S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand". Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

Made available in DSpace on 2015-02-24T17:42:52Z (GMT). No. of bitstreams: 1 ThalesRFP_DISSERT.pdf: 5363462 bytes, checksum: 16f2d3a123870a2d8c63de00ac4bf689 (MD5) Previous issue date: 2014-02-24 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Helicobacter pylori is the main cause of gastritis, gastroduodenal ulcer disease and gastric cancer. The most recommended treatment for eradication of this bacteria often leads to side effects and patient poor compliance, which induce treatment failure. Magnetic drug targeting is a very efficient method that overcomes these drawbacks through association of the drug with a magnetic compound. Such approach may allow such systems to be placed slowed down to a specific target area by an external magnetic field. This work reports a study of the synthesis and characterization of polymeric magnetic particles loaded with the currently used antimicrobial agents for the treatment of Helicobacter pylori infections, aiming the production of magnetic drug delivery system by oral route. Optical microscopy, scanning electron microscopy, transmission electron microscopy, x-ray powder diffraction, nitrogen adsorption/desorption isotherms and vibrating sample magnetometry revealed that the magnetite particles, produced by the co-precipitation method, consisted of a large number of aggregated nanometer-size crystallites (about 6 nm), creating superparamagnetic micrometer with high magnetic susceptibility particles with an average diameter of 6.8 ? 0.2 μm. Also, the polymeric magnetic particles produced by spray drying had a core-shell structure based on magnetite microparticles, amoxicillin and clarithromycin and coated with Eudragit? S100. The system presented an average diameter of 14.2 ? 0.2 μm. The amount of magnetite present in the system may be tailored by suitably controlling the suspension used to feed the spray dryer. In the present work it was 2.9% (w/w). The magnetic system produced may prove to be very promising for eradication of Helicobacter pylori infections / A Helicobacter pylori ? a principal causa de gastrites, ?lceras gastroduodenais e c?ncer g?strico. O esquema terap?utico de primeira escolha para a erradica??o desse pat?geno leva muitas vezes a elevado n?mero de rea??es adversas, baixa ades?o do paciente e consequentemente falha na terap?utica. A vetoriza??o magn?tica ? uma t?cnica bastante difundida na literatura que visa minimizar esses problemas, atrav?s da associa??o de f?rmacos a n?cleos magn?ticos direcionando para o local de a??o por interm?dio de campo magn?tico externo. O presente trabalho relata o estudo da s?ntese e caracteriza??o de part?culas polim?ricas magn?ticas contendo os mais frequentes antimicrobianos (amoxicilina e claritromicina) usados no tratamento de infec??es por Helicobacter pylori, objetivando a produ??o de um sistema para vetoriza??o magn?tica por via oral. Granulometria baseada no di?metro de Feret, microscopia eletr?nica de varredura e transmiss?o, difratometria de raio-x, isotermas de adsor??o/dessor??o de nitrog?nio e magnetometria de amostra vibrante revelaram que as part?culas de magnetita, produzidas pelo m?todo da coprecipita??o, consistem em grande n?mero de agregados de cristalitos de tamanhos nanom?tricos (da ordem de 6 nm) os quais formam part?culas microm?tricas superparamagn?ticas de alta susceptibilidade magn?tica, tendo formato irregular com di?metro m?dio de 6,8 ? 0,2 μm. Os n?cleos magn?ticos foram revestidos por pol?mero (Eudragit? S100) em conjunto com amoxicilina e claritromicina (forma polim?rfica II) sendo obtido micropart?culas n?cleo-camada de formato irregular, pela t?cnica de secagem por aspers?o (spray dryer), com um di?metro m?dio de 14,2 ? 0,2 μm. A quantidade de magnetita presente no sistema pode ser adaptada pelo controle da suspens?o inicial usada na alimenta??o do spray dryer. No presente trabalho o conte?do magn?tico final foi estimado em 2,9 % (p/p). Com base nos resultados obtidos, o sistema magn?tico produzido pode se tornar bastante promissor na erradica??o de infec??es por Helicobacter pylori

S100A12 porcina é um membro da família das proteínas S100, um grupo de pequenas proteínas ligantes de cálcio caracterizado pela presença de dois motivos “EF-hand”. Estas proteínas estão envolvidas em diversos eventos celulares, como a regulação da fosforilação protéica, atividade enzimática, tamponamento de Ca+2, processos inflamatórios e a polimerização de filamentos intermediários. Adicionalmente, algumas dessas proteínas podem ligar Zn+2, o qual pode afetar a ligação do íon Ca+2, particularmente para as proteínas S100. Neste trabalho, a seqüência gênica que codifica a proteína S100A12 porcina foi obtida por meio da construção de um gene sintético usando códons preferenciais para E.coli, permitindo a produção recombinante de grandes quantidades da proteína. Um estudo termodinâmico da estabilidade estrutural foi realizado, assim como a interação da proteína