O diabetes mellitus tipo 1 (DM-1) e a esclerose múltipla (EM) são doenças auto-imunes órgão-específicas, inflamatórias, mediadas por células T e B auto-reativas e caracterizadas pela destruição seletiva de células b pancreáticas produtoras de insulina e do sistema nervoso central, respectivamente. Acredita-se que a desregulação da expressão de genes reguladores da maquinaria apoptótica possa contribuir para o desenvolvimento da auto-imunidade, visto que algumas dessas moléculas participam nos processos de tolerância central e periférica de linfócitos auto-reativos. O objetivo deste projeto foi analisar a expressão de moléculas reguladoras das vias intrínseca, extrínseca e da Família de proteínas inibidoras da apoptose (IAP) em 33 indivíduos saudáveis, 15 pacientes com DM-1 e 18 com EM submetidos à terapia de imunossupressão em altas doses seguida do transplante autólogo de células-tronco hematopoéticas (IAD/TACTH). As células mononucleares (CMN) foram isoladas do sangue periférico dos controles e de pacientes nos períodos pré-mobilização (pré-mob), pré-condicionamento (pré-cond), D+180, D+360, D+540 e D+720 pós-transplante. As CMN foram utilizadas para extração de RNA, síntese de cDNA, quantificação da expressão por PCR em tempo real dos genes a1, bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bid, bik, bim, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 e cIAP-2 e protéica de Bcl-2, Bcl-xL, Bak, Bim e c-FLIPL por western-blotting. Os resultados de expressão gênica foram representados por unidades relativas de expressão em medianas nas diferentes amostras. Os pacientes com DM-1 apresentaram diminuição da expressão dos genes anti-apoptóticos bcl-2 (mediana: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) e cIAP-1 (1,24; p=0,003) nas CMN dos pacientes no período pré-mob em relação aos indivíduos saudáveis (medianas: bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), enquanto a expressão de cIAP-2 (60,8; p=0,0005) estava aumentada em relação aos controles (23,3). Foi observada redução significativa na expressão dos genes pró-apoptóticos bad (0,002; p<0,0001), bax (0,01; p=0,002) e fasL (1,66; p=0,001) no período pré-mob comparada aos controles sadios (bad: 0,23; bax: 2,79; fasL: 3,56). Os níveis de RNAm de bid (0,10; p=0,001) e bok (0,72; p=0,006) estavam elevados no pré-mob em relação ao grupo controle (bid: 0,004; bok: 0,31). As moléculas bcl-2, bcl-w, bcl-xL, mcl-1, bad, bax, bok, fasL e cIAP-1 atingiram níveis de RNAm similares aos controles após o TACTH. Foi verificado que a expressão de bcl-w, cIAP-1 e noxa estava maior nos pacientes com DM-1 em remissão quando comparados àqueles em recaída. A diminuição da expressão de a1, bcl-2 e bcl-w e o aumento de fas e noxa correlacionaram-se às porcentagens de hemoglobina glicosilada, concentração de auto-anticorpos GAD65, e aos níveis séricos de peptídeo-C após o transplante. Os pacientes com EM mostraram uma expressão reduzida dos genes anti-apoptóticos bcl-w (0,11; p=0,02) e cIAP-1 (1,87; p=0,04) no pré-mob comparada aos valores dos controles (bcl-w: 0,27; cIAP-1: 7,75) e maior expressão dos genes a1 (90,8; p=0,001) e cIAP-2 (58,8; p=0,009) em relação aos controles (a1: 12,7; cIAP-2: 22,3). As moléculas pró-apoptóticas bad (0,007; p=0,01) e bax (0,0007; p=0,004) mostraram menor expressão nas CMN no período pré-mob do que nos controles (bad: 0,27; bax: 1,24). Os genes bid (20,7; p=0,004), bik (0,84; p=0,02) e bok (1,77; p=0,0001) possuíam maior expressão no período pré-mob em relação aos indivíduos sadios (bid: 2,64; bik: 0,33; bok: 0,26). Não foram observadas diferenças significativas na expressão das moléculas da via extrínseca da apoptose nos pacientes com EM (p>0,05) nos períodos avaliados. Os valores de expressão de bcl-w, bak, bax, bik, bok e cIAP-1 atingiram níveis semelhantes aos controles após o transplante. A expressão dos genes bcl-2, cIAP-1, bad e bax estava maior nos pacientes em remissão da EM quando comparados àqueles em progressão neurológica. O aumento da expressão dos genes pró-apoptóticos bax, bak e bimEL correlacionou-se inversamente aos valores de EDSS dos pacientes com EM após o TACTH. Os resultados de expressão protéica foram equivalentes aos de expressão gênica nas duas doenças, com exceção dos dados das proteínas Bcl-2 e Bim. Em conjunto, os resultados demonstraram a desregulação da expressão de várias moléculas anti- e pró-apoptóticas nas CMN dos pacientes com DM-1 e EM. Esses achados sugerem a associação de alterações nos processos de apoptose celular com o surgimento e persistência de células auto-reativas no DM-1 e EM. Os dados indicam que essas alterações, principalmente a diminuição da expressão de moléculas pró-apoptóticas, como bak e bax, possam contribuir para a patogênese do DM-1 e EM. Além disso, a terapia de IAD/TACTH foi capaz de modular a expressão da maioria dos genes anormalmente expressos nas CMN dos pacientes com DM-1 e EM, já que esses atingiram níveis de expressão similares ao grupo controle após o transplante. Esta normalização da expressão de vários genes analisados correlacionou-se com a remissão clínica da doença na maioria dos pacientes / Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS) are inflammatory, organ-specific autoimmune diseases characterized by selective destruction of insulin-producing pancreatic -cells and central nervous system, respectively, by autoreactive B and T cells. Deregulation of apoptotic machinery is supposed to contribute to self-tolerance breakdown and autoimmune diseases pathogenesis, since apoptotic molecules have an important role in B and T lymphocytes central and peripheral tolerance mechanisms. The aim of this study was to evaluate the expression of pro and anti-apoptotic molecules from intrinsic and extrinsic apoptotic pathways and IAP Family members in 33 healthy individuals, 15 T1DM and 18 MS patients submitted to high-dose immunossupression therapy followed by autologous hematopoietic stem cell transplantation (HDI/AHSCT). Peripheral blood mononuclear cells (PBMC) were isolated from controls and patients at pre-mobilization (pre-mob), pre-conditioning (pre-cond), D+180, D+360, D+540 and D+720 post-transplantation. PBMC were used for RNA extraction, cDNA synthesis, gene quantification of a1, bcl-2, bcl-w, bcl-xL, bad, bak, bax, bid, bik, bimEL, bok, noxa, fas, fasL, c-FLIPL, cIAP-1 and cIAP-2 by Real Time PCR and Bcl-2, Bcl-xL, Bak, BimEL and c-FLIPL proteins detection by western-blotting. Results are expressed as median of relative expression units. Results from T1DM patients indicated that antiapoptotic molecules bcl-2 (median: 0,98; p=0,04), bcl-w (0,08; p=0,04), mcl-1 (1254; p=0,03) and cIAP-1 (1,24; p=0,003) were downregulated at pre-mob compared with healthy controls (medians bcl-2: 7,58; bcl-w: 0,52; mcl-1: 1659; cIAP-1: 14,5), while cIAP-2 (60,8; p=0,0005) gene expression was upregulated compared to healthy controls (23,3). We observed a significant decrease in proapoptotic bad (0,002; p<0,0001), bax (0,01; p=0,002) and fasL (1,66; p=0,001) genes expression in patients PBMC at pre-mob period compared to healthy subjects (bad: 0,23; bax: 2,79; fasL: 3,56). mRNA levels of bid (0.10; p=0.001) and bok (0.72; p=0.006) were elevated at pre-mob period when compared to control group (bid: 0.004; bok: 0.31). The bcl-2, bcl-w, bcl-xL, mcl-1, bad, bak, bax, bok, fasL and cIAP-1 mRNA levels reached controls levels after HDI/AHSCT. We observed that bcl-w, cIAP-1 and noxa gene expression were increased in T1DM patients in remission when compared to relapsed patients. The decreased antiapoptotic gene expression and increased in proapoptotic molecules correlated with decreased glicosilated hemoglobin percentages (Hb A1C) and anti-GAD65 antibodies and increased peptide-C levels. Results from MS patients showed decreased bcl-w (0,11; p=0,02) and cIAP-1 gene expression (1,87; p=0,04) in patients PBMC at pre-mob period compared to healthy controls (bcl-w: 0,27; cIAP-1: 7,75) and increased expression of a1 (90,8; p=0,001) and cIAP-2 (58,8; p=0,009) compared to controls (a1: 12,7; cIAP-2: 22,3). Proapoptotic molecules bad (0.007; p=0.01) and bax (0.0007; p=0.004) showed decreased gene expression at pre-mob compared to control group (bad: 0.27; bax: 1.24). bid (20.7; p=0.004), bik (0.84; p=0.01) and bok genes (1.77; p=0.0001) showed increased expression at pre-mob compared to healthy controls (bid: 2.64; bik: 0.33; bok: 0.26). Significant differences were not observed in the expression of the extrinsic pathway genes in pre-mob and healthy controls samples (p>0.05). bcl-w, bak, bax, bik, bok and cIAP-1 expression values reached healthy control values after transplantation. We observed that bcl-2, cIAP-1, bad and bax gene expression was increased in MS patients in disease remission when compared to patients with neurologic progression. Significant correlation of increased proapoptotic genes expression with decreased EDSS values in MS patients after HDI/AHSCT was observed. Results of protein quantification of apoptotic molecules in PBMC of T1DM and MS patients were similar to the gene expression results of these molecules, except for Bcl-2 and Bim proteins. Taken together, these data indicate a deregulated expression of anti- and proapoptotic genes in T1DM and MS patients PBMC. These data suggest an association of deregulated apoptosis with emergence and maintenance of autoreactive lymphocytes in analyzed patients. Based on these results, we suggest that this altered gene expression profile, mainly the decreased proapoptotic genes expression, as bak and bax, may contribute to T1DM and MS pathogenesis. Furthermore, we showed that the HDI/AHSCT therapy was able to modulate and normalize the expression of most genes abnormally expressed in T1DM and MS patients at pre-transplant period. Many analyzed genes achieved expression levels similar to healthy controls. The normalization of the expression of many evaluated genes correlated to disease remission in the majority of the patients.

INTRODUÇÃO: Transplantes parciais de fígado em crianças têm sido realizados com maior freqüência, enfatizando a importância do estudo da regeneração hepática, bem como dos efeitos de drogas imunossupressoras sobre a mesma. A regeneração do parênquima é o resultado do balanço entre multiplicação celular e apoptose, esta última definida como morte celular programada. Neste processo, estão envolvidas expressões de genes pró-apoptóticos (Bak e Bax) e anti-apoptóticos (Bcl-XL e Bcl-2). Dentre as proteínas relacionadas à proliferação hepatocitária, destaca-se a interleucina-6 (IL-6). Embora o modelo de ressecção de 70% da massa hepática de ratos adultos seja amplamente utilizado para estudos de regeneração, não há trabalhos com animais em crescimento. MÉTODOS: Na presente pesquisa, foi padronizado o modelo de hepatectomia parcial em ratos recém-nascidos e em recém-desmamados: realizou-se ligadura com fio de algodão do pedículo dos lobos esquerdo lateral, esquerdo medial e direito medial, seguida da ressecção do parênquima desses lobos. Os fígados remanescentes foram imediatamente pesados e comparados com os pesos dos fígados de animais controles. Para caracterização dos modelos de regeneração, 40 ratos recém-nascidos e 30 recém-desmamados foram submetidos à hepatectomia descrita e mortos nos dias subseqüentes (1, 2, 3, 4 e 7), e o fígado residual submetido a análises de peso e histologia convencional. A seguir, 36 animais (18 recém-nascidos e 18 recém-desmamados) foram divididos nos seguintes grupos: controle, cirurgia simulada e hepatectomia. Um dia após, utilizando métodos moleculares (técnica do RT-PCR), estudou-se a expressão do gene da IL-6, dos genes pró-apoptóticos e anti-apoptóticos nos fígados desses animais e, por meio de métodos imunoistoquímicos, analisou-se a presença de antígenos relacionados à proliferação celular (PCNA) e apoptose (TUNEL) em lâminas preparadas pela técnica do \"tissue microarray\". Em outros 36 ratos (18 recém-nascidos e 18 recém-desmamados), foram administradas drogas imunossupressoras (metilprednisolona, ciclosporina A ou tacrolimus, separadamente) no ato da hepatectomia, e o parênquima hepático remanescente submetido às mesmas análises moleculares e imunoistoquímicas, um dia após. RESULTADOS: A ressecção do parênquima hepático correspondeu a 70% da massa total do fígado. A mortalidade relacionada à hepatectomia nos animais recém-nascidos e recém-desmamados foi 30% e 0% respectivamente. Na análise histológica observou-se maior quantidade de mitoses em hepatócitos no terceiro dia nos recém-nascidos e no segundo dia nos recém-desmamados, com normalização da arquitetura do parênquima até o 7º dia e recuperação total do peso de ambos. No animal recém-nascido, notou-se intensa esteatose associada ao processo regenerativo. A hepatectomia provocou aumento na expressão do gene da IL-6 no fígado residual e diminuição da expressão dos genes pró-apoptóticos em ambos os modelos, além de aumento do anti-apoptótico Bcl-2 nos animais recém-desmamados. O estudo realizado sobre o efeito das drogas imunossupressoras mostrou resultados diferentes daqueles descritos em animais adultos, não havendo alteração no número de células em proliferação (PCNA positivas) ou apoptose (TUNEL positivas). As drogas não tiveram efeito sobre a expressão do gene da IL-6. Metilprednisolona e tacrolimus ocasionaram aumento da expressão do gene anti-apoptótico Bcl-2 em ambos os modelos; metilprednisolona e ciclosporina provocaram aumento na expressão do gene pró-apoptótico Bak nos ratos recém-nascidos. CONCLUSÕES: os modelos de regeneração hepática em ratos recém-nascidos e recém-desmamados foram exeqüíveis e adequados para a pesquisa; a hepatectomia promoveu estímulo da proliferação de hepatócitos com inibição da apoptose; as drogas imunossupressoras utilizadas não exerceram efeito sobre a proliferação de hepatócitos porém provocaram aumento da expressão de genes relacionados a apoptose. / INTRODUCTION: Partial liver transplantation has been performed in children with increasing frequency, and this emphasizes the importance of the studies of hepatic regeneration and the effects of immunosuppressive drugs on this phenomenon. Liver regeneration is controlled by the balance between cell proliferation and apoptosis (defined as a programmed cell death that results from the expression of pro-apoptotic genes - Bax and Bak - and anti-apoptotic genes - Bcl-2 and Bcl-XL). Among proteins related to hepatocyte proliferation, interleukin-6 is an important one. Although the adult rat model of 70% hepatectomy has been widely utilized for studies of liver regeneration, there are no studies using growing animals. METHODS: In the current paper, two experimental models were created utilizing newborn and weaning rats: using a cotton thread, the vascular hilum and the hepatic vein were ligated and the left lateral, left medial and right medial lobes were resected. The remaining liver was immediately harvested and weighted to be compared to control livers. The animals were sacrificed on days 1, 2, 3, 4, and 7 after the operation, the remnant livers were weighted and harvested for histological examinations. Then, 36 animals (18 newborn and 18 weaning animals) were divided into the following groups: control, sham and hepatectomy. One day after, the expressions of IL-6 gene, pro-apoptotic and anti-apoptotic genes were studied in the remnant livers. Immunohistochemical stainings for cell antigens related to cell proliferation (PCNA) and apoptosis (TUNEL) were also performed utilizing tissue microarray sections. In another group of 36 animals (18 newborn and 18 weaning animals), immunosuppressive drugs were administered just after the hepatectomy (methylprednisolone, cyclosporine A or tacrolimus separately), and the remnant liver submitted to the same molecular and immunohistochemical studies. RESULTS: The resected liver corresponded to 70% of the total liver weight. The mortality rates after hepatectomy were 30% and 0% for newborn and weaning rats, respectively. The histological examinations showed a great number of mitoses of hepatocytes on the third day in newborns and on the second day in weaning rats, and normalization of histological aspects by 7 days after hepatectomy and weight recuperation. In the newborn group liver regeneration was related to an intense steatosis. Hepatectomy promoted an increase in the expression of IL-6 gene of the remnant liver, a decreased expression of pro-apoptotic genes in both models, and an increased expression of anti-apoptotic Bcl-2 gene in weaning rats. The study of the effects of immunosuppressants showed different results from those described in adult animals, with no alterations in the number of cells in proliferation (PCNA positive) and apoptosis (TUNEL positive). Drugs had no effect in expression of IL-6 gene. Methylprednisolone and tacrolimus promoted an increased expression of anti-apoptotic gene Bcl-2. In addition, methylprednisolone and cyclosporine promoted an increase in the expression of the pro-apoptotic gene Bak in newborn rats. CONCLUSIONS: The experimental models were feasible and adequate for the current investigations; hepatectomy stimulated hepatocyte proliferation and inhibited hepatic cells apoptosis; the utilized immunosuppressant drugs did not affect hepatocyte proliferation although an increased expression of apoptosis-related genes was verified.

Dans le cadre d’un projet général concernant l’étude de la réactivité des acidesbenzoïques non protégés avec les organométalliques, la synthèse totale d’analoguesstructuraux de l’apogossypol mettant en jeu des réactions de métallation aromatique a étéétudiée ainsi que la réaction de substitution nucléophile aromatique des acides benzoïquesortho-fluorés et ortho-méthoxylés.Le gossypol, (1,1’,6,6’,7,7’-hexahydroxy-5,5’-di-iso-propyl-3,3’-diméthyl-2,2’-binaphtalène-8,8’-dicarboxaldéhyde), pigment principal des graines du cotonnier, existe sousla forme de deux atropoisomères et possède de multiples applications pharmaceutiques. Il estnotamment un inhibiteur efficace des protéines anti-apoptotiques de la famille Bcl-2. Legossypol déformylé ou apogossypol présente des propriétés similaires mais est plus stable,plus sélectif et moins toxique. Une méthode permettant de remplacer les groupes iso-propylesdu gossypol par des groupes structurellement proches a été mise au point. La stratégie retenuemet à profit les compétences du laboratoire dans le domaine des réactions de métallation etrepose sur la lithiation latérale de l’acide 4-hydroxy-6,7-diméthoxy-8-méthyl-2-naphtoïquepar le tétraméthylpipéridure de lithium. Divers dérivés 5,5’-didés-iso-propyl-5,5’-dialkylapogossypol racémiques ont été préparés selon cette méthode. La synthèse asymétriqued’analogues de l’apogossypol a également été étudiée et la voie de synthèse sélectionnéerepose sur le « concept lactone ». Un intermédiaire avancé de la synthèse, une lactonefonctionnalisée potentiellement réductible de façon asymétrique, a été préparée.La deuxième partie est consacrée à l’étude de la réaction de substitution nucléophilearomatique des acides benzoïques ortho-fluorés et ortho-méthoxylés (réaction SNArAB). Unerevue de la littérature des réactions de substitution nucléophile aromatique activée par lesesters est présentée. L’influence de substituants méthoxylés et halogénés (F, Cl, Br) sur lasélectivité SNAr/addition 1,2 a été évaluée. Il est montré que les acides 2-fluoro-6-halogénobenzoïques conduisent, par réaction avec les aryllithiens et les arylmagnésiens, auxproduits d’ipso-C2-substitution avec un excellent rendement et la réaction SNArAB permet unaccès efficace aux acides 3-halogéno-[1,1’-biphényl]-2-carboxyliques. Dans le cas de l’acide2,3-diméthoxybenzoïque, il a été découvert que la présence d’un substituant méthoxy enposition 3 permet de limiter la formation de cétone et le produit d’ipso-substitution est isoléavec un rendement correct. / As part of a program directed