Orientador: Carlos R. F. Grosso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-11T05:30:34Z (GMT). No. of bitstreams: 1 Correa_RenataMukai_D.pdf: 8022257 bytes, checksum: 6c8618cf4604060f0a0fddaede82dbb9 (MD5) Previous issue date: 2008 / Resumo: Este estudo teve como objetivo produzir micropartículas por gelificação iônica contendo, como recheios, constituintes nutricionalmente importantes para a alimentação de larvas de peixes. Na primeira parte deste estudo, foram desenvolvidos sistemas modelos compostos por micropartículas obtidas por gelificação iônica utilizando os polissacarídeos pectina e a mistura ternária: pectina/gelana/alginato e íons cálcio para formação das matrizes. Como conteúdo, foram incorporados compostos hidrofílicos (glicose e isolado protéico de soro de leite) na forma de micropartícula ou emulsão lipídica, utilizando uma mistura lipídica (gordura de peixe/ácido esteárico) contendo surfactantes (monoestearato de sorbitana+triesterato de sorbitana). Esses sistemas foram caracterizados em relação à eficiência de encapsulação, eficiência de inclusão, hidratação, tamanho médio e sua distribuição, morfologia e composição centesimal. Os sistemas preparados na forma de emulsão lipídica apresentaram uma distribuição mais homogênea do conteúdo das micropartículas e liberação de proteínas significativamente menor (p<0,05) do que os sistemas contendo micropartículas lipídicas, sendo os compostos por pectina/gelana/alginato, os que apresentaram a menor liberação observada (~5%). Na segunda parte deste estudo, os compostos hidrofílicos incorporados na forma de emulsão nos sistemas modelos foram substituídos por duas dietas experimentais. Seu preparo envolveu a obtenção e caracterização da gordura de peixe, concentrado protéico de peixe e dos náuplios de Artemia, utilizados na composição das dietas. Essas dietas foram otimizadas em relação ao conteúdo de proteínas, lipídeos e matéria seca, numa tentativa de alcançar o perfil nutricional dos náuplios de Artemia sp; e caracterizadas em relação à sua composição, tamanho, hidratação, morfologia, eficiência de encapsulação e liberação/retenção das proteínas. Ensaios in vivo foram feitos para avaliar a eficiência das dietas e in vitro para avaliar a digestibilidade dos compostos protéicos encapsulados. As dietas micropartículadas apresentaram perfis de composição protéica, lipídica e de matéria seca, próximos aos dos náuplios de Artemia (alimento vivo). A integridade foi mantida após a secagem, apresentando baixa liberação de proteína durante o período estudado (10% para micropartículas secas). Os ensaios in vivo e in vitro mostraram boa digestibilidade da massa protéica das dietas, manutenção da taxa de crescimento e razoáveis valores de sobrevivência (~50%), porém ainda não suficientes para suportar um crescimento similar ao alcançado quando foi utilizado alimento vivo / Abstract: The aim of this study was to produce microparticles by ionic gelation containing essential ingredients for larvae fish feeding. In the first part of this study two model systems of microparticles obtained by ionic gelation were developed. The wall materials used were pectin and one ternary mixture of polysaccharides: (pectin/gelan/alginate) and calcium ions. First, hydrophilic compounds (glucose and whey protein isolate) were used as core material to produce solid lipid microparticles or emulsions using the same lipid mixture (fish fat/stearic acid) plus surfactants (sorbitan monoestearate + sorbitan triestearate) and after the solid lipid microparticles and the emulsions were used as core to produce gelified microparticles. The systems were characterized with respect to encapsulation efficiency, inclusion efficiency, swelling capacity, size and distribution size, morphology and proximate composition The systems prepared with lipid emulsions presented more homogenous distribution of the core material and the protein release was lower than the systems containing lipid microparticles (p<0,05). The systems produced with ternary mixture presented the lowest protein release (5%). In the second part of the study the hydrophilic compounds incorporated as emulsions in the model systems were substituted by two experimental diets. The preparation of the diets included the extraction and characterization of fish fat, fish protein concentrate and Artemia nauplii. These materials were used in the diet formulations. Those diets were improved with