Mapeamento dos subsítios de α-amilase de Xanthomonas axonopodis pv. Citri envolvidos na interação com o substrato A família das enzimas α-amilases é um modelo experimental interessante para o estudo das interações entre os aminoácidos e seus ligantes, já que estas enzimas apresentam especificidade variável, são frequentemente alvos de estudos por mutagênese e há estruturas cristalinas disponíveis para alguns membros da família. A proposta deste trabalho foi o mapear subsítios da α-amilase de Xanthomonas axonopodis pv. citri (AXA) envolvidos na interação com substratos, através de comparações estruturais, mutagêneses sítio-dirigidas, análises de parâmetros cinéticos sobre amido e do padrão de clivagem sobre p-nitrofenil malto-oligossacarideos (PNPG7, PNPG5, PNPG4). Foi criado um modelo estrutural para AXA a partir da estrutura tridimensional da α-amilase de Alteromonas haloplanctis (Aghajari et al., 1998). O modelo de AXA foi sobreposto na estrutura da α-amilase pancreática de porco (Qian et al., 1994) e 11 resíduos foram selecionados e mutados para alanina. As α-amilases recombinantes mutantes e selvagem foram secretadas pela levedura Pichia pastoris GS115, apresentando uma massa molecular aparente de 45 kDa. Todos os mutantes analisados reduziram em maior ou menor grau a atividade catalítica da enzima sobre amido e p-nitrofenil maltooligossacarideos. Mutações dos resíduos H88, F136, D196, E223, D295 e N299, deletaram a atividade enzimática, indicando que suas cadeias laterais são essenciais para o desempenho catalítico da enzima. As análises cinéticas e estruturais sugerem fortemente que D196, E223 e D295 são os resíduos catalíticos. Substituições das cadeias laterais de C157, H200, G227, T230 e H294 reduziram a eficiência catalítica (kcat/Km) da α-amilase sobre o substrato amido para, respectivamente, 28%, 41%, 84%, 81% e 51%. As mutações em G227 e T230 foram menos importantes para a atividade da enzima e afinidade pelo amido, entretanto, estes resíduos mostraram-se importantes para a estabilização de complexos com substratos curtos (pNPG4). Os resultados indicam que o sítio ativo de AXA é formado por, no mínimo, seis subsítios. As interações dos anéis de glicose com os subsítios +2 e -2 são favorecidas em relação às interações nos subsítios -3 e +3, respectivamente, e a interação do anel de glicose no subsítio -3 é favorecida em relação à interação no subsítio +3. A enzima selvagem diva preferencialmente a terceira ligação glicosídica de p-nitrofenil maltooligossacarideos. Como produtos de hidrólise a enzima libera maltopentaose, maltotetraose, maltotriose, maltose e glicose. / The α-amylase family is an interesting group for structure/function relationship investigation, as this family exhibits a variable deavage patterm, several crystal structures are available, and its members were studied by mutagenesis. The aim of this study was the mapping of Xanthomonas axonopodis pv. Citri α-amylase (AXA) subsites involved in substrate binding, using structural comparison, site-directed mutagenesis and lcinetics analyses. A structural model for AXA was created from the three-dimensional structure of the α-amylase from Alteromonas haloplanctis (Aghajari et al., 1998). This model was superimposed on the structure ofthe pig pancreatic α-amylase, PPA (Qian et. al., 1994), and 11 residues were selected and changed to alanine. Wild type and mutant AXA were secreted by Pichia pastoris strain GS115 cells and showed apparent molecular mass of 45 kDa. All mutants have reduced α-amylase activity on starch and 4-nitrophenyl maltooligosaccharides (pNPG7, PNPG5 and PNPG4) at different levels. Mutation of residues H88, F136, D196, E223, D295 and N299 indicate their essential role by complete loss of activity. Kinetic and structural analyses strongly suggested that D196, E223 and D295 are the catalytic residues. The substitution of the side chain of C157, H200, G227, T230 and H294 reduced the catalytic efficiency (kcat/Km) of α-amylase on starch to respectively 28%, 41%, 84%, 81% and 51%. Although G227 and T230 were not much important for activity and binding on starch, these residues were important for stabilization of complexes with short substrates (PNPG4). The results indicate that AXA\'s active site is composed of at least six sugar binding subsites. The binding of the glucoses at subsites +2 and -2 are favored against binding at subsites -3 and +3, respectively. The binding of glucose at subsite -3 is favored against binding at subsite +3. The wild type enzyme primarily hydrolyzes the third glucosidic bond in PNPG7, PNPG5 and PNPG4 and the products of hydrolysis were maltopentaose, maltotetraose, maltotriose, maltose and glucose.

