observou estreita correlação significativa estatisticamente entre as três medidas. Quarenta porcento dos tumores eram gigantes (diâmetro maior ou igual a 4 cm). Consistência amolecida e invasão tumoral foram observadas em 87% e 67% dos tumores, respectivamente. Todos doentes foram operados pela via transesfenoidal, exceto um operado por craniotomia pterional. Complicações cirúrgicas ocorreram em cinco pacientes, três com fistula liquórica, dois com meningite e dois óbitos. A análise histológica confirmou o diagnóstico de adenoma hipofisário em todos os casos. A IHQ foi negativa para todos hormônios em 18 e positiva em 12 tumores (TSH, FSH, LH, GH ou ACTH). A IHQ para proteína P-53 foi negativa em todos os casos. A IHQ para KI-67 revelou ausência da proteína em 11, positividade em menos de 3% das células em 15 e em mais de 3% em 4 tumores. A IHQ para Bcl-2 foi positiva em apenas três pacientes. A análise molecular dos genes Bcl-2, Bax e Caspase-3 revelou expressão muito inferior nos tumores em relação à observada para um pool de hipófise normal. Observou-se correlação positiva estatisticamente significante entre os três genes porém não foi observada correlação entre os níveis destes três genes e nenhum fator de prognóstico tumoral estudado, quais sejam, idade, positividade para hormônios na IHQ, tamanho ou invasão tumoral / Pituitary adenomas are benign, slow-growing tumors that arise in the sella turcica and account for 10% to 15% of all intracranial tumors. Non-functioning pituitary adenomas (NFPA) account for around one third of all pituitary adenomas. NFPA do not clinically present as hormonal syndromes and are generally diagnosed as macroadenomas due to marked neurological and ophthalmologic symptoms and invasion of surrounding structures, beside hypopituitarism. Surgery is the gold standard to treat these tumors. It effectively relieves compressive symptoms but cure is uncommon. Despite benign in nature, NFPA usually show aggressive behavior. There are no hormonal markers and the follow-up usually is made only by magnetic resonance imaging. Apoptosis-related genes, Bcl-2, Bax, and caspase-3, were here studied in NFPA to assess their role as potential markers of tumor behavior. Out of 119 patients with pituitary adenomas treated by surgery, 30 patients (17 men, 13 women, median age 54.5 years old) harboring NFPA who underwent surgery in the Department of Functional Neurosurgery at Hospital das Clínicas Psychiatric Institute, University of S. Paulo Medical School, from August 2008 to July 2009, were studied. Information on gender, age, pituitary function, symptoms and their length was collected. Tumor dimensions were measured using magnetic resonance imaging of the sella turcica. The tumor volume was calculated by the following equation: anterior-posterior diameter x cranial-caudal diameter x lateral-lateral diameter x 0.5. Intra-operative information such as tumor invasion and consistence was recorded. Histological examination by hematoxylin-eosin staining and immunohistochemistry analysis of pituitary hormones, Ki-67, p53, and Bcl-2 were performed. The molecular analysis of Bcl-2, Bax, and caspase-3 genes was performed by real-time polymerase chain reaction (RT-PCR) in all tumor specimens collected during surgery and compared to a poll of normal pituitary gland. All patients had macroadenomas diagnosed due to visual loss (87%), headache (53%) and other neurological symptoms (17%) and one case was incidentally found. Hormonal deficits were seen in 92% of 26 cases; more than two axes were involved in half of these patients. There was found good correlation between tumor volume, largest diameter and the sum of the 3 diameters, and tumor volume was used to assess the correlations with other parameters. The median volume was 11.6 cm3. Giant tumors (4 cm) were diagnosed in 40% of the patients. Soft tumors and tumor invasion were observed in 87% and 67% of cases, respectively. A transsphenoidal approach was used in all patients, except one who had pterional craniotomy. Five patients presented post-operative complications: three had CSF leakage, two meningitis and two died. The histological examination confirmed pituitary adenoma in all cases, 18 of them were null cell and 12 showed a positive immunohistochemistry analysis for one or more hormones, mainly TSH. Immunohistochemistry analysis results for p-53 was negative in all cases; for Ki-67 was negative in 11, positive in less than 3% of the cells in 15 and positive in more than 3% of the cells in 4 cases; and for Bcl-2 was positive only in three patients. Bcl-2, Bax and caspase-3 molecular analysis revealed very low expression compared to normal pituitary values. There was found a positive correlation between these three genes but no correlation between them and age, tumor volume or invasion. The Bcl-2, Bax, and caspase-3 gene analysis by RT-PCR in NFPA did not evidence their potential as markers of tumor behavior

The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22334号 / 医博第4575号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM