Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / To determine the main factors influencing the biological behavior of 40 squamous cell carcinomas (SCC) of the upper digestive tract (UDT) of cattle associated with spontaneous ingestion of bracken fern (Pteridium aquilinum), morphological, including cell proliferation, and immunohistochemical aspects were studied. The aspects analyzed included anatomical localization of SCCs, degree of differentiation, occurrence and distribution of metastasis, intensity of the lymphoplasmocytic inflammatory infiltrate (LPII), of the desmoplastic reation, tumor-associated tissue eosinophilia, and the cell proliferation index evaluated through quantification of the argyrophilic nucleolar organizer regions (AgNORs). Forty two percent of SCCs were in the cranial region, 12.5% in the middle, and 45% in the caudal region of the UDT. The neoplasms were classified as well differentiated (WD-SCC; 67.5%), moderately differentiated (MD-SCC; 20%), or poorly differentiated (PD-SCC; 12.5%). When the degree of differentiation was correlated to the anatomical localization, it was observed in the cranial region that 88.2% were WD-SCC and 11.8% were MD-SCC. In the middle region, 60% were WD-SCC, 20% were MD-SCC, and 20% were PD-SCC. In the caudal region, 50% were WD-SCC, 27,8% were MD-SCC, and 22,2% were PD-SCC. Metastasis occurred in 57.75% of the cases, mostly to regional lymph nodes, and were observed in 58.82% of the cases with SCCs of the cranial region; in 40% of the middle region, and in 61.11% of the caudal region. Metastasis to regional lymph nodes and/or to distant organs were found in 44.44% of WDSCC, 75% of MD-SCC, and 100% of PD-SCC. Migration and invasion patterns were analyzed through the immunohistochemistry technique for cytokeratin. Islands and ribbons of neoplastic keratinocytes predominated in the WD-SCCs. The patterns varied greatly in the MD-SCCs, although small aggregates, ribbons, and cords predominated. PD-SCCS were characterized by small aggregates and individual cells. Lymphatic or hematogenous invasion were detected in 11/40 SCCs. There were SCCs originating from the ductal epithelium of the salivary glands. This finding was not previously reported. The intensity of the LPII was more accentuated in the BD-SCCs than in the MDs or PDs. The intensity of the desmoplastic reaction was quantified through the immunohistochemistry technique for vimentin, and was more severe in the PD-SCCs. The tumor-associated tissue eosinophilia (TATE) was measured in the SCCs and classified as mild, moderate, or severe. The only positive statistically significant association was established between TATE and LPII intensities. Cell proliferation was evaluated through quantification of the argyrophilic nucleolar organizer regions (AgNORs) on the neoplastic keratinocytes. AgNOR mean value and standard deviation (±sd) for WD-SCCs were 1.65 (±0.23), for MD-SCCs were 1.88 (±0.31), and for PD-SCCs were 2.39 (±0.26). The correlation between the AgNOR index and each histopathological grade was statistically significant. In conclusion, the factors which influenced the biological behavior of SCCs were the degree of cell differentiation, the patterns of migration and invasion, the intensity of LPII, TATE and desmoplastic reation, and the cell proliferation index measured through quantification of the AgNORs. / Aspectos morfológicos, incluindo proliferação celular, e imunoistoquímicos de 40 carcinomas de células escamosas (CCEs) do trato alimentar superior de bovinos que consumiram samambaia (Pteridium aquilinum) espontaneamente foram estudados, visando principalmente determinar os fatores que influenciam o comportamento biológico destes neoplamas. Os aspectos analisados incluíram localização anatômica dos CCEs, grau de diferenciação celular, ocorrência e distribuição de metástases, padrões de migração e invasão, intensidade do infiltrado inflamatório linfoplasmocítico (IILP), da reação desmoplásica e da eosinofilia tecidual associada a tumores (TATE), e o índice de proliferação celular avaliado através da contagem das regiões organizadoras nucleolares argirofílicas (AgNORs). Quanto a localização anatômica, 42% dos CCEs localizaramse na região cranial, 12,5% na média e 45% na caudal do trato alimentar superior (TAS). Os CCEs foram classificados quanto ao grau de diferenciação celular em bem (CCE-BD [67,5%]), moderadamente (CCE-MD [20%]) ou pouco diferenciados (CCE-PD [12,5%]). Quando relacionado o grau de diferenciação celular com a localização no TAS, verificou-se que na região cranial 88,2% eram CCEs-BD e 11,8% eram MD; na região média, 60% eram BD, 20% eram MD e 20% eram PD; na região caudal, 50% eram BD, 27,8% MD e 22,2% PD. Metástases ocorreram em 57,75% dos casos, principalmente para linfonodos regionais, e foram observadas em 58,82% dos CCEs na região cranial, em 40% dos da região média e em 61,11% dos da região caudal. Metástases para linfonodos regionais e/ou órgãos distantes foram encontradas em 44,44% dos CCEs-BD, em 75% dos MD e em 100% os PD. Foram analisados os padrões de migração e invasão com o auxílio da técnica de imunoistoquímica para citoqueratina. Nos CCEs-BD predominaram os padrões em ilhas e fitas de queratinócitos neoplásicos; nos MD os padrões variaram muito porém predominaram os agregados pequenos, fitas e cordões; nos PD predominaram os agregados e as células individuais. Invasão vascular linfática e/ou sangüínea foram observadas em 11/40 CCEs. Foram observados CCEs originando-se do epitélio dos ductos das glândulas salivares, aspecto este que não havia sido relatado anteriormente. Observou-se que a intensidade do IILP era muito mais acentuada nos CCEs-BD que nos MD e PD. A intensidade da reação desmoplásica foi quantificada através da imunistoquímica para vimentina e foi muito mais acentuada nos CCEs-PD. A TATE foi medida nos CCEs quanto à intensidade em leve, moderada ou acentuada. A única associação positiva estatisticamente significativa foi estabelecida entre a intensidade da TATE e a do IILP. A proliferação celular foi avaliada quantitativamente através da contagem das regiões organizadoras nucleolares argirofílicas (AgNORs) nos queratinócitos neoplásicos. A média e o desvio padrão (±DP) de AgNORs nos CCEs BD foi de 1,65 (±0,23), nos MD de 1,88 (±0,31) e nos PD foi de 2,39 (±0,26). A correlação entre o índice de AgNOR e cada grau de diferenciação celular foi estatisticamente significativa. Concluiu-se que os fatores que influenciaram no comportamento biológico dos CCEs foram o grau de diferenciação celular, os padrões de migração e invasão, a IILP, a TATE e da reação desmoplásica e o índice de proliferação celular avaliado através da contagem das AgNORs.

Abstract Incidence of esophageal adenocarcinoma is rising rapidly in Western countries. The main risk factor for esophageal adenocarcinoma is Barrett’s esophagus. Barrett’s esophagus results from long-term gastroesophageal reflux disease. The gastrointestinal tract is colonized by bacteria, fungi and viruses forming the alimentary tract microbiome. Microbiome transformation is involved in pathogenesis of alimentary tract cancer and also in the development of Barrett’s metaplasia. Toll-like receptors (TLR) are molecules of the innate immune system and they are involved in bacterial and viral recognition and regulation of immune functions in the host and cancer cells. This thesis examined the effect of alimentary tract microbiome and cancer- on the function of TLRs in normal gastrointestinal epithelial cells. An additional focus of the thesis was also to assess the carcinogenetic effect of TLRs 1–9 in Barrett’s esophagus metaplasia – dysplasia – carcinoma sequence. Study material consisted of: a patient cohort, organ donors, conventional and germ-free mice. TLRs are expressed also in a “microbe-free” gut. There were significant differences in all TLRs between small- and large intestine of conventional mice and in humans. In germ-free mice that difference was not observed. Normal tissue sampled adjacent to the tumors of cancer patients can be used as controls in immunohistochemical TLR studies in gastrointestinal cancer Clinical data indicate that TLRs linearly increase toward dysplasia in Barrett’s esophagus. High cytoplasmic and nuclear TLR4 expression and TLR1 and 8 nuclear immunoreactivity in esophageal adenocarcinoma are associated with metastatic disease and poor prognosis. Based on our results, bacteria seem to downregulate TLR expression of the intestine. TLRs 1–9 apparently have a role in malignant progression of Barrett’s dysplasia. TLR1, TLR4 and TLR8 may represent a novel therapeutic target in esophageal adenocarcinoma. / Tiivistelmä Ruokatorven adenokarsinooma on länsimaissa nopeasti yleistyvä syöpätyyppi. Tämän syöpätyypin tärkein riskitekijä on Barrettin ruokatorvi, joka kehittyy pitkään jatkuneen gastroesofageaalisen refluksitaudin pohjalta. Ruuansulatuskanavassa on suuri määrä bakteereja, sieniä ja viruksia, jotka muodostavat yhdessä ruuansulatuskanavan mikrobiomin. Normaalin mikrobiomin muutokset ovat yhteydessä usean eri ruuansulatuskanavan syövän patogeneesiin ja myös Barrettin ruokatorven muodostumiseen. Tollin kaltaiset reseptorit ovat luontaisen immuniteetin molekyylejä, jotka osallistuvat bakteerien ja virusten tunnistukseen ja sääntelevät immuunivastetta sekä normaalitilanteessa että syövissä. Väitöskirjassa tutkitaan ruuansulatuskanavan mikrobiomin ja syövän vaikutuksia normaalien epiteelisolujen TLR:ien toimintaan. Lisäksi selvitetään TLR:ien karsinogeneettisiä vaikutuksia Barrettin ruokatorven metaplasia- dysplasia -adenokarsinoomasekvenssissä. Tutkimusmateriaalina käytetään potilaskohortista ja elinluovutuksista peräisin olevia potilasnäytteitä sekä normaalien ja bakteerittomien hiirien näytteitä. Tuloksemme osoittavat, että TLR:t ilmentyvät myös bakteerittomassa ruuansulatuskanavassa, ja TLR:en ilmentyminen oli merkittävästi voimakkaampaa ohutsuolessa kuin paksusuolessa normaaleilla hiirillä ja ihmisillä. Tätä eroa ei havaittu bakteerittomilla hiirillä. Ruuansulatuskanavan syöpien viereistä ja sen altistamaa tervettä kudosta voidaan käyttää terveenä kontrollina immunohistokemiallisissa TLR- tutkimuksissa. Kliinisessä aineistossa TLR:ien ilmentyminen kasvaa lineaarisesti kohti dysplasiaa Barrettin ruokatorvessa. TLR4:n korkea ilmentyminen solulimassa ja tumassa sekä TLR8:n ilmentyminen tumassa ovat yhteydessä metastaattiseen tautiin ja huonoon ennusteeseen. Tulosten perusteella bakteerit näyttävät heikentävän TLR:ien toimintaa suolistossa. Lisäksi kaikilla tutkituilla TLR:illä (1–9) näyttää olevan osuutta Barrettin dysplasian etenemisessä kohti syöpää. TLR1, TLR4 ja TLR8 ovat mahdollisia terapeuttisia kohteita ruokatorven adenokarsinoomassa.

Submitted by Pedro Mizukami (pedro.mizukami@fgv.br) on 2010-09-09T19:11:42Z No. of bitstreams: 1 DMPPJ - MARCIA REGINA LEAL CAMPOS.pdf: 587709 bytes, checksum: 77bdbd307302576b017ee42c06fa25ab (MD5) / Approved for entry into archive by Pedro Mizukami(pedro.mizukami@fgv.br) on 2010-09-09T19:34:53Z (GMT) No. of bitstreams: 1 DMPPJ - MARCIA REGINA LEAL CAMPOS.pdf: 587709 bytes, checksum: 77bdbd307302576b017ee42c06fa25ab (MD5) / Made available in DSpace on 2010-09-09T19:34:53Z (GMT). No. of bitstreams: 1 DMPPJ - MARCIA REGINA LEAL CAMPOS.pdf: 587709 bytes, checksum: 77bdbd307302576b017ee42c06fa25ab (MD5) / Public Administration enjoys procedural prerogatives, such as special deadlines for presenting defenses and appeals, a situation that occurs from the public supreme interest principle over the private interest. The Labor Procedure excels for simplicity and celerity of the lawsuit, mostly due to the nature of the quotas under discussion: its alimentary nature. The worker usually remains unbalanced in the material and procedural relationships, and he is not able to survive with dignity, without the strength of his work. Hence the need to simplify the labor lawsuit procedures pattern and to reduce their lasting period. Such is the approach of this dissertation: to discuss the consistency of the special deadlines assured to Public Administration, whenever we are dealing with labor lawsuits, when, on the other side of procedural relationship, stands an unbalanced and weak worker, in need of a fast and effective judicial solution. Mainly since the new interpretations of Administrative Law, after the 1988 Constitution, which adopted the human being dignity basis. / A Administração Pública goza de prerrogativas processuais, dentre elas prazos especiais para apresentação de defesas e recursos, que decorrem do princípio da supremacia do interesse público sobre o interesse privado. O Processo do Trabalho prima pela simplicidade e celeridade dos procedimentos judiciais, principalmente em razão da natureza das parcelas discutidas: natureza alimentar. O trabalhador, em geral parte hipossuficiente nas relações material e processual, não sobrevive dignamente, sem os recursos advindos de sua força de trabalho. Por isso, a necessidade de simplificar a forma dos processos trabalhistas e diminuir seu tempo de duração. Esta é a abordagem desta dissertação: questiona-se a compatibilidade dos prazos especiais assegurados à Administração Pública, quando se cuida de ações trabalhistas, em que, do outro lado da relação processual, há um trabalhador hipossuficiente, carente da solução judicial célere e efetiva. Principalmente a partir das novas interpretações do Direito Administrativo, pós Constituição de 1988, que adota como fundamento a dignidade da pessoa humana.

Made available in DSpace on 2014-07-29T15:22:58Z (GMT). No. of bitstreams: 1 Lorena santana.pdf: 473142 bytes, checksum: de4188354e54c06e706ab6ee23e80538 (MD5) Previous issue date: 2008-01-28 / The objective of this work was to investigate the feasibility of the pell and flesh of the baru, while ingredient in the development of bread-form. From the formulation of a bread standard were developed four types of breads so using to peel and flesh of baru in different proportions (PCB25 - 25.00%, PCB50 - 50.00%, PCB75-75, 00% and PCB100 - 100.00%) to replace the farelo of wheat. The peel and flesh of baru were analyzed for microbiological quality, levels of moisture, protein, lipids, carbohydrates, fiber, ash, pH, content of soluble solids. With the exception of the last two analisys, the others have been held for breads, even to the sensory analysis, specific volume and density. The results were analyzed by the coefficient of variation, analysis of variance, test Tukey (p <0.05) and correlation of Pearson. The analysis of the composition centesimal of the peel and flesh of the baru revealed a high content of carbohydrates (65.00%) and fiber (4.00%), a low concentration of protein and lipid (4.00% and 3.00%, respectively). The peel and flesh of baru presented concentration of soluble solids with a value of 5.00 ° Brix and pH equal to 5.40. The breads have developed an average 34.00% moisture, 2.00% ash, 2.00% of lipids, 13.00% of protein, 42.00% of carbohydrates and 6.00% of total fiber food. There was an increase in up to 58.20% in the content of FAT with increasing proportion of the peel and flesh of the baru. All samples were accepted on the attributes appearance, texture and flavor, and is considered low in total fat, not showing significant difference between them on the content of protein and moisture. The results of microbiological analysis was favorable. The survey showed that the peel and flesh of baru are feasible for implementing technological ingredient in breads, giving improves the nutritional characteristics and sensory attributes. / O objetivo deste trabalho foi investigar a viabilidade da casca e polpa do baru, enquanto ingrediente, no desenvolvimento de pães do tipo forma. A partir da formulação de um pão de forma integral padrão foram desenvolvidos quatro tipos de pães de forma utilizando-se a casca e polpa do baru em diferentes proporções (PCB25 25,00%, PCB50 50,00%, PCB75 75,00% e PCB100 100,00%) em substituição ao farelo de trigo. A casca e polpa do baru foram analisadas quanto à qualidade microbiológica, teores de umidade, proteínas, lipídios, carboidratos, fibras, cinzas, pH, teor de sólidos solúveis. Com exceção das duas últimas análises, as demais também foram realizadas para os pães, além da análise sensorial, volume específico e densidade. Os resultados foram analisados pelo coeficiente de variação, análise de variância, teste de Tukey (p< 0,05) e correlação de Pearson. A análise da composição centesimal da CPB revelou um elevado teor de carboidratos (65,00%) e fibras (4,00%), uma baixa concentração de proteína e lipídios (4,00% e 3,00%, repectivamente). A casca e polpa do baru apresentaram concentração de sólidos solúveis com valor de 5,00°Brix e pH igual a 5,40. Os pães desenvolvidos apresentaram em média 34,00% de umidade, 2,00% de cinzas, 2,00% de lipídios, 13,00% de proteínas, 42,00% de carboidratos e 6,00% de fibra alimentar total. Observou-se um acréscimo em até 58,20% no teor de FAT com o aumento da proporção da casca e polpa do baru. Todas as amostras foram aceitas quanto aos atributos aparência, textura e sabor, sendo considerados com baixo teor de gorduras totais, não apresentando diferença significativa entre si quanto ao teor de proteína e umidade. O resultado das análises microbiológicas foi favorável. A pesquisa realizada demonstrou que a casca e polpa do baru constituem ingrediente viável para aplicação tecnológica em pães, conferindo melhora das características nutricionais e atributos sensoriais.