Background: The rapid diffusion of Novel Psychoactive Substances (NPS) constitutes an important challenge in terms of public health and a novelty in clinical settings, where these compounds may lead to erratic symptoms, unascertained effects and multi-intoxication scenarios, especially in emergency situations. The number of NPS available on the illicit drug market is astonishing: official reports suggest the appearance of a new drug every week. NPS may be enlisted in many different families such as synthetic phenethylamines, tryptamines, cathinones, piperazines, ketamine-like compounds, cannabimimetics and other plant-derived, medical products and derivatives. Therefore, healthcare services and professionals are often called to face this unknown 'galaxy' where NPS users seem to perceive traditional services 'unfitting' for their needs, requiring an attention which is quite different from known classic drug abusers. In this context, the Recreational Drugs European Network (ReDNet), a research project funded the European Commission and led by the University of Hertfordshire, aimed to explore the NPS galaxy and develop information tools for vulnerable individuals and professionals working with them. This initiative reported specific Technical Folders on new drugs and disseminated the collected information through innovative communication technologies (e.g. multimedia tools, social networking and mobile phone services) internationally. Aim and objectives: The aim of this work is to evaluate and contribute to expand the knowledge of health professionals on NPS. The key objectives are: 1) to assess the level of knowledge on NPS amongst a sample of Italian healthcare professionals; 2) to evaluate the effectiveness of dissemination tools developed by ReDNet, including an SMS-Email/mobile service (SMAIL); 3) to understand the clinical impact of NPS by providing four Technical Folders and collecting two clinical cases on NPS. Methodology: According to the objectives, the methodological approach has been articulated in the following three phases. Phase 1: investigating knowledge and preferred channels of information via an online survey among health professionals in Italy. This first Italian study on NPS awareness had been online from February to July 2011, recruiting participants from Departments of Addiction, Psychiatry and other services. Phase 2: evaluating the ReDNet initiative. An evaluation questionnaire was designed and disseminated online to assess the various resources provided by ReDNet project; it had been online from April to July 2013, targeting professionals registered to ReDNet services. This phase also investigated the SMAIL service, a mobile application that was the latest technological tool developed by ReDNet team. Phase 3: promoting evidence based work in clinical practice through the preparation of four Technical Folders and two case reports. Technical Folders followed the methodology optimised during the ReDNet experience, organising NPS data under specific headings, measured for the need of health professionals. Case reports were collected in a Dual Diagnosis Unit in Italy ('Casa di Cura Parco dei Tigli'); assessed patients revealed for the first time the use of NPS; clinical interviews were conducted to collect a full anamnesis while for the first time psychopathological characteristics were measured in NPS abusers, using a psychometric instrument (MMPI-2). Results: In Phase 1 Italian services, in particular interviewees (n=243) from Departments of Psychiatry and Addiction, showed a strong interest for the subject but a poor understanding of NPS: 26.7% of respondents did not know if their patients ever used NPS; at the same time they considered this phenomenon as very relevant to their profession (e.g. psychomotor agitation [75.7%], errors in the assessment [75.7%], management of the clients [72%]); in addition less of a quarter of them had reliable information on new substances. Interviewees also reported the need for easily accessible channels of information to expand their expertise in the field (including emails [70%] and dedicated websites [51.9%]). The ReDNet initiative (Phase 2) reached professionals (n=270) from European countries and various other regions; they appreciated the website above all (48.5%), which provided access to other information (in form of academic papers, news, technical folders, etc.). The integration of technological-based and classic educational resources was used to self-educate professionals (52.6%) and supply information for research (33.7%) with up-to-date and 3 reliable information; in the same Phase the SMAIL service was analysed in its first 557 searches: in the pilot period 122 professionals used SMS inquiries (95%), asking information on NPS while highlighting the increasing number of NPS available on the market. Technical folders (Phase 3) described two new phenethylamines (Bromo-dragonfly and 25I-NBOMe), a novel ethno drug (Kratom) and a new synthetic cathinone (alpha-PVP) whose severe effects were also described in one of the clinical cases. The first case report (Alice) involved a clubber who used mephedrone and other NPS with a severe worsening of her psychiatric disturbances; the second one (Marvin) described a patient who was referred by a psychiatric service and revealed himself as a 'psychonaut' with an intense abuse of alpha-PVP. Conclusions: The exploration of the NPS galaxy is a new challenge for healthcare professionals. In this study, Italian services seemed to be unprepared to face the emergency and requested rapid access to reliable information; the ReDNet project provided both technology-based and traditional resources to expand knowledge on NPS, making professionals more aware of emerging issues and helping especially clinicians working in the field (e.g. via SMAIL service and Technical Folders). Overall, it can be observed that effective information services on NPS targeted at professionals initiatives should include an online interface integrating up-to-date information, describing NPS through specific Technical Folders and disseminating scientific literature; the use of technological tools, including mobile applications, is an important strategy to support health professionals in their activity. Finally, more 'visual' guidelines, possibly in the form of a 'map' of these heterogeneous compounds, could be a useful framework to describe NPS to physicians and other professionals who are often unprepared and unconfident to face such an expanding galaxy.

Soils contain highly diverse consortia of bacteria making them very attractive starting points for both culture-dependent and metagenomic discovery efforts. The present diploma thesis analyses the composition of the microbial community from pharmaceutically polluted soil, with the employment of next-generation Illumina sequencing of 16S rDNA region. This analysis revealed high complexity of the soil microbial environment and confirmed that anthropogenic activity (represented by production of beta- lactam antibiotics) influences the variability and abundance of the species, yet without reducing the microbial diversity. In the second part of the thesis, isolation and heterologous expression of a novel gene encoding alpha-amino acid ester hydrolase (AEH) from a cultivable soil microorganism B. cereus is described. AEHs possess industrial potential for biocatalytic synthesis of semi-synthetic beta-lactam antibiotics, which are presently of great clinical importance. Powered by TCPDF (www.tcpdf.org)

Nesta tese de doutorado foram desenvolvidos e caracterizados nanomateriais eletroativos, explorando estratégias de estabilização e os efeitos sinérgicos em nanomateriais multifuncionais baseados em alfa-hidróxido de níquel, com potencial aplicação nas mais essências e urgentes áreas de atuação científica e tecnológicas, como por exemplo na conversão e armazenamento de energia, e sensores amperométricos. Nesse sentido, foram desenvolvidos nanopartículas (NPs) de alfa-hidróxidos de níquel (α-Ni(OH)2) estabilizados pela formação de nanocompósitos com óxido de grafeno (GO), denominados de α-Ni(OH)2@GO. Analogamente, foram preparados NPs de sais de hidróxido duplo de níquel e cobalto (α- NiCo(OH)2), estabilizados pela incorporação cátions Co2+ na estrutura do α-Ni(OH)2, e o correspondente nanocompósito com óxido de grafeno reduzido (rGO) denominado de α- NiCo(OH)2@rGO. De modo geral, os nanocompósitos exibiram alta capacidade de carga em elevadas densidades de corrente, alta capacidade de retenção de carga e elevada estabilidade como consequência da interação sinergística de seus componentes, com potencial aplicação em dispositivos de armazenamento de energia de alto desempenho, como por exemplo em supercapacitores híbridos. Por outro lado, um dos principais desafios no campo da conversão de energia, concentra-se no desenvolvimento de eletrocatalisadores eficientes e robustos para impulsionar a cinética intrinsecamente lenta da reação de evolução de oxigênio (OER), que envolve etapas de transferência de elétrons acopladas a de prótons. Para isso, foram preparados nanocompósitos ternários baseados em NPs de αNi(OH)2 de diferentes tamanhos, octacarboxiftalocianina de ferro (FeOCPc) e rGO (α-NiFeOCPc@rGO). As NPs de α- Ni@rGO-K maiores e mais cristalinas e seus respectivos nanocompósitos mostraram propriedades eletrocatalíticas superiores para a OER quando comparados com os respectivos derivados de α-Ni(OH)2-Na, indicando a relevância do tamanho do nanocristal de α-Ni(OH)2 na estrutura do nanocompósito e consequente efeitos sinérgicos nas propriedades eletroquímicas e eletrocatalíticas dos nanocompósitos ternários. Além disso, foram desenvolvidos eletrodos modificados com NPs de hidróxido duplo lamelar de NiCe (α-NiCe) como sensores de alta sensibilidade, e de fundamental importância para detecção e quantificação de prednisona, uma droga proibida pelo Comitê Olímpico Internacional (COI). Em resumo, a incorporação de 20% em mols de íons Ce(III/IV) em nanopartículas de α- Ni(OH)2 aumentou a estabilidade deste material na fase alfa, conferindo melhores propriedades eletrocatalíticas responsáveis pela rápida oxidação da prednisona e de seus subprodutos de degradação, permitindo a preparação de sensores amperométricos com elevada sensibilidade e baixo limite de detecção (LOD) em comparação aos eletrodos modificados já reportando na literatura. Em suma, as estratégias no design de nanomateriais foram exploradas com sucesso para gerar novos nanomateriais e nanocompósitos com propriedades eletrocatalíticas e de armazenamento de carga aprimoradas, com potencial de aplicação em sensores e supercapacitores híbridos. / In this doctoral thesis, nanomaterials design strategies were explored in order to improve the stability of alpha-nickel hydroxide nanoparticles while developing synergistic effects, generating multifunctional electrochemically active nanomaterials and nanocomposites with potential application in amperometric sensors and energy conversion and storage devices. In this context, the α-Ni(OH)2 NPs were stabilized by generating double hydroxide salts, by interaction with octacarboxyphthalocyanine molecules and graphene oxide (GO) generating hybrid and nanocomposite materials, for example α-Ni(OH)2@GO. Nickel hydroxide NPs was stabilized by incorporation of Co2+ cation to produce α-NixCoy(OH)2 double hydroxide salts and the corresponding nanocomposite with reduced graphene oxide (rGO) designated - NiCo(OH)2@rGO. In this way, nanocomposites with potential application in high performance energy storage devices such as hybrid supercapacitors were produced, since exhibited large charge capacities at high current densities and great stability as a consequence of the synergistic interaction of their componentes. One of the main challenges in the field of energy conversion, the development of efficient and robust electrocatalysts to boost the intrinsically slow multielectronic multiprotonic transfer kinetics of the oxygen evolution reaction (OER) was also addressed using ternary nanocomposites based on α-Ni(OH)2 NPs, iron octacarboxyphthalocyanine (FeOCPc) and rGO (α-NiFeOCPc@rGO). The more crystalline - Ni@rGO-K NPs and their respective nanocomposites showed superior electrocatalytic properties when compared to the respective α-Ni(OH)2-Na derivatives, indicating the relevance of the α-Ni(OH)2 nanocrystal size and synergistic effects on their electrochemical and electrocatalytic properties. In addition, amperometric sensors based on NiCe layered doublehydroxide NPs (α-NiCe) were developed for determination of prednisone, a drug forbidden by the International Olympic Committee (IOC). The incorporation of 20 m% of Ce3+/4+ ions in α- Ni(OH)2 nanoparticles increased its stability in the alpha phase, conferring improved electrocatalytic properties responsible for the rapid oxidation of prednisone and its degradation byproducts, responsible for the higher sensitivity and lower detection limits (LOD) than similar devices reported in the literature. In short, nanomaterials design strategies were successfully explored to generated new nanomaterials and nanocomposites with enhanced electrocatalytic and charge storage properties with potential application in sensors and hybrid supercapacitors.

Cardiovascular diseases represent the most important health risk factors because they are responsible for more than 50% of total mortality. Among them, the ischemic heart disease is leading cause of mortality. From the whole spectrum of different cardioprotective phenomena we have selected: 1) adaptation to chronic normobaric hypoxia (CNH) as the traditional experimental model in our laboratory area and 2) protective effect of exercise which in recent years represents promising and clinically relevant protective mechanism. The whole thesis is based on two studies. Aim of the first study was to characterize the expression of the main pro-inflammatory cytokine, TNF-α, in hearts of rats adapted to CNH. Chronic TNF-α inhibition by infliximab was used for discovering of certain role of TNF-α in CNH. We showed that increased myocardial level of TNF-α during adaptation to CNH was contributed via its receptor TNFR2 and nuclear factor κB-dependent activation of protective redox signalling with increased antioxidant defence. This adaptive pathway participates on the infarct size-limiting effect of CNH. Aim of the second study was find out whether exercise training and CNH could play synergy in cardiac protection in rats model. We reported that CNH and exercise reduced infarct size but their combination...

A síndrome de McCune Albrigth (SMA) é uma doença genética não hereditária, com incidência estimada entre 1/100.000 e 1/1.000.000 casos/ano. A SMA caracteriza-se clinicamente pela tríade: displasia óssea fibrosa (FD), manchas cutâneas café-com-leite e hiperfunção endócrina tais como: síndrome de Cushing, pseudo-puberdade precoce, hipertiroidismo, acromegalia. O diagnóstico da SMA clássica é usualmente baseado no quadro clínico associado a dosagens hormonais e exames de imagem, principalmente cintilografia do esqueleto. No entanto, quadros atípicos e formas parciais muitas vezes dificultam o diagnóstico preciso da síndrome. O objetivo deste estudo foi padronizar dentre as técnicas de PNA (peptide nucleic acid) e PCT em Tempo Real, para a detecção de polimorfismos de base única (SNPs), a técnica mais sensível para a discriminação das mutações ativadoras da subunidade da proteína G. Para este estudo foram selecionados 32 pacientes, 1 masculino e 31 femininos, com SMA, todos em seguimento no Hospital das Clínicas da Faculdade de Medicina da USP. Como resultado positivo, apresentamos nesse trabalho pela primeira vez o uso do RT-PCR genotipagem na detecção das mutações ativadoras da proteína G, em DNA extraído de tecidos afetados e em leucócitos de sangue periférico, sendo a técnica considerada sensível o suficiente para discriminar de forma simples e rápida as mutações ativadoras da PGs. Sugerimos nesse estudo o uso da técnica de discriminação alélica pelo sistema Taqman. Essa técnica possibilita a detecção destas mutações gsp no sangue periférico mesmo numa baixa porcentagem, uma vez que nem sempre o tecido afetado (gônada, osso, hipófise) é disponível. / The McCune-Albright Syndrome (MAS) is a genetic disease, with incidence estimated at 1/100.000 and 1/1000000 cases per year. MAS is clinically characterized by the triad: bone fibrous dysplasia (FD) café-au-lait skin spots and endocrine hyperfunction, such as: precocious puberty (PP), Cushing's syndrome, hyperthyroidism and acromegaly. The diagnosis of MAS is originally based on clinical characteristics associated with hormonal and imaging studies. However, atypical and partial forms often hamper the accurate diagnosis of the syndrome. For this study we selected 32 patients, 1male and 31 females, all being treated in Hospital das Clínicas, School of Medicine, University of São Paulo. As a positive result, we showed for the first time the use of Real Time PCR/genotyping for the detection of activating mutations of the stimulatory G protein, using blood leucocytes DNA. This technique was sensible and can bring fast results for the patient and the physician, making the diagnosis easier. Our study proposes the use of allelic discrimination by Taqman system, which can be used as a probe that allows the identification of specific genotypes. These techniques could help detect these mutations in peripheral blood when the affected tissue is not available.