respect to protein content, lipids, and dry matter, trying to mimetic the nutritional profile of Artemia náuplii and characterized as a function of composition, size, swelling, morphology, encapsulation efficiency and protein release. In vivo assay were made in order to evaluate diets efficiency and in vitro assay to evaluate protein digestibility. The content of protein, lipid and dry matter of the microparticles similar as observed for Artemia náuplii (live feed). Integrity was kept after dryer showed low protein release during the release period studied (10% to dry microparticles). In vivo and in vitro assays showed razonable digestibility, growing rates and also survival rates. However the microparticles could not sustained the same growing profile obtained when Artemia was used as the source of feed (alive feed). The results indicated that these particles can be improved as substitutes for live food (Artemia) / Doutorado / Consumo e Qualidade de Alimentos / Doutour em Alimentos e Nutrição

O objetivo desta Tese foi a produção, caracterização e avaliação da digestibilidade in vitro de géis biopoliméricos de isolado proteico de soro de leite (IPSL) e goma xantana (GX), incorporados por dispersão de micropartículas lipídicas sólidas (MLS) encapsulando curcumina. Formulações de MLS compostas poróleo de babaçu e triestearina com 2 % (MLS-2) ou 4 % (MLS-4) de tensoativos (Tween 60 e Span 80) encapsulando curcumina foram produzidas por ultra-agitação e a estabilidade das MLS foram avaliadas durante 60 dias. As formulações de géis foram definidas variando-se as concentrações de IPSL, GX e de sal e o processo de gelificação foi conduzido a 90 °C por 30 min. As propriedades mecânicas e reológicas dos géis sem sal incorporados de diferentes concentrações de MLS-2 ou MLS-4 foram avaliadas. Os géis não-carregados e carregados com MLS e diferentes concentrações de sais foram caracterizados quanto às propriedades mecânicas, reológicas, umidade expremível (UE), microscopia eletrônica de varredura (MEV) e confocal (CLSM) e estabilidade da cor. Ensaios de digestibilidade in vitro foram conduzidos para os géis carregados e não-carregados e para as dispersões de MLS usando dois protocolos - Infogest e redução gradual do pH da fase gástrica (RG) - e a bioacessibilidade do curcuminoide foi avaliada. As dispersões MLS-2 e MLS-4 permaneceram estáveis durante 60 dias de armazenamento para as características avaliadas. As composições dos géis escolhidos foram de 12 % de IPSL, 0,2 % GX, nas condições sem sal; 0,1 M NaCl ou 0,1 M CaCl2. As diferentes concentrações de MLS incorporadas ao gel influenciaram de maneiras distintas as propriedades mecânicas e reológicas e a concentração de 88 % m/m de MLS foi escolhida para ser incorporada aos géis. As imagens de MEV e de CLSM mostraram géis do tipo particulado, porosos eas MLS homogeneamente distribuídas na rede gelificada. A rigidez do gel carregado diminui com presença de sal e aumentou com a incorporação de MLS-2 no ensaio mecânico, mas nos ensaios reológicos a presença das MLS e a de sal não contribuíram com o reforço estrutural do gel. Os parâmetros colorimétricos dos géis carregados permaneceram estáveis após 30 dias. O processo de digestibilidade in vitro das amostras foi influenciado pela heterogeneidade da matriz gelificada, pela presença de sal, pelo tipo de MLS incorporada ao gel e pelo o protocolo utilizado nos ensaios. Os géis carregados apresentaram maior liberação de ácidos graxos livres (AGL) durante a fase duodenal que as dipersões de MLS, mas os resultados de bioacessibilidade do curcuminoide foram pouco afetados pela incorporação das MLS à matriz gelificada, independemente do protocolo. Os géis carregados com MLS-4 apresentaram resultados superiores de liberação de AGL e de bioacessibilidade, nos dois protocolos. Porém, o tipo de protocolo teve pouco impacto sobre os comportamentos das amostras ao final da fase intestinal. Concluiu-se que a composição de tensoativos na MLS pode influenciar tanto nas propriedades mecânicas e reológicas quanto no processo de digestão simulada dos géis carregados. Após os resultados de digestibilidade, definiu-se a MLS-4 a formulação mais indicada para ser incorporada aos géis biopoliméricos, os quais podem ser empregados na produção de alimentos carregadores de compostos bioativos lipofílicos. / This Thesis aimed the production, characterization and in vitro digestibility evaluation of biopolymer