Este trabalho descreve a ocorrência da doença meningocócica na região de abrangência da Divisão Regional de Saúde de Sorocaba-SP, no período de 1999 a 2008. Fundamentado em dados fornecidos pelo Instituto Adolfo Lutz e pelo Grupo de Vigilância Epidemiológica, a incidência e a letalidade foram calculadas, para toda a população e por faixa etária. Os valores obtidos foram comparados com os dados do Estado de São Paulo e do Brasil. Além disso, foram obtidas as distribuições de ocorrência por manifestação clínica e de critérios diagnósticos utilizados, permitindo a análise da situação epidemiológica da doença meningocócica, na região em estudo. Ao verificar os resultados relativos aos sorogrupos, sorotipos e soross ubtipos identificados, foi possível estabelecer o fenótipo das cepas que, predominantemente, causam a doença na região. Em relação à incidência, conclui-se que, durante praticamente todo o período em estudo, é maior do que os valores endêmicos encontrados nos países desenvolvidos. A faixa etária mais atingida, tanto do ponto de vista da incidência como da letalidade é a de 0 a 4 anos, indicando a necessidade de incremento e continuidade dos programas de vacinação relativos a esse grupo populacional. Em relação às cepas circulantes, os fenótipos B:4,7:P1.19,15 e C:23:P1.14-6 predominam, fato coerente com os resultados obtidos para a Grande São Paulo e Baixada Santista / This work describes the meningococcical disease occurrence in the area related to the Health Regional Division of Sorocaba-SP, between 1999 and 2008. Based on data available at Adolfo Lutz Institute and Epidemiological Vigilance Group, the incidence and the lethality were calculated, for the whole population and for age groups. The obtained values were compared with the data related to São Paulo State and Brazil. Besides, the clinical manifestation and diagnosis criteria distributions were obtained, allowing the analysis of the meningococcical disease epidemiological situation in the studied region. Verifying the results related to the identified serogroups, serotypes and serosubtypes, it was possible to establish the phenotype of the strains that, primarily, cause the disease in the region. Related to the incidence, it can be concluded that, in almost all the study period, it is greater than the endemic values found for developed countries. The more affected age group, concerning either incidence or lethality, is the 0 to 4 years old, indicating the necessity of increment and continuity of the vaccination programs. Related to the circulating strains, the phenotypes B:4,7:P1.19,15 e C:23:P1.14-6 are the more frequent, coherently with the obtained results for the Great São Paulo and Baixada Santista

This thesis’s main focus is a CO2 capture technology known as chemical looping combustion (CLC). The technology is a novel form of combustion and fuel processing that can be applied to gas, solid and liquid fuels. By using two interconnected fluidised-bed reactors, with a bed material capable of transferring oxygen from air to the fuel, a stream of almost pure CO2 can be produced. This stream is undiluted with nitrogen and is produced without any direct process efficiency loss from the overall combustion process. The heart of the process is the oxygen carrier bed material, which transfers oxygen from an air to fuel reactor for the conversion of the fuel. Oxygen carrier materials and their production should be of low relative cost for use in large-scale systems. The first part of this research centres on development and investigative studies conducted to assess the use of low-cost materials as oxygen carriers and as supports. Mixed-oxide oxygen carriers of modified manganese ore and iron ore were produced by impregnation. While copper (II) oxide supported on alumina cement and CaO have been produced by pelletisation. These oxygen carriers were investigated for their ability to convert gaseous fuels in a lab-scale fluidised bed, and characterised for their mechanical and chemical suitability in the CLC process. The modified ores and pelletised copper-based oxygen carriers’ mechanical properties were enhanced by their production methods and in the case of the modified iron ore, significant oxygen uncoupling was observed. The copper-based oxygen carriers particularly those containing alumina cement showed high conversion rates of gaseous fuels and improved mechanical stability. The second part of this research thesis focuses on the design philosophy, commissioning and operation of a dual-fast bed chemical looping pilot reactor. Based on the operational experience, recommendations for modifications to the CLC system are discussed. In support, a parallel hydrodynamic investigation has been conducted to validate control and operational strategies for the newlydesigned reactor system. It was determined that the two fast bed risers share similar density and pressure profiles. Stable global circulation rate is flexible and could be maintained despite being pneumatically controlled. Reactor-reactor leakage via the loop-seals is sensitive to loop seal bed-height, and inlet fluid velocity but can be maintained as such to ensure no leakage is encountered.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Radka Vaňková Supervisor: doc. PharmDr. Martin Beránek, Ph.D. Title of diploma thesis: Influence of Carrier Molecule Addition on Plasma Free Circulating Nucleic Acid Extraction Outline: The aim of this thesis was to compare the yields of cell-free DNA (cfDNA) extracted from blood plasma specimens with the addition of commercially available carrier molecules. According to the real-time polymerase chain reaction (qPCR) results, we chose the most optimal carrier molecules for routine isolation of cfDNA from blood plasma samples. Methods: cfDNA was isolated from aliquots of a pooled blood plasma by spin column extraction method NucleoSpin Plasma XS (Macherey-Nagel). We used 7 different types of carrier molecules as RNA carrier (Qiagen), Glycogen (Invitrogen), Poly (A) Polyadenylic acid (Roche), Linear Acrylamide (Invitrogen), Yeast transfer RNA (Ambion), Salmon sperm DNA (Invitrogen), Herring sperm DNA (Promega). After the extraction, plasmatic DNA with the addition of carrier molecules was quantified by spectrophotometry, fluorimetry and qPCR. Results: The best results were achieved with the addition of polyadenylic acid with final concentration of 1.55 μg/ml. The addition of carrier RNA and...

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

MicroRNAs (miRNAs) are a class of small, single-stranded, non-protein coding RNA molecules that regulate cellular messenger RNA (mRNA) and protein levels by binding to specific mRNAs. Aberrant miRNA expression has been shown to be implicated in several diseases, including cancer. Extracellular miRNAs have been found to circulate in the bloodstream and some of their levels have been associated with different diseases. Furthermore, they hold promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Blood-based biomarkers are attractive for cancer screening due to their minimal invasiveness, relatively low cost and ease of reproducibility. New miRNA analysis techniques will add toward the understanding of their biological functions. In this thesis, I investigate the utility of capillary electrophoresis (CE) and mass spectrometry (MS) for analysis of miRNAs through proof-of-concept experiments. In the fi rst part of this work, we developed a Protein-Facilitated Affinity Capillary Electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum (see List of publications (6)). We also implemented a capillary electrophoresis with laser induced fluorescence detection (CE-LIF) method with online sample pre-concentration for detection of endogenous microRNAs in human serum and cancer cells. 3' modification of miRNA is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species. Recent studies have shown that post-transcriptional addition of nucleotides to the 3' end of miRNAs is a mechanism for miRNA activity regulation. For example, such modifications in plants and C. elegans influence miRNA stability. In humans, effects on miRNA stability and on mRNA target repression have both been observed. Thus, there is a need for miRNA detection techniques which are direct and multiplexed, require minimal sample preparation and provide qualitative information regarding these modifications. We developed a multiplexed miRNA detection technique based on capillary electrophoresis coupled on line with electrospray ionization mass spectrometry (CE-ESI-MS). This method allowed a label-free, direct detection of multiple miRNAs extracted from cancer serum as well as their post-transcriptional modifications with a high mass accuracy.

Cerebral small vessel pathology is now known to be associated with the development of cognitive impairment and mild motor impairments such as gait disturbance in a variety of neurodegenerative diseases. This dissertation explores the hypothesis that blood brain barrier dysfunction is an early event in cerebral ischemia and contributes to the development of cerebral small vessel disease (CSVD). A common rodent model of CSVD is permanent bilateral common carotid artery occlusion in the rat. This model was used to study several aspects of the progression of CSVD including the timecourse of blood brain barrier permeability changes following the onset of ischemia, gait disturbance, the expression of tight junction proteins and cytokine expression. It was determined that BBB permeability was elevated for 2 weeks following BCCAO and ischemic rats displayed lower gait velocity. There was no change in expression of TJ proteins. However, ischemic rats had higher levels of some proinflammatory cytokines and chemokines in brain tissue with no obvious changes in plasma levels. The mechanisms underlying the increase in BBB permeability were studied in vitro using artificial barriers made of confluent rat brain microvascular endothelial cells. Cerebral ischemia has been reported to cause an increase in plasma toxicity, likely by elevating the numbers of circulating microparticles (MPs). MPs isolated from the plasma of ischemic rats were applied to artificial barriers where it was found that they act mainly as vectors of TNF-α signaling. MPs induce activation of caspase-3 and the Rho/Rho kinase pathways. It is concluded that most of the increase in barrier permeability is due to apoptosis and disassembly of actin cytoskeleton and disruption of adherens junctions IV and not an increase in transcellular transport. The effects of treatment with the type III phosphodiesterase inhibitor cilostazol on dye extravasation in the brain, glial activation, white matter damage and motor performance were evaluated. It was determined that cilostazol could improve the increased BBB permeability and gait disturbance and microglial activation in optic tract following BCCAO. Also, the effects of treatment with cilostazol on plasma toxicity in vivo (24h and 14d following BCCAO) and artificial barriers (in vitro) were assessed. It was found that cilostazol could reduce plasma toxicity at 24h and improve increased endothelial barrier permeability that is induced by MP treatment respectively. In summary BBB dysfunction occurs in the rat model of chronic cerebral hypoperfusion with no differences in expression of TJ proteins. There is a mild motor disturbance in the form of lower gait velocity following BCCAO. Cytokines released in brain tissue may be associated with pathological consequences following BCCAO while there is no significant difference in plasma levels and circulating MPs may play a role in BBB dysfunction.

Background: Angiogenesis is the process by which new blood vessels develop from existing vasculature and is a critical step in all tumours to facilitate growth beyond a few millimetres. As this process is largely inactive in physiological circumstances in adults, it represents an attractive therapeutic target in oncology. Drugs that target the angiogenic process are classified as anti-angiogenic agents. The first anti-angiogenic drug to be approved by the FDA was bevacizumab; a recombinant humanized monoclonal antibody against VEGF. Randomised studies in colorectal cancer (and other solid malignancies) have reported prolonged progression free survival and overall survival for bevacizumab. However, standard radiological criteria, Response Evaluation Criteria In Solid Tumours (RECIST), although widely employed to assess response to therapy in clinical trials, are generally insensitive to the predominantly cytostatic effects of anti-angiogenic and other targeted therapies. Alternative methods of predicting or assessing early response to such agents are needed, particularly given the cost and toxicity implications of such treatments. However, biomarkers to aid selection of patients for anti-angiogenic therapies, including bevacizumab, remain elusive. Purpose: To investigate Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI), Diffusion Weighted Imaging (DWI) and circulating angiocytokines, measured using an ELISA multiplex, as prognostic markers in patients with metastatic colorectal cancer treated with bevacizumab and chemotherapy. Results: Seventy patients were treated. DCE-MRI and DWI parameters showed good reproducibility with coefficient of variation between 3.7 to 23% for parameters. The median progression free survival, the primary end point of the trial, was 9.3 months. The overall response rate was 44%. The clinical variables which were significant for progression free survival on univariate analysis were: performance status (p=0.005), CEA (p=0.04) and serum LDH (p=0.005). Biomarkers which were significant for progression free survival on univariate analysis were serum VEGF-A (p=0.02), serum HGF (p=0.005), sVEGFR-2 (p=0.02). In each case, low values of the biomarker were associated with improved outcome. Multivariate analysis identified Ktrans (p=0.015), performance status (p=0.008) and serum HGF (p=0.003) as the most significant predictors of progression free survival. A prolonged progression free survival was associated with a good ECOG performance status, high Ktrans and low serum HGF.Conclusions: Whilst these results are encouraging, future work is required to establish whether HGF and Ktrans are prognostic markers for metastatic colorectal cancer and their precise role in the prediction of patients likely to benefit from treatment with bevacizumab.

O tratamento padrão para pacientes com câncer de reto localmente avançado consiste no uso de quimioradioterapia neoadjuvante (QRTn), seguida por cirurgia. Uma fração significativa dos pacientes responde completamente ao tratamento e no momento da reavaliação não apresenta evidência clínica nem radiológica de doença. Uma abordagem alternativa, Watch and Wait, propõe não operar imediatamente esses pacientes e submetê-los a um protocolo de observação frequente, a fim de evitar as morbidades associadas à cirurgia. No entanto, a avaliação da resposta ao tratamento ainda é um desafio, devido à subjetividade da avaliação clínica e a ausência de exames radiológicos suficientemente sensíveis e específicos para garantir a ausência de células tumorais residuais ou capazes de detectar a recorrência precoce da doença. DNA circulante contendo alterações genéticas específicas do tumor (ctDNA) pode ser encontrado na fração livre de células do sangue e tem sido utilizado para monitorar a dinâmica tumoral em tumores sólidos. Avanços recentes das tecnologias de sequenciamento permitem a identificação eficiente e rápida e a um custo relativamente baixo de alterações genéticas em tumores individuais, superando o problema imposto pela ausência de alterações genéticas recorrentes nesses tumores. Essas alterações podem ser utilizadas como biomarcadores personalizados para monitorar a resposta ao tratamento, detectar doença residual e a recidiva precoce do tumor. O objetivo deste trabalho foi identificar e estudar biomarcadores personalizados em pacientes com câncer de reto localmente avançado tratados com QRTn e avaliar a capacidade desses biomarcadores para monitorar a dinâmica tumoral, e auxiliar na definição da conduta cirúrgica e na detecção da recidiva precoce da doença. Biópsias de seis pacientes com adenocarcinoma de reto distal (cT2- 3N0-1M0), foram coletadas prospectivamente pré-tratamento. O DNA genômico extraído a partir das biópsias foi