Este trabalho descreve detalhadamente a implantação de rotinas experimentais para a determinação das atividades de 238U, 234U e 230Th pela técnica de espectrometria alfa. As melhores condições foram obtidas utilizando-se traçadores de 233U, 232U e 229Th, simulando algumas situações comumente encontradas no processamento de silicatos. A qualidade das determinações foi avaliada efetuando-se testes com os padrões geológicos brasileiros BB-1 (basalto) e GB-1 (granito). O processamento radioquímico consta das seguintes etapas: adição de traçadores radioativos e dissolução das amostras através de digestão ácida, pré-concentração de U e Th por co-precipitação, separação e purificação desses elementos por cromatografia de troca iônica e eletrodeposição em discos de aço inoxidável. Os rendimentos químicos para a determinação de urânio e tório, para ambos os padrões, são da ordem de 60% e 70%, respectivamente. Esta metodologia fornece resultados com elevados níveis de precisão (precisão relativa inferior a 4%) e de exatidão (da ordem de 1%), que são fundamentais para aplicações em estudos petrogenéticos. A espectrometria alfa, juntamente com os métodos de análise por ativação neutrônica e espectrometria gama natural, foram aplicados ao estudo do desequilíbrio radioativo nas rochas vulcânicas da Ilha da Trindade e em um fonólito de Martin Vaz. Com isso, foram determinadas as razões de atividades (234U/238U), (238U/232Th) e (230Th/232Th), como também as atividades dos nuclídeos 234Th, 214Pb, 214Bi, 235U, 228Ac, 212Pb, 212Bi e 208Tl. As análises químicas de elementos maiores e menores, obtidas por meio da técnica de fluorescência de raios X, auxiliaram na interpretação desses resultados. No estudo da série do 232Th, verificou-se que a condição de equilíbrio radioativo foi atingida, indicando que o sistema permaneceu fechado para todos os seus membros nos últimos 40 anos. As rochas mais antigas da ilha (Complexo Trindade e Seqüência Desejado) encontram-se em equilíbrio radioativo secular na série do 238U, conforme esperado. Por outro lado, os resultados da análise das rochas pertencentes aos três últimos episódios vulcânicos (Formação Morro Vermelho, Formação Valado e Vulcão do Paredão) ocorridos em Trindade, mostram que a série do 238U encontra-se em desequilíbrio radioativo entre 230Th e 238U. Além disso, as únicas 3 amostras da Formação Morro Vermelho analisadas por espectrometria gama mostraram desequilíbrio entre 226Ra e 238U. As determinações das razões de atividades (238U/232Th) e (230Th/232Th) permitiram estimar a idade da Formação Morro Vermelho em cerca de 140.000 anos. No caso de Martin Vaz, uma única amostra disponível (fonólito peralcalino) mostrou um forte desequilíbrio entre 226Ra e 238U, o qual foi interpretado como resultante de uma perda substancial de Ra, ocorrida no intervalo de 8.000 e 40 anos atrás. / This work describes the implementation of experimental procedures for alpha spectrometry measurement of 238U, 234U and 230Th activities in silicates. The best experimental conditions were defined using 233U, 232U and 229Th radioactive tracers and simulating the usual conditions found in processing silicates. The chemical procedures consists of the following steps: radioactive tracer addition and sample dissolution by acid digestion, U and Th pre-concentration by co-precipitation, element separation and purification by ion exchange chromatography and electrodeposition in inox steel disks. In order to evaluate its effectiveness, the procedure was applied to the Brazilian geological standards BB-1 (basalt) and GB-1 (granite). The obtained chemical yields for uranium and thorium are of about 60% and 70%, respectively, for both matrices. The described methodology furnishes activity measurements with less than 4% relative precisions and accuracies of about 1%, that are essential for petrogenetic applications. The 238U and 232Th series disequilibrium conditions were investigated by alpha spectrometry, together with neutron activation analysis and natural gamma-ray spectrometry. 