toward the study of the reactivity of unprotected benzoicacids with polar organometallics, the total racemic synthesis of apogossypol analogues bymetalation reactions was studied as well as the aromatic nucleophilic substitution reaction ofortho-fluoro- and ortho-methoxybenzoic acids (SNArAB reaction).Gossypol (1,1’,6,6’,7,7’-hexahydroxy-5,5’-di-iso-propyl-3,3’-diméthyl-2,2’-binaphtalène-8,8’-dicarboxaldéhyde) which is the main pigment of cotton seed, displaysmultiple pharmacological applications. It is a potent anti-apoptotic Bcl-2 protein inhibitor.The racemic route developed herein allows the replacement of the iso-propyl groups byvirtually any alkyl groups, providing a series of 5,5’-dides-iso-propyl-5,5’-dialkylapogossypolderivatives. Lateral metalation of 4-hydroxy-6,7-dimethoxy-8-methyl-2-naphthoic acid withLTMP is the key step of the synthesis. Atroposelective synthesis of apogossypol analoguewas also examined. The strategy relies on the “lactone concept” and involves a functionalizedlactone as a key intermediateThe influence of halogen atoms (F, Cl, Br) and methoxy groups on the 1,2-addition/SNArAB selectivity was examined. Treatment of 2-fluoro-6-halobenzoic acids withorganolithiums or Grignard reagents gives ipso-substituted products in excellent yields. Themethod allows the efficient preparation of 3-halo-[1,1’-biphenyl]-2-carboxylic acids and doesnot require protection of the carboxylate. Interestingly, the presence of an additional methoxyin C3 reduces the nucleophilic addition of the organometallic species to the carboxylate and2,3-dimethoxybenzoic acid affords ipso-substituted products in good yields.

INTRODUÇÃO: O linfoma difuso de grandes células B, sem outras especificaçoes (LDGCB, SOE) é uma neoplasia agressiva caracterizada pela heterogeneidade morfológica, imunofenotípica e molecular, porém o atual tratamento padrão utilizando imunoquimioterapia (R-CHOP) não considera tal diversidade. Há percentual significativo de pacientes que são refratários à terapia de primeira linha e alguns que apresentam recidiva precoce ou tardia, os quais representam as vítimas desta doença. O estudo imuno-histoquímico (IHQ), que é um método simples e universalmente disponível, vem sendo utilizado para reconhecer a diversidade biológica do LDGCB, SOE, identificando biomarcadores e subgrupos distintos da doença, que poderiam predizer a resposta terapêutica ao tratamento padrão e apontar possíveis candidatos a novas estratégias terapêuticas. OBJETIVOS: Este estudo avalia o valor prognóstico de cinco algoritmos para classificação do LDGCB segundo a célula de origem (COO) e da expressão de três biomarcadores (BCL2, CD30 e MYC) tendo como endpoint a sobrevida global. MÉTODOS: Foi realizado estudo retrospectivo com setenta e nove pacientes com LDGCB,SOE de novo tratados com imunoquimioterapia padrão, estadiados e acompanhados protocolarmente. Os casos foram classificados como subgrupo célula B centrogerminativa símile (GCB) ou como subgrupo célula B não-centrogerminativa símile (NGCB), de acordo com três algoritmos IHQ (Hans, Choi, e Visco-Young) pareados com estudo do perfil de expressão gênica (PEG) e dois algoritmos IHQ não-PEG pareados (Muris e Nyman). Foi estimado o valor prognóstico destes algoritmos e também avaliado a concordância entre eles. O valor prognóstico da expressão do BCL2, CD30 e MYC utilizando IHQ também foi analisado. RESULTADOS: Os algoritmos IHQ PEG pareados revelaram maior concordância entre si, porém nenhum deles revelou força prognóstica. A expressão do CD30 mostrou tendência a melhor prognóstico, porém a expressão de BCL2 e MYC avaliados isoladamente não revelaram impacto prognóstico. Contudo, a coexpressão do BCL2 e MYC, denominado como fenótipo linfoma duplo-expressor (LDE), revelou-se importante marcador prognóstico desfavorável. Foram identificados três subgrupos de risco baseado no fenótipo LDE e o Índice Prognóstico Internacional (IPI). CONCLUSÃO: Em pacientes com LDGCB, SOE de novo tratados com esquema terapêutico padrão, a pesquisa da expressão do fenótipo LDE é mais relevante do ponto vista prognóstico que a classificação em subgrupo GCB ou NGCB. Além disso, a expressão do CD30 pode ser relevante tanto para identificar subgrupo com tendência a melhor prognóstico como para identificar possíveis candidatos a nova terapia alvo / BACKGROUND: Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is an aggressive neoplasm characterized by morphological, phenotypic and molecular heterogeneity, but the current standard therapy using immunochemotherapy (R-CHOP) does not consider such diversity. There is a significant percentage of patients who are refractory to first-line therapy and those with early or late recurrence, whose represent the victims of this disease. Immunohistochemistry (IHC), a simple and universally available method, has been used to recognize the biological diversity of DLBCL, NOS, to identify biomarkers and distinct subgroups of the disease, which would predict the