usado para construir bibliotecas tipo mate-pair para o sequenciamento do genoma completo, utilizando a plataforma SOLiD. Rearranjos inter e intracromossômicos foram identificados utilizando programas computacionais desenvolvidos pelo nosso grupo de pesquisa e em seguida foram validados utilizando PCR e sequenciamento Sanger. Foram validadas, pelo menos, três variações estruturais para cada paciente. Amostras de plasma foram coletadas no momento do diagnóstico, depois da QRTn e durante o seguimento. DNA circulante total foi extraído a partir das amostras de plasma e ensaios personalizados foram desenvolvidos para monitorar a presença de variações estruturais através de PCR Digital. ctDNA foi detectado em todas amostras de plasma pré-tratamento de pacientes com tumores T3. A detecção desses biomarcadores apresentou boa correlação com a resposta ao tratamento, no entanto, esta abordagem não foi sensível o suficiente para detectar doença residual. Para dois pacientes que desenvolveram doença metastática foi verificado um aumento nos níveis de ctDNA com pelo menos 36 semanas antes do diagnóstico clínico de doença metastática, sendo possível correlacionar os níveis de ctDNA detectados em coletas subsequentes com a resposta ao tratamento sistêmico de segunda linha. Este estudo, embora de caráter exploratório, gerou dados relevantes e suficientes para justificar a realização de estudos adicionais para avaliar a aplicação dos biomarcadores personalizados na definição da conduta cirúrgica e no acompanhamento de pacientes com câncer de reto tratados com QRTn. / The standard treatment for patients with locally advanced rectal cancer comprises in neoadjuvant chemo radiotherapy (nCRT), followed by surgery. A significant fraction of these patients show complete response to the treatment and at the time of reassessment, there are no clinical and nor radiological evidence of residual tumor. An alternative approach, Watch and Wait, proposes not to immediately operate these patients, but to submit them to a protocol of frequent observation in order to avoid the morbidities associated with radical surgery. However, assessment of treatment response remains a significant challenge due to the subjectivity of the clinical examination and to the lack of sufficiently sensitive tools to ensure the absence of tumor cells or to detect early disease recurrence. Circulating DNA carrying tumor-specific genetic alterations (circulating tumor DNA - ctDNA) can be found in the cell-free fraction of the blood and has been successfully used to monitor the tumor dynamics in solid tumors. Recent advances in sequencing technologies have enabled the rapid and cost effective identification of genetic alterations in individual tumors, overcoming the problem imposed by the absence of recurrent genetic alterations in these tumors. These alterations can be used as personalized biomarkers to monitor treatment response, detect residual disease and early tumor recurrence. The purpose of this work was to identify and validate the use of personalized biomarkers for patients with locally advanced rectal cancer treated with nCRT and to evaluate the ability of these biomarkers to monitor the tumor dynamics, to define surgical approach and to detect early recurrence of the disease. Pre-treatment biopsies from 6 patients with cT2-3N0-1M0 distal rectal adenocarcinoma were prospectively collected. Genomic DNA extracted from the biopsies was used to construct mate-pair libraries for whole genome sequencing using SOLiD platform. Inter and intrachromosomal rearrangements were identified using an in-house bioinformatics pipeline and validated using PCR amplification and Sanger sequencing. At least three structural variations were validated for each patient. Plasma samples were collect at diagnosis, after nCRT and follow-up. Circulating DNA was obtained from the plasma samples and personalized assays were designed to monitor the presence of structural variations using Droplet Digital PCR. ctDNA was detected in all pre-treatment plasma samples for patients with T3 tumors. The detection of these biomarkers showed a good correlation with the treatment response, nonetheless, the approach was not sensitive enough to detect residual disease. In two patients who developed metastatic disease, an increase in ctDNA levels was observed at least 36 weeks before clinical detection of metastatic disease, and it was possible to correlate the level of ctDNA in subsequent plasma samples with response to the second-line treatment. This study, although exploratory, generated relevant and sufficient data to support additional studies to evaluate the use of personalized biomarkers in the surgical management and follow-up of rectal cancer patients treated with nCRT.