234U/238U, 238U/232Th and 230Th/232Th activity ratios and the 234Th, 214Pb, 214Bi, 235U, 228Ac, 212Pb, 212Bi and 208Tl specific activities were obtained. These results were interpreted with the help of additional constraints given by the larger and smaller elements concentrations, measured by X-ray fluorescence. The 232Th series is in secular radioactive equilibrium in all analysed samples. In the case of the 238U series, the equilibrium condition was verified, as expected, in the oldest rocks from the Trindade Island (Trindade Complex and Desejado Sequence). On the other hand, the results show that, in the samples from the last three volcanic episodes in the island (Morro Vermelho Formation, Valado Formation and Vulcão do Paredão), the 230Th and 238U are not in equilibrium. Furthermore the three Morro Vermelho Formation samples analysed by gamma spectrometry showed the disequilibrium between 226Ra and 238U. The obtained (238U/232Th) and (230Th/232Th) activity ratios allowed to estimate a 140,000 y age for the Morro Vermelho Formation. In the case of the Martin Vaz sample (peralkaline phonolite) a strong disequilibrium between 226Ra and 238U was observed, suggesting a substantial loss of 226Ra between 8,000 y and 40 y ago.

Introdução. O câncer é responsável por uma das principais causas de morte entre mulheres e nota-se um aumento na sua incidência. A linhaça tem sido preconizada como capaz de reduzir o risco do câncer de mama. Objetivo. Verificar o efeito de dietas ricas em linhaça marrom e dourada na evolução do câncer de mama. Métodos. Células cancerosas mamárias humanas (tipo MCF-7) foram inseridas no tecido subcutâneo de camundongos fêmeas nude. Quando os tumores já puderam ser claramente identificados, após 7 semanas, os animais constituíram 3 grupos, um com dieta controle e os outros dois com as variedades de linhaça marrom e dourada. As sementes de linhaça foram moídas para produção de farinhas que foram utilizadas na suplementação das dietas oferecidas aos animais. O crescimento dos tumores implantados nos animais foi acompanhado por oito semanas. Resultados. As linhaças das variedades marrom e dourada mostraram-se eficazes na diminuição da taxa de crescimento dos tumores MCF-7, não sendo verificada nenhuma diferença entre elas. Conclusão. Ambas variedades de linhaça reduziram o crescimento dos tumores. Assim, o consumo de linhaça deve ser estimulado a fim de diminuir a incidência do câncer de mama. / Introduction. Breast cancer is one of the main causes of death among women and an increase in its incidence has been noticed. There is evidence that flaxseed intake could reduce breast cancer risk. Objective. To verify the effect of brown and golden flaxseed rich diets on breast cancer. Methods. Cancer cells of the MCF-7 line were injected subcutaneously into athymic mice. After seven weeks, when tumors were already established, the animals were randomly divided in three groups: (1) control, fed on a basal diet; (2) and (3) mice fed on the basal diet supplemented with 10% brown and with 10% golden flaxseed, respectively. Flaxseed was freshly ground for production of the diets offered to the animals. Tumor growth was monitored weekly for eight weeks. Results. The two varieties of flaxseed inhibited tumor growth, without any difference between them. Conclusion. Both flaxseed varieties were able to reduce tumor growth. Therefore, their intake should be stimulated with the aim to reduce breast cancer incidence.