recombinante com íons divalentes usando técnicas de dicroísmo circular (CD) e fluorescência extrínseca. A desnaturação e renaturação induzidas por uréia ou temperatura indicam que se trata de um processo reversível e que a ligação dos íons Zn+2 e ou Ca+2 à rS100A12 aumenta sua estabilidade. A interação da sonda ANS com a proteína na presença de seus ligantes expõe superfícies hidrofóbicas podendo assim facilitar sua interação com macromoléculas alvo. Analisados em conjunto, os resultados obtidos indicam que S100A12 porcina é capaz de assumir diferentes conformações as quais podem estar correlacionadas com sua função fisiológica. / Porcine S100A12 is a member of S100 family, a small acidic calcium-binding proteins group characterized by the presence of two EF-hand motifs. These proteins are involved in many cellular events as the regulation of protein phosphorylation, enzymatic activity, Ca+2 homeostasis, inflammatory processes and intermediate filament polymerization. In addition, some of these proteins can bind Zn+2, which can affect the binding of Ca+2 particularly to S100 proteins. In this study, the gene sequence encoding S100A12 was obtained by the synthetic gene approach using E. coli codon bias allowing the recombinant production of large amounts of the protein. We report here a thermodynamic study on the structural stability of this recombinant protein and its interaction with divalent ions using circular dichroism and extrinsic fluorescence. The folding/unfolding induced by urea or temperature indicated a reversible process and the binding of Zn+2 or Zn+2 and Ca+2 to S100A12 increasing its stability. The interaction of the ANS probe with the protein in the ligant presence can lead to exposition of hydrofobic regions allowing its interaction with target macromolecules. Taken together, the results indicated that porcine S100A12 may assume different conformations that could be correlated to its physiological function.

Trauma de crânio leve (TCE leve) é definido como um déficit neurológico transitório que ocorre após um trauma incluindo história de náuseas, vômitos, cefaléia ou tontura acompanhada de alteração ou perda da consciência com duração inferior a 15 minutos, amnésia pós traumática e Escala de Coma de Glasgow entre 13 e 15. Apesar da alta taxa de sobrevida, pode cursar com alguma morbidade, principalmente nos três primeiros meses posteriores ao trauma e cerca de 18 % dos pacientes desenvolvem pelo menos uma síndrome psiquiátrica no primeiro ano após o acidente. O diagnóstico ainda é um desafio no sentido de minimizar-se gastos desnecessários com exames subsidiários entretanto, intervenções precoces podem evitar seqüelas. Nosso objetivo foi verificar o impacto do TCE leve na qualidade de vida de suas vítimas e diagnosticar as várias alterações neuropsicológicas que podem advir deste trauma. Esses alterações podem ser verificadas através de instrumentos de pesquisa. Inicialmente, foram avaliadas cinqüenta vítimas com TCE leve, atendidas no Hospital João XXIII, em Belo Horizonte - MG, as quais foram submetidas a dosagem de proteína S100B e tomografia de crânio (TCC) na admissão. Nessa fase, verificou-se que a proteína S100B tem valor preditivo negativo de 100%. Dezoito meses após o trauma, esses pacientes foram procurados em suas residências, quando foi lhes solicitado para que respondessem a quatro instrumentos de pesquisa [dois para diagnóstico de qualidade de vida (World Health Organization WHOQOL-100), e o Short Form-36 (SF-36), um para análise da presença de ansiedade e depressão (Escala Hospitalar de Ansiedade e Depressão (EHAD) e o Questionário de Sinais e Sintomas (QSS), baseado no Post Concussion Questionnaire] com o objetivo de pesquisar a presença de sinais e sintomas da síndrome pós-concussão. Também foram pesquisados aspectos sociodemográficos, como idade, escolaridade, estado civil, renda pessoal e origem desta renda. Os mesmos questionários foram preenchidos por um grupo de controle composto, necessariamente, por coabitantes dos pacientes, sem história de trauma craniano de qualquer gravidade e com idade a mais próxima possível da do paciente. Na avaliação pelo WHOQOL-100, pacientes apresentaram qualidade de vida inferior nos domínios nível de independência, ambiente e no total de domínios (p< 0,05). Na avaliação do SF-36, pacientes revelaram qualidade de vida inferior nos domínios capacidade funcional, vitalidade, saúde mental (p<0,001), dor, estado geral de saúde e aspectos mentais (p<0,05). Pacientes apresentaram mais ansiedade e estavam uma classe acima de seus controles pela EHAD. Pacientes referem ainda número maior de sinais e sintomas da síndrome pós-concussão do que seus respectivos controles. Não verificamos correlação entre a qualidade de vida, classificação na EHAD ou número de sinais e sintomas da SPC com as dosagens de proteína S100B ou com a presença de lesão na TCC realizadas na admissão / Mild head trauma (MHT) is defined as a transitory neurological deficit that happens after the trauma and includes a history of nausea, vomiting, headache or dizziness and loss or alteration of consciousness (less than 15 minutes), post-trauma amnesia, and Glasgow Coma Scale (GCS) at admission between 13 and 15. Despite the high survival rates, some morbidity has been observed in the three month period after this trauma. Approximately 18% of head trauma patients develop at least one psychiatric syndrome in the first year after the accident. The diagnostics difficulty and the risks of complications after the MHT continue to be a relevant problem at the emergency departments around the world. Limitations of active participation in daily life are alterations that influence life quality. Several of these alterations may be diagnosed through Interview Instruments. Our study was divided in two phases. In the first phase, 50 MHT patients admitted at Hospital João XXIII, Belo Horizonte-MG, Brazil, had protein S100B dosing and head CT taken at admission. Concentration values of S100B lower than 0.01 g/l were considered negative once this was the lowest value found in patients who did not show brain injuty signs in the CT scan. In that study it was found that protein S100B has 100% negative predictive value. In this second phase of the study, 18 months after the trauma, these patients were contacted at their homes and asked to answer four self- assessment questionnaires: two for quality of life diagnostic - World Health Organizations WHOQOL-100 and the Short Form-36 (SF36); one for the analysis of anxiety and depression - Hospital anxiety and depression scale-HADS; and one instrument developed by the author based on the Rivermead Post Concussion Questionnaire to evaluate the presence of post-concussion syndrome signs and symptoms. Several socio-demographic aspects were also analyzed, including income, source of income, means of transportation used, etc. The same questionnaires were filled by a control group formed necessarily by patients co-inhabitants, with no history of head trauma of any severity, and with closest age as possible to the patients. In the WHOQOL assessment patients showed a lower quality of life in the independence, environment, as well as in the total domains (p< 0,05). In the SF 36 assessment patients showed a lower quality of life in the functional capacity, vitality, and mental health domains (p<0,001); and also in pain, general health situation, and mental aspects (p<0,05). Patients showed more anxiety and, in the HADS Scale, showed at least a level higher, on average, than their controls. Patients also showed a higher number of post-concussion signs and symptoms than their respective controls. We did not find correlation between the later quality of life and protein S100B dosing at admission. We were not able to find correlation between the protein concentrations with the presence of brain lesions in the CCT scans taken at patients admission in the emergency department

A proteína S100<font face=\"symbol\">b no cérebro é produzida e secretada pela célula da glia astrócito, e exerce de acordo com sua quantidade extracelular, ação trófica ou tóxica sobre os neurônios. Investigamos a distribuição da proteína S100<font face=\"symbol\">b, no animal em condição basal, realizando o mapeamento em diferentes áreas do encéfalo, com a técnica imuno-histoquímica, explorando a hipótese do aparecimento de S100<font face=\"symbol\">b em áreas encefálicas preferenciais. A distribuição da proteína foi analisada pela técnica do imuno-histoquímica, com utilização de anticorpo anti-S100 (<font face=\"symbol\">b subunidade). O mapeamento da proteína S100<font face=\"symbol\">b ao longo do eixo AP, permitiu observar marcação de elementos gliais distribuídos pelo telencéfalo, diencéfalo, e tronco encefálico, onde a proteína apresentou-se preferencialmente distribuída, na comparação dos animais. Nossos resultados sugerem que a proteína pode estar relacionada ao fato dessa distribuição ser conservada como padrão dentro da espécie. / S100<font face=\"symbol\">b protein is expressed primarily by astroglia in the brain, and practice functional implication of S100<font face=\"symbol\">b secretion by astrocytes into the extracellular space is scant but there is substantial evidence that secreted glial S100<font face=\"symbol\">b exerts trophic or toxic effects depending on its concentration. We provide here a detailed description of the distribution of the calcium-binding protein S100<font face=\"symbol\">b in and glial elements in the encefalo of rats. The distribution of S100-like immunoreactivity was analyzed by antisera: monoclonal, the b subunit (S100b) of this protein. All sera showed glial positive elements, which were more abundant in the brainstem than in the prosencephalon. S100-immunoreactive was detected in glial elements, in different regions of the telencephalon, diencephalon and mesencephalon. This distribution appears very similar to that, as well as to sparse observations on different vertebrates. Therefore, our results suggest that the distribution pattern of this protein in glial elements is highly conserved between the species.

Diabetes melito ou intolerância à glicose estão presentes em até 80% dos pacientes com adenocarcinoma de pâncreas. Portadores desta neoplasia têm resistência à insulina e alteração na secreção de insulina em resposta à glicose, o que pode levar ao aparecimento ou piora de diabetes. Para identificar genes diferencialmente expressos, que podem representar fatores diabetogênicos produzidos pelo adenocarcinoma de pâncreas, utilizou-se a comparação de microarranjos de oligonucleotídeos hibridizados com RNA complementar (cRNA) de tumores pancreáticos de pacientes com e sem diabetes melito no pré-operatório. Uma lâmina foi hibridizada com cRNA de dois pacientes portadores de diabetes melito, e outra com cRNA de dois pacientes com tolerância normal à glicose pelo teste oral. Considerando a expressão ajustada para os controles internos dos microarranjos, 293 genes estavam duas ou mais vezes mais expressos na lâmina dos portadores de diabetes melito; destes, 25 genes estavam pelo menos cinco vezes mais expressos. Duzentos e noventa e sete genes estavam pelo menos duas vezes mais expressos na lâmina dos pacientes com tolerância normal à glicose, dos quais 54 genes estavam cinco ou mais vezes mais expressos nestes indivíduos. Dos genes mais expressos nos tumores dos indivíduos portadores de diabetes melito, três deles, FAM3D, do inglês Family with Sequence Similarity number 3 member D, neuropeptídeo Y (NPY), e proteína de ligação do cálcio S100A8, foram estudados por reação em cadeia da polimerase em tempo real. A expressão do FAM3D foi 4070 (1000-37588) nas amostras de tumores de pacientes com diabetes melito, contra 109 (10-1112) nas de pacientes não-diabéticos (com intolerância à glicose ou com tolerância normal à glicose) (p<0,05). A expressão do NPY foi 0,46 (0,19-0,91) nos tumores dos portadores de diabetes, contra 0,32 (0,21- 0,58) nos tumores dos não-diabéticos (p = NS). Quanto à expressão de S100A8, foi 0,52 (0,27-0,60) nos tumores dos diabéticos, e 0,34 (0,16-1,44) nos não-diabéticos. Estudo imunohistoquímico mostrou que o FAM3D está expresso no núcleo e no citoplasma de células de tumores pancreáticos, tanto de indivíduos com diabetes melito quanto de não-diabéticos, assim como no citoplasma de células de ilhotas pancreáticas e de células ductais normais do pâncreas. Concluímos que o FAM3D é uma proteína expressa em tecido pancreático normal e tumoral, e que existe maior conteúdo do mRNA do FAM3D nos adenocarcinomas de pâncreas de portadores de diabetes melito do que nos de não-diabéticos. / Pancreatic ductal adenocarcinoma is closely related to diabetes mellitus; up to 80% of pancreas adenocarcinoma patients have diabetes or impaired glucose tolerance. Pancreas adenocarcinoma patients have both insulin resistance and altered insulin secretion in response to glucose, and impaired glucose metabolism has been reported in muscle of tumor patients, involving glycogen metabolism and post-receptor insulin signaling. But despite progress in research about this issue, precise mechanisms responsible for the interaction of pancreatic adenocarcinoma and diabetes mellitus remain unknown. The aim of this study was to identify differentially expressed genes between pancreas adenocarcinoma of patients who had and who did not have diabetes mellitus before surgery. Clinical and laboratorial data of 33 patients with pancreatic adenocarcinoma were evaluated, and tumor gene expression was analyzed by microarray method between two patients who had diabetes mellitus and two who did not have glycemic homeostasis impairment, and later used quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in twelve tumor fragments mRNA to confirm obtained data. Pancreatic adenocarcinoma patients who had diabetes mellitus had higher HOMA-IR (p < 0.05) and a trend to lower HOMA-beta indexes than non-diabetic patients. icroarray revealed 293 genes twice more expressed in the pool of diabetic patients as compared to the pool of normal glucose tolerance patients. Of these, 25 were five times more expressed in diabetic patients? pancreatic adenocarcinomas. Three genes were chosen for RT-qPCR: Family with Sequence Similarity number 3 member D (FAM3D), neuropeptide Y (NPY), and calcium-binding protein S100A8. FAM3D expression was 4070 (1000-37588) in diabetic patients tumors versus 109 (10-1112) in non-diabetic (impaired glucose and normal glucose tolerance) patients? tumors (p<0.05). NPY expression was 0.46 (0.19- 0.91) in diabetic patients and 0.32 (0.21-0.58) in non-diabetic patients? tumors (p=NS). Calcium-binding protein S100A8 expression was 0.52 (0.27-0.60) in diabetic and 0.34 (0.16-1.44) in non-diabetic patients (p=NS). Immunohistochemistry revealed that FAM3D protein was expressed in pancreatic adenocarcinoma cells in a diffuse nuclear and cytoplasmic pattern. It was also expressed in the cytoplasm of islets of Langerhans and normal pancreatic ducts cells. The present study indicates that cytokine-like FAM3D protein is expressed in normal and tumoral pancreatic tissue, and that FAM3D mRNA content is higher in pancreatic adenocarcinoma in diabetic than in non-diabetic patients.