Les lésions musculaires sont des événements fréquents. Le diagnostic repose sur la mesure de biomarqueurs sanguins. Les outils actuels présentent des limites qui justifient la recherche de nouveaux candidats biomarqueurs. Récemment, des petits ARNs non codants, les microARNs (miARNs), ont été identifiés. Détectables dans le plasma, certains sont spécifiques d’un tissu et ont été proposés comme de potentiels biomarqueurs de lésions tissulaires. Toutefois, leur intérêt dans le diagnostic des lésions musculaires chez l’individu sain n’est pas connu. Le but de ce travail était d’identifier et de caractériser la réponse des miARNs circulants à des lésions musculaires chez le rat.Nous avons premièrement étudié les profils plasmatiques des miARNs en réponse à des lésions musculaires myotoxiques chez le rat sain pour identifier des candidats biomarqueurs et leurs cinétiques de détection. Un criblage par RT-qPCR nous a conduits à identifier l’augmentation importante des niveaux plasmatiques de miARNs spécifiques du tissu musculaire, miR-1-3p, -133a-3p, -133b-3p, -206-3p, -208b-3p et -499-5p avec un pic de détection à 12 h. De plus, deux miARNs non spécifiques du muscle, miR-378a-3p et miR-434-3p, avaient des profils comparables. L’évaluation des performances diagnostiques a montré que les miARNs sélectionnés pouvaient discriminer les rats lésés des rats non lésés avec peu d’erreurs et une approche combinatoire nous permettait d’améliorer encore ces performances. Ces résultats ont été confirmés chez des rates femelles et des rats mâles âgés. Par ailleurs, nous avons évalué la robustesse des miARNs que nous avons sélectionnés. Malgré des profils d’expression différents des miARNs dans les fibres lentes ou rapides, le phénotype du muscle lésé avait une influence limitée sur la réponse des miARNs. Nous avons ensuite observé que la lésion d’une masse musculaire croissante ne s’accompagnait pas d’une réponse proportionnelle des miARNs circulants. Les miARNs sélectionnés n’augmentaient pas en réponse à des lésions musculaires traumatiques. Néanmoins, nous avons observé que miR-133a-3p et -133b-3p pourraient être des marqueurs intéressant pour détecter un remodelage musculaire précoce après des lésions neurologiques. Enfin, l’hémolyse et la contamination plaquettaire, deux paramètres préanalytiques connus pour induire des modifications des profils circulants, n’avaient pas d’effet sur les miARNs que nous avions identifiés.Pris dans leur ensemble, nos résultats montrent que les miARNs circulants spécifiques du muscle ainsi que miR-378a-3p et miR-434-3p, sont des biomarqueurs robuste et prometteur de lésions musculaires aigues chez le rat. / Skeletal muscle damage is an often-occuring event. Diagnosis is based on blood biomarkers assessment. Yet, the markers currently available suffer limitations and new biomarker candidates are needed. Recently, small non-coding RNA, microRNAs (miRNAs), were identified. Detectable in plasma, some miRNAs are tissue-specific and have been proposed as biomarkers of tissue damage. However, their relevance as biomarkers of skeletal muscle damage in healthy individuals is unknown. The aim of this work was to identify and characterize the circulating miRNAs response to muscle damage in rats.First, we studied circulating miRNAs response to myotoxic muscle damage in healthy rats in order to identify biomarker candidates and their detection kinetics. RT-qPCR profiling led to the identification of muscle-specific miRNAs that subtantially increased in plasma in response to muscle damage, namely miR-1-3p, -133a-3p, -133b-3p, -206-3p, -208b-3p, and -499-5p with a peak value at 12 h. Two non-muscle-specific miRNAs, miR-378a-3p and miR-434-3p, had similar profiles. The evaluation of the diagnostic accuracy has shown that selected miRNAs were able to discriminate damaged from non-damaged rats with almost no error and a combinatory approach was able to further increase this accuracy. Similar results were found in female and aged rats. Moreover, we sought to evaluate the robustness of selected miRNAs. Despite diferente expression of selected miRNAs in slow and fast fibers, the phenotype of injured muscle had a very limited influence on the plasma miRNA response. Then, we induced muscle damage in an increasing muscle mass and we observed that damage responsive miRNA response was not proportional to the extent of muscle damage. Selected miRNAs did not increased in response to traumatic muscle damage. However, we observed that miR-133a-3p et -133b-3p could be useful markers to detect an early muscle remodeling following neurologic damage. Finally, hemolysis and platelet contamination, two pre-analytical factors known to affect circulating miRNA profiles, had no effect on the miRNAs we selected.Taken together, our results show that circulating muscle-specific miRNAs as well as miR-378a-3p and miR-434-3p, are robust and promising biomarkers of acute muscle damage in rats.