O receptor CD40 e seu ligante CD40L possuem um papel importante na interface entre a resposta imune inata e a adaptativa. Disfunções desta via de sinalização são descritas em doenças de origem inflamatória e autoimunes. Em Lúpus eritematoso sistêmico (LES) foi descrito um aumento nos níveis séricos de CD40L solúvel, que participa na produção de autoanticorpos. Receptores ativados por proliferadores de peroxisomos (PPARs) são fatores de transcrição que inicialmente foram descritos como envolvidos apenas no metabolismo lipídico, mas que atualmente são também descritos como atuantes no controle da resposta imune. Com isso, nosso objetivo é determinar se a ativação dos PPARs modula o processo inflamatório através da interação com CD40/CD40L in vitro ou in vivo. Células de linhagem monocítica humana THP-1 foram tratadas por 24 horas com forbol-éster (PMA, 40 nM) e posteriormente estimuladas com CD40L recombinante (rhCD40L, 1 g/ml) por diferentes períodos. Transcritos de mRNA foram analisados por real time PCR e os resultados expressos como razão da expressão do gene housekeeping GAPDH. As células THP-1 apresentam um aumento na expressão de PPAR e após 16 e 2 horas de estímulo com rhCD40L, respectivamente. Estas células também foram estimuladas com LPS (10 g/ml) e LPS+rhCD40L para sabermos se a resposta obtida anteriormente era específica ao estímulo com rhCD40L. O resultado mostra que há uma diminuição na expressão de PPAR e após o estimulo com LPS ou LPS+rhCD40L, indicando que nessas condições a modulação da expressão de PPARs é especifica para a via de sinalização CD40/CD40L. Foi medida também a expressão de CD36, que é descrito na literatura como um indicador da atividade de PPARs. O resultado mostra que o estímulo com CD40L promove um aumento de CD36, o que indica indiretamente que o PPAR estava ativo neste modelo experimental. Para mostrar a interação direta destas duas vias de sinalização, silenciamos o gene de PPAR por siRNA e posteriormente anlisamos a expressão de CD80, cuja expressão encontra-se aumentada logo após a ativação do CD40 de acordo com a literatura. O resultado mostra que, com o silenciamento de PPAR , há um aumento de CD80 logo após a ativação do CD40, evidenciando assim a interação entre essas duas vias de sinalização. A fim de verificar se os achados encontrados in vitro poderiam ser observados in vivo, foi isolada a fração mononuclear de sangue periférico de pacientes com LES com a doença em atividade (n=17), a doença inativa (n=21) ou doadores saudáveis (n=12) e foi medida a expressão de PPAR e por real time PCR. PPAR apresenta um aumento em pacientes com a doença ativa ou inativa em comparação aos doadores saudáveis. Já a expressão de PPAR apresenta aumento apenas em lúpicos em atividade quando comparados com lúpicos inativos ou doadores saudáveis. Quando considerado nesta análise o efeito do tratamento dos pacientes com corticosteróides nos níveis de PPAR, obsevou-se que a expressão de PPAR apresenta o mesmo padrão anterior. Estes resultados sugerem a hipótese de que PPAR seja um possível marcador de atividade de LES. Para confirmar esta especificidade, foram adicionadas à analise células mononucleares retiradas de pacientes com tuberculose e com infecções agudas. Os dados mostram que os níveis elevados de PPAR se mantém apenas em pacientes com lúpus ativo, o que confirma nossa hipótese. Nossos achados sugerem que PPAR e são regulados especificamente em reposta a ativação da via do CD40/CD40L, em monócitos em cultura e em células obtidas de pacientes com LES. Podemos também sugerir que PPAR possa ser um marcador para a atividade de LES. Estes resultados podem representar um novo mecanismo de controle da via de sinalização do CD40/CD40L, participando no controle da resposta inflamatória em cultura e em células de pacientes lúpicos / The membrane receptor CD40 and its ligand CD40L play an important role in the interface between innate and acquired immunity. Dysfunction of this signaling pathway was described in inflammatory and autoimmune diseases. In systemic lupus erythematosus (SLE), increased serum levels of soluble CD40L have been detected, where it plays a significant role in the generation of auto-antibodies. Peroxisome proliferator activator receptors (PPARs) are transcription factors originally described in lipid metabolism. More recently, they were also characterized as inflammatory modulators. Therefore, our objective was to determine whether the activation of PPARs may modulate the inflammatory process through interaction with the CD40/CD40L signaling pathway in vitro and in vivo. Macrophages derived from the human monocytic cell line THP-1 by 24h-treatment with PMA (40 nM) were stimulated with human recombinant CD40L (rhCD40L, 1 g/ml) for different periods. Messenger RNA (mRNA) transcripts for PPAR , and were determined by real time PCR and expressed as a ratio of the housekeeping gene GAPDH transcripts. THP-1 cells express a basal level of PPAR and gene transcription, which is increased 16 and 2 hours after exposure to rhCD40L, respectively. We also stimulated the THP-1 cells with LPS (10 g/ml) and LPS+rhCD40L to see if the increase of PPAR was a response specific to the rhCD40L stimuli. The data show that there is a decrease in PPAR and genes expression upon LPS or LPS+rhCD40L stimulation, indicating that in these times (2 and 16 hours) the response is specific for the CD40/CD40L signaling pathway. Increased expression of CD36 is known as an indicator of PPARs activity. We measured CD36 and saw an increase