L’objectif de ce projet doctoral a été de coupler la chromatographie en phase supercritique (SFC) avec un spectromètre de masse haute résolution pour l’analyse de substances naturelles apolaires. La SFC est une technique dite « verte » contrairement à la chromatographie liquide en phase normale (NPLC), très consommatrice de solvants organiques toxiques pour l’environnement, puisque la phase mobile est principalement constituée de CO2. Le CO2 ayant une faible viscosité, cela implique une diffusivité, des débits élevés et des temps d’analyse courts. Notre attention a été focalisée sur des molécules apolaires : les lipides. Le but était de mettre au point des dosages dans des matrices alimentaires et biologiques et de débuter une approche lipidomique d’étude de la maladie de Parkinson. La première partie a été dédiée au développement du système SFC avec une détection UV, prêté par le constructeur Agilent Technologies. La première étude s’est portée sur 6 composés de la famille des vitamines A. Une phase d’optimisation a été réalisée afin d’obtenir une séparation satisfaisante des composés, en testant différents paramètres chromatographiques comme le type de phase stationnaire ou encore la composition de la phase mobile, afin d’obtenir une résolution optimale. Ensuite, des études de linéarité et de répétabilité ont été réalisées et des limites de détection et de quantification ont été déterminées afin d’obtenir une méthode fiable et robuste. Une deuxième partie a concerné la mise en place du couplage entre la SFC et un spectromètre de masse de type quadripôle-temps de vol (Q-TOF), afin d’améliorer la spécificité et la sensibilité des analyses. Différentes sources d’ionisation ont été utilisées : ESI, APCI et APPI. Chacune des sources présente des modes d’ionisation différents, qui permettent de pouvoir balayer une large gamme de polarité des analytes. Nous avons choisi 8 dérivés de la vitamine E, composés apolaires pour lesquels la SFC paraît être la technique d’analyse idéale. La séparation de ces composés a été optimisée de façon à obtenir une bonne résolution chromatographique et un temps d’analyse minimal. L’ionisation des composés est réalisée avec les 3 sources disponibles en faisant varier les paramètres de sources ou encore le solvant « make-up », de façon à obtenir une sensibilité optimale. La source APPI a été finalement choisie après une étude sur les performances de la méthode. Cette source présente une bonne répétabilité, linéarité et des limites de détection de l’ordre de celles retrouvées dans la littérature par HPLC-MS. Nous avons ensuite réalisé la quantification des ces composés dans 2 types de matrices alimentaire et biologique : l’huile de soja et le plasma de rat. Une troisième partie a été débutée sur le profilage de lipides à polarités variées par SFC-MS. Cette technique se révèle idéale de par la faible polarité de ces composés et leur absence d’absorbance dans le domaine UV. En effet, l’intégrité des lipides peut être altérée suite aux dommages causés par les radicaux libres, qui sont potentiellement impliqués dans de nombreuses maladies neurodégénératives. Il parait primordial de développer des outils analytiques présentant une haute sensibilité et résolution et la possibilité d’accéder aux informations structurales. La source d’ionisation ESI nous a permis de détecter 12 lipides sur les 20 sous-classes analysées en mode positif et 8 lipides en mode négatif. Une application a été réalisée sur un échantillon de plasma humain. Il serait intéressant à l’avenir d’effectuer cette étude en utilisant la source APPI, source propice à l’analyse structurale de lipides et présentant une bonne sensibilité et répétabilité. Ce couplage SFC-MS, présentant une bonne sensibilité et répétabilité, sera par la suite étendu à l’analyse de lipides dans diverses matrices biologiques et pourra à l’avenir être appliqué à l’étude de nouveaux biomarqueurs et au screening rapide d’un grand nombre d’échantillons / The aim of this PhD project was to couple supercritical fluid chromatography (SFC) with a high resolution mass spectrometer for apolar natural compounds analysis. Because mobile phase is principally constituted of CO2, SFC is called “green technic” contrary to normal phase