therapeutic response to standard treatment and point possible candidates for novel therapeutic strategies. OBJECTIVES: The current study was conducted to evaluate the prognostic value from five algorithms for classification of DLBCL based on cell of origin (COO) and the expression of three biomarkers (BCL2, CD30 and MYC) with overall survival (OS) as an endpoint. METHODS: We retrospectively evaluated seventy nine patients with de novo DLBCL, NOS treated with R-CHOP-like immunochemotherapy. The cases were assigned as germinal center B-cell like (GCB) or non-GCB subgroup (NGCB) according to five different IHC algorithms, including three algorithms based on gene expressing profile study (GEP), proposed by Hans, Choi, and Visco-Young, and two non-GEP based algoritms proposed by Muris, and Nyman. We evaluated their prognostic relevance and the concordance between these algorithms. The prognostic power of BCL2, CD30 and MYC expression were also assessed by IHC. RESULTS: None of the profiles assessed by IHC algorithms was able to predict overall survival (OS). The positive expression of CD30 showed a trend toward a better outcome. Neither the positive expression of BCL2 nor the positive expression of MYC were associated with outcome. However, the double-expressor lymphoma phenotype (DEL), represented by the concurrent expression of MYC and BCL2, exhibited a negative prognostic impact. Three different risk subgroups were identified based on the DEL phenotype and the International Prognostic Index (IPI) score. CONCLUSIONS: These data suggest that the DEL, rather than the cell of origin classification based on IHC, is a better predictor of OS in patients with DLBCL treated with R-CHOP-like immunochemotherapy. Besides, the CD30 expression may be a useful prognostic marker and a possible therapeutic target

Para verificar a expressão das proteínas Fas e Bcl-2 em linfócitos e suas correlações com a atividade da doença foram avaliados 38 pacientes com lúpus eritematoso sistêmico de início na infância e 25 controles sem doença autoimune. Observou-se que as porcentagens de linfócitos T CD3+ e CD8+ e linfócitos B que expressavam a proteína Fas e a intensidade média de fluorescência da proteína Bcl-2 nos linfócitos T CD3+, CD4+ e CD8+ dos pacientes com lúpus foram significativamente maiores quando comparadas aos controles. Os pacientes com doença ativa apresentavam porcentagens de linfócitos B que expressavam a proteína Fas significativamente maiores que os pacientes com doença inativa e os controles e houve correlação direta entre estas porcentagens e o SLEDAI (p=0.02, r=0.38) / In order to verify the expression of Fas and Bcl-2 proteins on lymphocytes and their relationship with disease activity 38 patients with juvenile-onset systemic lupus erythematosus and 25 healthy controls were studied. The measurements showed that percentages of lymphocytes T CD3+ and CD8+ and B lymphocytes positively stained for Fas antigen and mean fluorescence intensity of Bcl-2 on CD3+, CD4+ and CD8+ T cells from lupus patients were significantly increased compared to healthy controls. Lupus patients with active disease presented percentages of lymphocytes B positive for Fas antigen significantly higher compared to patients with inactive disease and healthy controls and there was a statistically significant direct correlation between these percentages and SLEDAI score (p=0.02, r=0.38).

Para verificar a expressão das proteínas Fas e Bcl-2 em linfócitos e suas correlações com a atividade da doença foram avaliados 38 pacientes com lúpus eritematoso sistêmico de início na infância e 25 controles sem doença autoimune. Observou-se que as porcentagens de linfócitos T CD3+ e CD8+ e linfócitos B que expressavam a proteína Fas e a intensidade média de fluorescência da proteína Bcl-2 nos linfócitos T CD3+, CD4+ e CD8+ dos pacientes com lúpus foram significativamente maiores quando comparadas aos controles. Os pacientes com doença ativa apresentavam porcentagens de linfócitos B que expressavam a proteína Fas significativamente maiores que os pacientes com doença inativa e os controles e houve correlação direta entre estas porcentagens e o SLEDAI (p=0.02, r=0.38) / In order to verify the expression of Fas and Bcl-2 proteins on lymphocytes and their relationship with disease activity 38 patients with juvenile-onset systemic lupus erythematosus and 25 healthy controls were studied. The measurements showed that percentages of lymphocytes T CD3+ and CD8+ and B lymphocytes positively stained for Fas antigen and mean fluorescence intensity of Bcl-2 on CD3+, CD4+ and CD8+ T cells from lupus patients were significantly increased compared to healthy controls. Lupus patients with active disease presented percentages of lymphocytes B positive for Fas antigen significantly higher compared to patients with inactive disease and healthy controls and there was a statistically significant direct correlation between these percentages and SLEDAI score (p=0.02, r=0.38).