Resumo não disponível.

<p>The present thesis addresses various means of reducing inflammatory responses associated with cardiopulmonary bypass (CPB) and retransfusion of pericardial suction blood (PSB) during cardiac surgery.</p><p>Four (I-IV) prospective randomised controlled clinical trials comprising 475 patients were performed in the following areas: effects of heparin coating on measures of clinical outcome and memory function (I, II), inflammatory reactions in PSB and its systemic effects after retransfusion using cardiotomy suction or cell salvage (III) and effects of retransfusion of PSB on memory function and release patterns of protein S100B (IV).</p><p>The use of heparin coated CPB-circuits was associated with a decrease of postoperative blood loss (I, II), transfusion requirements (II), shorter stay in hospital (I) decreased postoperative ventilator time (I), lower incidences of atrial fibrillation (II) and neurological deviations (I), reduction in releases of protein S100B (I, II) and lower postoperative creatinine elevation (I, II).</p><p>PSB contained high concentrations of cytokines, complements, myeloperoxidase, free plasma haemoglobin and protein S100B (III, IV). Retransfusion using cardiotomy suction increased the systemic concentrations of free plasma haemoglobin and protein S100B, whereas retransfusion using cell salvage caused no detectable systemic effects (III, IV). CPB was associated with a small but significant release of protein S100B, despite elimination of PSB-contained protein S100B using cell salvage (IV).</p><p>Subtle signs of impaired memory function were identified that were not associated with the use of heparin coated CPB-circuits (I, II) or retransfusion of PSB (IV).</p><p>Key words: cardiopulmonary bypass, oxygenators, heparin, S100 proteins, blood loss, haemostasis, memory, outcome and process assessment.</p>

The present thesis addresses various means of reducing inflammatory responses associated with cardiopulmonary bypass (CPB) and retransfusion of pericardial suction blood (PSB) during cardiac surgery. Four (I-IV) prospective randomised controlled clinical trials comprising 475 patients were performed in the following areas: effects of heparin coating on measures of clinical outcome and memory function (I, II), inflammatory reactions in PSB and its systemic effects after retransfusion using cardiotomy suction or cell salvage (III) and effects of retransfusion of PSB on memory function and release patterns of protein S100B (IV). The use of heparin coated CPB-circuits was associated with a decrease of postoperative blood loss (I, II), transfusion requirements (II), shorter stay in hospital (I) decreased postoperative ventilator time (I), lower incidences of atrial fibrillation (II) and neurological deviations (I), reduction in releases of protein S100B (I, II) and lower postoperative creatinine elevation (I, II). PSB contained high concentrations of cytokines, complements, myeloperoxidase, free plasma haemoglobin and protein S100B (III, IV). Retransfusion using cardiotomy suction increased the systemic concentrations of free plasma haemoglobin and protein S100B, whereas retransfusion using cell salvage caused no detectable systemic effects (III, IV). CPB was associated with a small but significant release of protein S100B, despite elimination of PSB-contained protein S100B using cell salvage (IV). Subtle signs of impaired memory function were identified that were not associated with the use of heparin coated CPB-circuits (I, II) or retransfusion of PSB (IV). Key words: cardiopulmonary bypass, oxygenators, heparin, S100 proteins, blood loss, haemostasis, memory, outcome and process assessment.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.