gels of whey protein isolate (WPI) and xanthan gum (XG) incorporated with solid lipid microparticles (SLM) encapsulating curcumin. Two formulations of SLM composed by babacu oil and triestearin with 2 % (SLM-2) or 4 % (SLM-4) surfactants (Tween 60 and Span 80) and 0.03 % of curcumin were producted by ultra turrax and the stability of SLM for 60 days were evaluated. Gels formulations were determined using different compositions of WPI, XG and salt and gelation processes were carried out at 90 °C for 30 min. Mechanical and rheological properties of gels filled with different concentrations of SLM-2 and SLM-4 were investigated. Non-filled gels and gels filled with SLM-2 and SLM-4 and different salt concentrations were characterized as the mechanical and rheological properties, water holding capacity, scanning electron (SEM) and confocal (CLSM) microscopy and color stability. in vitro digestibility assays were carried out for filled and non-filled gels and for SLM dispersions using two protocols - Infogest and gradual reduction of gastric pH (GR) - and curcumin bioaccessibility were accessed. SLM-2 and SLM-4 dispersions were stable during 60 days for the parameters investigated. The chosen gels compositions were 12 % WPI, 0.2 % XG and without salt, 0.1 M NaCl or 0.1 M CaCl2. Filled gels with different concentrations of SLM showed different mechanical and rheological behavior and the concentration of 88 % m/m SLM was chosen to be incorporated in gels. SEM and CLSM imagens showed particulate and porous gels structures with SLM homogeneously distributed in the gelled network. Salt decreased and SLM-2 increased gel rigidity, but in rheological measurements salt and SLM did not reinforce gels. The colorimetric parameters of filled gels were stable after 30 days. The in vitro digestibility processes of samples were affected by gelled matriz heterogeneity, salt concentration, type of SLM and by the digestibility protocol. Filled gels released more free fat acids (FFA) during duodenal step than SLM, but the incorporation of SLM in gelled matrix did not promote higher curcumin bioaccessibility results, independently of the protocol. Gels filled with SLM-4 showed the highest FFA release and curcumin bioaccessibility results for both protocol. However, the type of protocol did not affect the samples behavior at the end of intestinal step. It was concluded that the different surfactant concentrationsin SLM could change the mechanical and rheological properties as well as the in vitro digestion processes of filled gels. After digestion results, SLM-4 was the most indicated to be incorporated in mixed gels. The incorporation of SLM in mixed biopolymer gels could be used for production of foods as lipophilic bioactive compound carriers.

Orientador: Lireny Aparecida Guaraldo Gonçalves / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-19T08:12:03Z (GMT). No. of bitstreams: 1 DiazGamboa_OscarWilfredo_D.pdf: 1876345 bytes, checksum: 56e9f8f4f7650f56a9f3dd8d60c68957 (MD5) Previous issue date: 2011 / Resumo: O presente projeto visou estabelecer condições ideais de obtenção de um produto que poderá ser disponibolizado para comercialização no Brasil tendo como foco a encapsulação para aplicação na área alimentícia. Tocoferóis são antioxidantes naturais que podem ser utilizados para enriquecimento de alimentos. Contudo há a necessidade de proteção desse agente ativo por métodos especiais como a microencapsulação. No presente estudo foram desenvolvidos sistemas compostos por micropartículas obtidas por ¿spray chilling" utilizando lipídios interesterificados sem isômeros trans com óleo de soja totalmente hidrogenado na relação de 70:30% m/m respectivamente, com ponto de fusão na faixa de 40-65 °C para formação das matrizes. Alfa-tocoferol foi utilizado como principio ativo a ser encapsulado. Para a obtenção das micropartículas matrizes lipídicas foram fundidas e mantidas em banho à temperatura de 65 °C . O a-tocoferol foi adicionado nas misturas lipídicas que em seguida foram homogeinizados em ultraturrax. As soluções foram pulverizadas em atomizador duplo fluido aquecido também a 65 °C e pressão de ar de 0,25 MPa, com a a tomização efetuada dentro de uma câmara resfriada a 10 °C. Foi realizado um p lanejamento DCCR (Delineamento Central Rotacional) com 2 variáveis independentes: a velocidade de homogeneização (3000 a 11000 rpm) e a concentração de tocoferol (5-25 g/100g). A quantificação do princípio ativo encapsulado foi realizada utilizando técnica isocrática de cromatografia líquida de alta eficiência (CLAE). Os tratamentos foram caracterizados em relação à eficiência de encapsulação, morfologia, tamanho médio, estabilidade e liberação do princípio ativo. Para o estudo da estabilidade os tratamentos foram submetidos à estocagem por 180 dias em três diferentes temperaturas (ambiente 25°C ±5 °C, em estufa 22 °C e freezer a -18°C). Medidas de difração de raios-X (0 , 60, 120, 180 dias) e medidas calorimétricas (tempo zero) foram efetuadas. Foi realizada a incorporação das micropartículas em um produto comercial (Iogurte), que foi avaliado sensorialmente. De forma geral, as partículas lipídicas obtidas neste trabalho apresentaram bons resultados quanto à eficiência de encapsulação e apresentando forma esférica, com paredes contínuas, porém rugosas. Os termogramas, obtidos por calorimetria diferencial de varredura (DSC), em tempo zero, não apresentaram diferenças entre os ensaios. Os difratogramas foram muito semelhantes entre os tratamentos e constatou-se a presença de 3 picos principais que parecem estar associados à forma polimórfica ß. As micropartículas de liberação apresentaram boa capacidade de controle da liberação do composto ativo, mostrando-se potenciais para o uso futuro na indústria de alimentos. Finalmente os resultados da análise sensorial de aceitação não foram estatisticamente significativos para os atributos avaliados / Abstract: This project aimed to establish optimal conditions for obtaining a product that will be commercially available in Brazil, focusing on encapsulation for use in the food industry. Tocopherols are natural antioxidants that can be used for food enrichment.However, there is the need for protection of active agent by special methods, such as microencapsulation. The present study developed systems composed of microparticles obtained by "spray chilling" using an interesterified fat with no trans isomers with fully hydrogenated soybean oil in the ratio of 70:30% w/w respectively, with a melting point in the range of 40-65°C for the formation of matrices to encapsulate a-tocopherol as active principle.To obtain small particles a lipid matrices were melted in a water bath at a temperature of 65°C. The a-tocopherol was added to the lipid mixtures and then homogenized in Ultra Turrax for 5 min.The solutions were sprayed in double-fluid atomizer also heated to 65°C and air pressure of 0.25 MPa, the atomization performed inside a chamber cooled to 10°C. Were conducted a CCR design (Central Compo site Rotational Design) with two independent variables: the speed of homogenization (3000 to 11000 rpm) and the concentration of tocopherol (5-25 g/100 g).The quantification of the active ingredient encapsulated was performed using isocratic HPLC technique. The treatments were characterized with respect to encapsulation efficiency, morphology, average size and stability of the microparticles and release of active ingredient.For the stability study treatments were subjected to storage for 180 days at three different temperatures (ambient 25°C ± 5°C , 22°C in an oven and freezer -18°C).Since x-rays diffraction measurements (0, 60, 120, 180 days) and calorimetric measurements (time zero) were made. Were performed the incorporation of the microparticles in a commercial product (yogurt), which was evaluated using the sensory evaluation.In general, the lipid particles studied in this work showed good results in terms of encapsulation efficiency showing spherical shape, with solid rough walls. The thermograms obtained by DSC at time zero did not differ between trials.The XRD patterns were very similar among treatments and found the presence of three major peaks that are associated with the polymorphic form ß. The microparticles showed good ability to control the release of the active principle, showing potential for future use in the food industry. Finally the results of an smooth sensory acceptance indicated that the tested samples did not differ statistically from the standard sample for evaluated attributes / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos

Nos travaux montrent qu’au cours de la fibrillation atriale (FA), les microparticules (MP) reflètent et contribuent à un état d’hypercoagulabilité et pro-inflammatoire. Leurs concentrations similaires dans les deux oreillettes de patients en FA témoignent d’une absence de différence de statut pro-thrombotique entre ces deux cavités cardiaques. Au cours des procédures d’ablation de FA, les concentrations de MP évoluent parallèlement à l’augmentation de l’activation cellulaire et plaquettaire. Nous avons également montré dans l'altération tissulaire des oreillettes en FA, l'importance de la sénescence qui évolue avec la progression du trouble du rythme. Nous avons caractérisé un modèle cellulaire de sénescence réplicative de cellules endothéliales auriculaires de porc permettant d'identifier l'apparition d'un phénotype pro-thrombotique, pro-inflammatoire, pro-adhésif et de mieux comprendre la physiologie de la cellule endothéliale atriale sénescente et le rôle majeur du système rénine-angiotensine dans ces mécanismes. / Our data evidence that during atrial fibrillation (AF), microparticles (MP) contribute to an enhanced hypercoagulable and pro-inflammatory state. Similar concentrations of MP measured in left and right atria of AF patients highlight the absence of chamber-specific enhanced thrombogenic status. During AF ablation procedures, MP concentrations progress in parallel with cell and platelet activation. We also showed that AF progression is strongly related to human atrial senescence burden pointing toward a possible network that links in human atrium, senescence burden, endothelial dysfunction, thrombogenicity and atrial remodeling. We also developed a model of left atrium endothelial cell replicative senescence providing compelling evidences indicating that atrial endothelial senescence promotes thrombogenicity, inflammation and proteolysis. These data underline the major role of renin-angiotensin system in endothelial atrial cell senescence.

L’arthrose et la polyarthrite rhumatoïde sont deux atteintes ostéo-articulaires qui affectent les tissus cartilagineux et osseux. Elles représentent un réel problème de santé publique de par leur prévalence et le manque de traitements curatifs. Des approches innovantes de thérapie cellulaire sont en cours d’évaluation dans ces maladies. Elles sont basées sur l’utilisation des cellules souches mésenchymateuses (CSM), qui possèdent des propriétés immunosuppressives et régénératrices, et pourraient apporter de nouveaux espoirs aux patients. Ces cellules sécrètent des vésicules extracellulaires, moyen de communication intercellulaire puissant, qui sont classées en 3 populations : exosomes, microparticules (MPs) et corps apoptotiques. Dans cette étude, nous proposons d’étudier et de comparer les effets in vitro et in vivo des exosomes et des MPs sécrétés par les CSMs. Nous montrons pour la première fois que les exosomes et les MPs de CSMs exercent in vitro des effets anti-inflammatoires, anti-apoptotiques et chondroprotecteurs similaires. In vivo, seuls les exosomes exercent un effet thérapeutique dans un modèle expérimental de polyarthrite rhumatoïde. En revanche dans un modèle expérimental d’arthrose, les exosomes et les MPs protègent le cartilage et l’os de la dégradation et ce, de manière équivalente. Cette étude est la première preuve de concept que des exosomes et/ou des MPs isolés de CSMs ont un effet thérapeutique dans des maladies rhumatismales. / Osteoarthritis and rheumatoid arthritis are osteoarticular diseases affecting primarily cartilage and bone. They are a high public health problem because of their prevalence and absence of curative treatment. Innovating cell therapy approaches are being evaluated in the clinic for treating these diseases. They rely on the use of mesenchymal stem cells (MSCs), which display immunosuppressive and regenerative functions and could provide new hope for patients. These cells release extracellular vesicles, which are very powerful tools for intercellular communication and are classified into 3 populations: exosomes, microparticles (MPs) and apoptotic bodies. In the present study, we characterize and compare the in vitro and in vivo effects of MSC-derived exosomes and MPs. We show for the first time that exosomes and MPs exert in vitro anti-inflammatory, anti-apoptotic and chondroprotective functions. In vivo, only exosomes exert a therapeutic effect in an experimental model of inflammatory arthritis. However in a model of osteoarthritis, both exosomes and MPs protect cartilage and bone from degradation, in a similar fashion. This study provides the proof-of-concept that MSC-derived exosomes and/or MPs exert a therapeutic effect in rheumatic diseases.