BH3-only Proteine, eine pro-apoptotische Untergruppe der Bcl-2 Proteinfamilie, sind zentrale Mediatoren von apoptotischen Signalen durch die Regulierung intrinsischer Apoptose-signalwege. Unsere Arbeitsgruppe hat vor kurzem gezeigt, dass Apoptose, die durch den p14ARF Tumorsuppressor induziert wird über die p53-abhängige Aktivierung des BH3-only Proteins Puma/Bbc3 vermittelt wird. Interessanterweise induziert p14ARF aber auch in p53 defizienten Zellen Zellzyklusarrest und Apoptose. Die dahinterliegenden Signalwege sind jedoch nicht bekannt. In dieser Arbeit berichten wir, dass das BH3-only Protein Bmf (Bcl-2 modifying factor) beim p14ARF-induzierten Zelltod in p53 defizienten Zellen eine wichtige Rolle spielt. Expression von p14ARF führt zu einer Induktion der PERK Kinase, daran anschließender Phosphorylierung von eIF2α sowie Aktivierung der stromabwärts liegenden Transkriptionsfaktoren ATF4 und CHOP. Diese Signalkaskade ist normalerweise Teil einer zellulären Antwort auf fehl- oder ungefaltete Proteine im Endoplasmatischen Retikulum (ER), der sogenannten ‘unfolded protein response’ (UPR), die zum einen durch verminderte Translationsinitiation und Hochregulierung von Chaperonen die Menge der fehlgefalteten Proteine reduzieren soll. Allerdings induziert p14ARF keinen ER Stress, sondern den PERK‒CHOP Signalweg. Die Transkriptionsfaktoren ATF4 und CHOP binden direkt in der Promotorregion von bmf und sind für dessen transkriptionelle Regulation verantwortlich. Unsere Daten zeigen, dass der PERK‒eIF2α‒ATF4‒CHOP Signalweg eine wesentliche Rolle bei der Induktion von Apoptose durch p14ARF spielt. Dieser Weg könnte ein Sicherungsmechanismus sein, der es den Zellen auch nach Verlust von p53 erlaubt Apoptose einzuleiten, nachdem p14ARF durch Onkogene hochreguliert wurde. / BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family of proteins, are central mediators of apoptosis signals by regulating the intrinsic apoptosis pathway. We have recently shown, that apoptosis triggered by the p14ARF tumour suppressor protein is mediated by the p53-dependent activation of the BH3-only protein Puma/Bbc3. Nevertheless, expression of p14ARF in p53-family deficient cells is capable of inducing both cell cycle arrest and apoptosis, but the signalling pathways initiated remain elusive. Here, we report that the BH3-only protein Bmf (Bcl-2 modifying factor) is involved in cell death in p53-deficient cells triggered by p14ARF. Expression of p14ARF leads to the induction of the PERK kinase, subsequent phosphorylation of eIF2α and activation of transcription factors ATF4 and CHOP. This signalling cascade is usually part of the ‘unfolded protein response’ (UPR), which is activated upon ER stress to reduce the amount of misfolded proteins by reduction of global protein translation initiation and upregulation of chaperones. Of note, p14ARF does not induce ER stress but activates the PERK‒CHOP pathway. ATF4 and CHOP transcription factors directly bind to the promotor region of bmf and induce its transcription. These data suggest that the PERK‒eIF2α‒ATF4‒CHOP signalling pathway may play a substantial role in mediating p14ARF-triggered apoptosis. This pathway could play the role of a ‘fail-safe’ mechanism that allows cells, even after loss of p53, to undergo apoptosis induced by upregulation of p14ARF by oncogenes.