liquid chromatography (NPLC), which uses lot of organic solvents toxic for environment. The CO2 presents a low viscosity, in this way high diffusivity and flow rate, and lower analysis times are obtained. Our work was focused on apolar molecules: the lipids. The aim was to quantify molecules in alimentary and biological matrices and to a lipidomic approach to study Parkinson disease. The first part was to develop the system SFC with a UV detection on a system on loan by Agilent Technologies. This first study was carried out on 6 vitamin A compounds. An optimization of chromatographic parameters has been realized in order to obtain a good separation of the compounds. Then, linearity, repeatability, detection and quantification limits have been determined in order to have a reliable and robust method. A second part concerned the coupling of SFC and a quadrupole time-of-flight mass spectrometer (Q-TOF), in order to improve specificity and sensitivity of analysis. Different ionization sources have been tested: ESI, APCI and APPI. Each ion source presents different ionization mode, which permits to analyze a wide range of polarities of compounds. We have chosen 8 vitamin E derivatives, which are apolar compounds for which SFC seems to be well suited. Separation compounds have been optimized in order to have a good chromatographic resolution and a short analysis time. This compounds ionization is realized with the 3 sources, varying ionization parameters and make-up solvent, to have an optimal sensitivity. The APPI source has been chosen after a performance evaluation method. This source presents a good repeatability, linearity and detection limit in the same order of magnitude than those found in the literature by HPLC-MS. Then we have quantified these compounds in alimentary and biological matrices: a soya oil and plasma rat. A third study has been started on lipid profiling with various polarities by SFC-MS. This technic is well suited because of the low polarity of this molecules and their lack of absorbance in the UV range. The integrity of lipids can be altered with damages caused by free radicals, and are potentially involved in neurodegenerative diseases. It is essential to develop analytical systems with a high sensitivity and resolution and the possibility to access to structural information. The ESI source permits to detect 12 lipids on the 20 sub-classes analyzed in positive ion mode and 8 lipids in negative mode. An application has been realized on human plasma. In the future, it will be interesting to analyze these lipids with the APPI source, which is good choice for structural analysis of lipids, with good sensitivity and repeatability. Studies with this SFC-MS system, presenting good sensitivity and repeatability, will be extended to lipid analysis in biological matrices and could be applied to new biomarkers study and for fast screening of a large number of samples

This diploma thesis is focused on the evaluation of six selected zoonoses with the occurrence in South Bohemia (campylobacteriosis, salmonellosis, listeriosis, tick-borne meningoencephalitis, Lyme borreliosis, tularemia) between the years 2003-2013. Campylobacteriosis and salmonellosis were evaluated the most common zoonoses in the South Bohemian Region during the monitored period. Their common feature is the alimentary transmission. Between 2003 to 2008 salmonellosis recorded the highest number of reported cases, on the contrary, campylobacteriosis has reported the highest number of occurrences since 2008. District of Czech Budweis showed the highest incidence of both zoonoses during the monitored period (4,139 cases of salmonellosis and 4,924 cases of campylobacteriosis). Another but not less important zoonotic is tick-borne meningoencephalitis, which incidence had the highest number in South Bohemian Region of all the regions of Czech Republic. The second part of the thesis is based on questionnaire studies focused on awareness of zoonoses and the issue of resistance and overuse of antibiotics. In total 479 questionnaires were evaluated. The survey findings may be equally described as interesting and positive, since the respondents had considerable awareness of the issue of antibiotic resistance.