Les études de libération de substance active à partir de système polymériques tels que des microparticules à base d’acide poly(lactique-co-glycolique) (PLGA) ont été largement explorées au cours de ces dernières décennies . L’objectif principal de ce travail consiste à mieux comprendre les mécanismes de transport de masse contrôlant la libération de substance active à partir des microparticules de PLGA. Un nouvel aperçu devait être acquis sur la base de suivi expérimental de la cinétique de gonflement de microparticules. Dans un premier temps, des microparticules à base de PLGA chargées de différents types de substances actives (acide, base et neutre), tels que kétoprofen, prilocaine base libre et dexamethasone ont été préparées par simple émulsion (huile dans eau) en utilisant une méthode d'extraction/évaporation du solvant. Les microparticules obtenues avaient des taux d’efficacité d’encapsulation qui sont variables selon la substance active utilisée. Une caractérisation des propriétés clés des microparticules obtenues a été réalisée en utilisant différentes techniques (microscopie optique, microscopie électronique). La chromatographie par permeation de gel a été utilisé pour déterminer le poids moléculaire du PLGA après exposition des microparticules au milieu de libération à différents temps afin d’évaluer la cinétique de dégradation du polymère. La diffraction des rayons X et la calorimétrie différentielle à balayage étaient utilisés pour étudier l’état physique du polymère, de la substance active pure ainsi que les microparticules chargées en substance active. Les études de libération ont montré deux types de profils de libération : un profil tri-phasique et un profil plus ou moins mono-phasique. Le profil tri-phasique observé est constitué de trois phases : une phase de libération initiale rapide suivie d’une libération constante qui est suivie ; à son tour ; par une seconde phase de libération rapide. En revanche, les différentes phases étaient difficilement distinguées pour le deuxième type de profil obtenu, du fait de la libération rapide de substance active ce qui permet de dire que les profils obtenus étaient plus ou moins mono-phasique. L’élucidation des mécanismes de libération de substance active était basée sur le suivi expérimental de la cinétique de gonflement des microparticules. Comme pour les cinétiques de libération obtenues à partir des microparticules à base de PLGA, différentes phases peuvent être distinguées pour les profils de gonflement. Les transitions d’une phase à une autre semblent s’accorder entre le profil de libération et celui du gonflement. Ainsi, le gonflement des microparticules pourrait contribuer au contrôle de la libération de la substance active à partir des microparticules à base de PLGA. / The drug release studies from polymeric system such as Poly(lactic-co-glycolic) acid (PLGA)-based microparticles have been widely investigated during recent decades. The main objective of this work is to better understand the mass transport mechanisms controlling the drug release kinetics from PLGA microparticles. New insight was to be gained based on the experimental monitoring of the swelling kinetics of single microparticle. Initially, PLGA microparticles containing different type of drugs (acidic, basic and neutral), such as ketoprofen, prilocaine free base and dexamethasone were prepared using simple oil in water emulsion extraction/evaporation solvent technique. The characterization of the key properties of microparticles was performed using different techniques (optical microscopy, electron microscopy). The gel permeation chromatography was used to determine the molecular weight of PLGA following exposure of microparticles to the release medium at various times to assess the kinetic degradation of the polymer. The X-ray diffraction and differential scanning calorimetry were used to study the physical state of the polymer, drug and drug-loaded microparticles. Release studies have shown two types of release profiles: tri-phasic and more or less mono-phasic profile. The tri-phasic profile is composed of three phases: an initial rapid release phase followed by a constant release which is followed by a second phase of rapid release. In contrast, at the investigated higher initial drug loadings, different release phases could hardly be distinguished: The profiles were more or less mono-phasic. The elucidation of drug release mechanisms was based on the experimental results of the swelling kinetics of single microparticles. As for drug release, distinct phases can be distinguished for microparticles swelling. The transition from one phase to another seem to coincide for microparticle swelling and drug release. Thus also microparticle swelling might contribute to a significant extent to the control of drug release.