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text

蛋白激酶 CK2 是一種具有多功能的絲胺酸/蘇胺酸蛋白激酶，大量表現於哺乳類動物的腦中，對於調控細胞週期的發展、基因表現、訊息傳遞以及抗細胞凋亡機制扮演相當重要的角色。許多研究顯示 CK2 也參與調節許多神經系統功能，包括神經保護及神經存活，但是其中調控機制目前尚未釐清。DARPP-32 (dopamine- and cAMP- regulated phosphoprotein with a molecular mass of 32 kDa) 主要表現在紋狀體中型多刺狀神經元中，過去研究已證實 DARPP-32 Ser102 胺基酸是CK2 的磷酸化作用受質。雖然DARPP-32 被發現主要透過抑制蛋白磷酸水解酶 PP1 參與藥物成癮的細胞調控機制，但近年研究指出DARPP-32 也參與抗細胞凋亡作用。PP1 是真核細胞的絲胺酸/蘇胺酸磷酸水解酶，能調節多種細胞功能，如轉錄、細胞訊息傳遞及細胞凋亡。過去文獻已指出 PP1 可以調節 Bcl-x 基因的 pre-mRNA 選擇性剪切，再經由轉譯過程合成抗細胞凋亡 Bcl-xL 異構蛋白，研究也發現抑制 PP1 可以防止細胞週期的停滯及細胞凋亡，強調細胞在壓力的情況下，PP1 扮演了相當關鍵性的角色。因此論文以人類神經母細胞瘤 SH-SY5Y 為實驗模式，探討透過 CK2 調控 DARPP-32 Ser102 的磷酸化是否具有抑制 PP1 的活性並促進細胞存活的作用。實驗結果顯示，抑制 CK2或DARPP-32 蛋白含量會導致細胞存活率下降，轉染 CK2 siRNA 會降低 DARPP-32 Ser102 的磷酸化現象、Bcl-xL 的蛋白質表現；轉染DARPP-32 siRNA 及突變型DARPP-32 S102A DNA 質體也會降低 Bcl-xL 的蛋白質表現，PP1 活性則會因轉染突變型DARPP-32 S102A DNA 質體而增加；此外，給予 PP1 抑制劑的實驗結果發現會促進 Bcl-xL/Bcl-xS mRNA 的比例以及 Bcl-xL 的蛋白質表現量。利用過氧化氫誘導細胞造成氧化壓力狀況下，同時給予 PP1 抑制劑，發現 Bcl-xL 的蛋白質表現量會回復以及促進細胞存活。轉染 CK2-EGFP 或 DARPP-32 S102D DNA 質體可以顯著回復Bcl-xL 的蛋白質表現量及Bcl-xL/Bcl-xS mRNA 的比例，轉染 DARPP-32 S102D DNA 質體亦可降低 PP1 的活性。論文的實驗結果提供 CK2 調節抗細胞凋亡基因表現的新機制，是經由促進 DARPP-32 Ser102 磷酸化作用進而抑制 PP1 活性，此條細胞訊息傳遞路徑將可提供應用於在氧化壓力下提升神經存活的臨床治療。 / Protein kinase casein kinase II (CK2) is a multifunctional serine/threonine protein kinase and is highly abundant expression in the mammalian brain. CK2 plays an important role in the regulation of the cell cycle, gene expression, signal transduction and anti-apoptotic mechanisms. A number of studies have indicated that CK2 is involved in several neuronal functions including the neuroprotection and neuron survival, but its cellular mechanisms are not well-studied. The DARPP-32 (dopamine- and cAMP-regulated phosphoprotein with a molecular mass of 32 kDa) is highly enriched in the striatal medium spiny neurons and the Ser102 residue is identified as the phosphorylation site for CK2. Although DARPP-32 is known as a prominent cellular mediator of drug abuse through the inhibition of protein phosphatase 1 (PP1), the recent studies indicate that DARPP-32 may also be involved in the anti-apoptotic effects. Protein phosphatase PP1 is a major eukaryotic serine/threonine phosphatase that regulates diverse cellular functions such as transcription, cell signaling and apoptosis. PP1 is indicated to regulate the pre-mRNA alternative splicing of Bcl-x gene to encode the anti-apoptotic Bcl-xL isoform. Inhibition of PP1 prevents the induction of cell cycle arrest and apoptosis, underlines the crucial role of PP1 in the cellular response to the stress. In my thesis study, the neuroblastoma SH-SY5Y cell line system was used to investigate whether the promotion of cell survival by PP1 inhibition is through the signaling pathway of DARPP-32 Ser102 phosphorylation by CK2. The current results reveals that the cell viability is decreased under down-regulations of CK2 and DARPP-32. The Ser102 phosphorylation status of DARPP-32, Bcl-xL mRNA and protein level are decreased by CK2 siRNA transfection. Transfection of either DARPP-32 siRNA or mutant DARPP-32 S102A plasmid DNA decreased the Bcl-xL protein level. The PP1 activity was increased by mutant DARPP-32 S102A plasmid DNA transfection. Furthermore, the PP1 inhibitor treatment increased the Bcl-xL/Bcl-xS mRNA ratio and Bcl-xL protein level. Under oxidative stress, inhibition of PP1 activity can reverse the H2O2-induced decrease in Bcl-xL protein level and promote the cell viability. The transfection of CK2-EGFP or DARPP-32 S102D plasmid DNA both can antagonize the effects of H2O2 on Bcl-xL protein level and the Bcl-xL/Bcl-xS mRNA ratio. The DARPP-32 S102D plasmid DNA transfection also attenuated the induction of PP1 activity under oxidative stress. These findings provide another insight for the regulation of anti-apoptotic gene expression by inhibition of PP1 activity through DARPP-32 phosphorylation on Ser102 by CK2. This signaling pathway might be applied in the clinical therapy for neuronal survival under oxidative stress.

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