of this receptor after rhCD40L stimulus, indicating indirectly that PPARs were active in this experimental model. To prove the direct interaction between CD40/CD40L and PPAR , we silenced the PPAR gene by siRNA and analyzed the expression of CD80, which is known to increase after CD40 activation. The results show an increase in CD40L-stimulated CD80 expression upon silencing of PPAR , showing that there is an interaction between these signaling pathways. To confirm whether these findings also occur in vivo, mononuclear cells were isolated from whole blood samples from SLE patients with active (n=17) and inactive disease (n=21), and healthy donors (n=12). The mRNA transcripts for PPARs were detected by real time PCR. In both active and inactive SLE patients, monocytes show an increase in PPAR mRNA expression, as compared to healthy donors. PPAR mRNA is increased only in active patients when compared to healthy donors and inactive lupus patients. Further in this analysis, when we separated the patients with and without the administration of corticosteroids, PPAR displayed the same pattern as above. These results suggested that PPAR may be a marker for lupus activity. To validate this hypothesis, we compared the results obtained from patients with tuberculosis and acute infections. Results showed that only active-lupus patients have an increase in PPAR , confirming the specificity of this phenomenon and hence our hypothesis Our findings suggest that PPAR and are up-regulated specifically in response to CD40/CD40L activation, in both cultured macrophages and in monocytes obtained from SLE patients. We could also suggest that PPAR may be marker for lupus activity. Our results may represent a new control mechanism of the CD40/CD40L signaling pathway and seem to be implicated in the control of the inflammatory response in both human macrophages in vitro and SLE patients

A mucosite oral (MO) constitui uma condição dolorosa que se desenvolve entre 47% e 100% dos pacientes submetidos a transplante de células-tronco hematopoiéticas (TCTH), impactando enormemente em sua qualidade de vida. Investigar fatores preditivos para MO por meio de exames não invasivos faz-se necessário, visando melhorar a qualidade de vida dos pacientes. O objetivo do estudo foi investigar a relação dos regimes de condicionamento e dos níveis salivares de Interleucina 6 (IL- 6) e Fator de Necrose Tumoral alfa (TNF-?) com a MO, bem como investigar o impacto destes na qualidade de vida. Foram selecionados 82 pacientes submetidos a TCTH, que foram avaliados em 4 momentos diferentes: no início do condicionamento para o TCTH (M1), no dia da infusão das células (M2), após 12/20 dias do início do condicionamento para transplante autólogo e alogênico, respectivamente (M3), e após 30 dias ou na alta hospitalar para ambos (M4). Nestes momentos, foi avaliado clinicamente o grau de MO segundo critérios da Organização Mundial da Saúde (OMS), coletada saliva total e aplicados 3 questionários de avaliação de qualidade de vida em relação à MO e à saúde bucal: PROMS, OHIP-14 e OMQoL. As informações clínicas e laboratoriais foram correlacionadas através do STATA 13.0 com 5% de nível de significância. Verificou-se que a maior incidência e intensidade de MO, os piores índices de qualidade de vida e os maiores níveis de IL- 6 e TNF-? foram registrados no M3, porém não houve correlação entre as citocinas e graus de MO. Houve associação entre altos níveis salivares de IL-6 e maiores pontuações no PROMS. O regime de condicionamento mieloablativo (ML) foi relacionado à MO intensa (graus 3 e 4) e à maiores pontuações nos 3 questionários de qualidade de vida, e os escores dos questionários foram maiores conforme maior foi intensidade da MO (p<0,05). / Oral mucositis (OM) is a painful condition that develops between 47% and 100% of patients undergoing hematopoietic stem cell transplantation (HSCT), impacting greatly on their quality of life. Investigation of predictive factors for OM through noninvasive exams is necessary, in order to improve the quality of life of patients. The aim of the study was to investigate the relationship of conditioning regimens and salivary levels of Interleukin 6 (IL-6) and Tumor Necrosis Factor alpha (TNF-?) with OM, and to investigate their impact on quality of life. We selected 82 patients undergoing HSCT, which were assessed at four different moments: at the start of conditioning for HSCT (M1), on the cell infusion day (M2), after 12/20 days of the start of conditioning for autologous and allogeneic transplantation, respectively (M3), and after 30 days or at hospital discharge for both (M4). In these moments, it was clinically evaluated the degree of OM according to criteria of the World Health Organization (WHO), collected whole saliva and applied 3 questionnaires of assessment of quality of life related to OM and oral health: PROMS, OHIP-14 and OMQoL. Clinical and laboratorial data were correlated using STATA 13.0 in a 5% significance level. It was found that the highest incidence and intensity of OM, the worst indices of quality of life and higher IL-6 and TNF-? levels were found in M3, but there was no correlation between cytokines and levels of OM. There was an association between high levels of salivary IL-6 and higher scores in PROMS. The myeloablative conditioning regimen (ML) was related to intense OM (grades 3 and 4) and to highest scores in the 3 questionnaires of quality of life, and the scores of questionnaires were higher as higher was the intensity of OM (p<0,05).