Le cancer colorectal (CRC) est le plus fréquent des cancers digestifs. Cependant, il est moins grave et moins fréquemment associé à une complication thrombo-embolique que le cancer du pancréas (PC). Les microparticules (MPs) sont de petites vésicules produites par bourgeonnement de la membrane cellulaire. Les MPs sont impliquées dans la croissance tumorale, le développement de métastases et l’activité pro-coagulante associée aux cancers. Les objectifs de ces travaux étaient d’identifier et de caractériser les différentes MPs dans le CRC and le PC, afin de décrire une signature microparticulaire, puis d’évaluer leur implication dans la survenue de complications thrombo-emboliques. Nous avons donc rapporté une signature microparticulaire spécifique dans le CRC et le PC par rapport à des pathologies bénignes colorectales et pancréatiques, ainsi que des sujets sains, que nous avons appelé “microparticulosome”. Le microparticulosome se modifie avec l’évolution de la maladie, pour se rapprocher des formes bénignes voire des sujets sains, en cas de rémission du CRC. De plus,il varie en présence ou non d'une complication thrombo-embolique. Nous avons également rapporté le cas d’un cancer du sein diagnostiqué grâce à des taux élevés de MPs exprimant la fibrine. En conclusion, les MPs pourraient constituer de nouveaux bio-marqueurs pertinents en cancérologie, dans le diagnostic, le pronostic de survie et de survenue d’une complication thrombo-embolique. La connaissance des interactions des MPs avec le microenvironnement tumoral permettra de mettre au point des thérapeutiques adaptées et efficaces dans la croissance tumorale et l’extension métastatique. / Colorectal cancer (CRC) is the most common gastrointestinal cancer. It is less serious and less frequently associated with thrombo-embolic event than pancreatic cancer (PC). Microparticles (MPs) are small vesicles produced and released by exocytic blebbing of the activated and apoptotic cell membrane from most, if not all, types of cells. They are known to be implicated in the tumor growth, the development of metastases and the cancer-associated procoagulant activity. Our objectives were to identify and to characterize the different concentrations of circulating MPs in CRC and PC, in order to describe a MPs hallmark, and to evaluate their implication in the occurrence of a venous thromboembolism. We have thus reported a specific hallmark of MPs in CRC and PC, comparing to benign colorectal and pancreatic diseases and healthy subjects, so-called the “microparticulosome”. We have observed that microparticulosome changed with the evolution of the disease, and tended to the signature observed for benign diseases or healthy subject in case of CRC remission. We also reported variations in the microparticulosome in case of an occurrence of a thrombo-embolic event.In conclusion, MPs may constitute new pertinent biomarkers in cancers, in the diagnosis, the survival prognostic and the prognostic of the occurrence of thrombo-embolic events. Understanding the interactions of MPs with tumor environment will allow to find efficient treatments against tumor growth and metastases development.

The thesis were focused on the optimalization of micromethod for isolation of DNA in quality for polymerase chain reaction (PCR) using magnetic microparticles from plant food products. There were chosen a red beetroot (fresh, frozen, dried and sterilized) for the analysis and food products containing red beetroot. Different approaches of processing of homogenates were compared and optimized. The homogenates were prepared in lysis buffer with cetyltrimethylammonium bromide (CTAB) with different amounts of NaCl with or without addition of organic extraction agents chloroform-octanol and isopropanol. Microisolation of DNA was performed using magnetic particles P(GMA). The concentration of NaCl and polyethylene glycol (PEG) 6000 in separation mixtures was tested. The influence on quantity and purity of isolated DNA was compared and the optimum amounts of NaCl in CTAB buffer and optimal concentration of PEG 6000 in separation mixtures were compared. The optimized separation mixture for the DNA isolation from red beetroot was applied to food products containing red beetroot. Amplifiability of DNA was tested in conventional PCR using specific primers for plant DNA. PCR products of length 700 bp were detected by agarose gel electrophoresis.

Different species of genus Bifidobacterium are part of human and animal intestinal flora. These bacteria have benefit effects and therefore they are used in foods and pharmaceutical products as probiotics. Cheese is now suitable as a probiotic matrix except yoghurts and fermentated milks. This diploma thesis was focused on optimalization of DNA isolation from bacteria of genus Bifidobacterium. Magnetic microparticles (P(HEMA-co¬-GMA)) were used for DNA isolation in presence of 8% polyethyleneglycol PEG 6000 and 5 M sodium chloride. Phenol extraction weas also used as an isolation method. Isolated DNA was used for amplification in domain, genus and species specific PCRs. Optimized method was tested for detection of bacteria of genus Bifidobacterium in experimentaly prepared probiotic cheeses. These cheeses contained potential probiotic bacteria from Laktoflóra collection. Bacteria were identified into species using species specific PCR. Species Bifidobacterium animalis was identified in all samples of probiotic cheeses.

The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.