A produção e liberação de substâncias vasodilatadoras pelas células endoteliais requer uma elevação sustentada na concentração intracelular de cálcio; essa elevação é consequente a um influxo de cálcio. Porém, a identidade do (s) canal (is) envolvido (s) nesse influxo ainda não foi (ram) determinada (s) conclusivamente. Existem evidências de que o gene TRPV4 (que codifica uma proteína permeável a cátions, inclusive ao cálcio) é expresso em células endoteliais. Porém, a falta de agentes que modulem especificamente a função dessa proteína não permitiu que o papel do TRPV4 no controle da função endotelial pudesse ser elucidado. Recentemente foram descritos dois novos compostos, o GSK1016790A (GSK) e o HC-067047 (HC), com ação ativadora e bloqueadora seletiva desse canal, respectivamente. Consequentemente, nesta dissertação descrevemos e interpretamos os resultados obtidos em experimentos concebidos para caracterizar o efeito do GSK1016790A (e com fins comparativos o efeito do 4PDD) em artérias isoladas de várias espécies. Para isso, empregamos anéis de artérias suspensos em cubas para órgão isolado para registro da tensão desenvolvida por esses anéis durante a contração isométrica provocada pela adição de fenilefrina; todos os experimentos foram realizados com solução de Krebs contendo diclofenaco (10 M). Inicialmente verificamos mediante imunohistoquímica a presença de imunorreatividade para o TRPV4 no endotélio da aorta torácica de rato. A adição de concentrações isoladas ou cumulativas de GSK produziu relaxamentos dependentes da concentração na aorta torácica de rato (CE50=0,5 nM; IC95%=0,35-0,72 nM; n=7); o 4PDD (1-10 µM), em concentrações isoladas, também produziu relaxamentos na aorta torácica de rato. Resultados semelhantes foram observados para o GSK na aorta torácica de coelho (CE50= 4,3 nM; IC95%=3,58-5,14 nM; n=5), de camundongo (CE50=1,4 nM; IC95%=0,85-2,24 nM; n=3) e de cobaia (CE50=0,2 nM; IC95%=0,12-0,22 nM; n=4). GSK relaxou também a aorta abdominal (CE50=6,5 nM; IC95%=3,71-11,3 nM; n=3) e a artéria femoral de coelho (CE50=17 nM; IC95%=16,8-18,7 nM; n=4); Os relaxamentos produzidos por ambas as drogas apareceram 1-2 min após a adição e atingiram o máximo em 5-8 min, foram reversíveis e não apresentaram taquifilaxia. Em todas as artérias os relaxamentos foram estritamente dependentes de endotélio e da presença de cálcio no meio extracelular. Na aorta torácica de rato, a pré-incubação com HC (5 minutos) aboliu o efeito do GSK sem afetar os relaxamentos produzidos pela acetilcolina. Em todas as artérias testadas os efeitos do GSK e do 4PDD foram revertidos completamente pelo HC (1-3 µM) ou pelo vermelho de rutênio (aorta torácica de rato e artérias de coelho, 1µM, VR). Esses resultados demonstram que os canais TRPV4 estão presentes na célula endotelial e que a sua ativação leva à produção de fatores relaxantes. Como corolário, esses resultados constituem indícios de que os canais TRPV4 podem participar da regulação da função das células endoteliais em situações fisiológicas e/ ou fisiopatológicas. / Production and release of vasodilator substances by endothelial cells require a sustained elevation of intracellular calcium which depends on calcium influx. The identity of the channels involved in this influx remains to be established. There is evidence that the TRPV4 gene (which encodes for a cation permeable channel including calcium) is expressed in endothelial cells; the lack of pharmacologic agents that selectively modulate the activity of TRPV4 channels has hindered the elucidation of its function in endothelial cells. Recently two new compounds, GSK1016790A (GSK) and HC-067047 (HC), which selectively activate or block TRPV4 channels, respectively, were described. This dissertation consists in the description and interpretation of results from experiments conceived to characterize the effect of GSK (and of 4PDD for comparison) in isolated arterial rings from several animal species. To this aim we used arterial rings mounted in isolated organ chambers; we recorded continuously the tension developed by them during isometric contractions elicited by phenylephrine (Phe); all the experiments were conducted using Krebs solution containing diclofenac (10 µM). Initially, we confirmed by immunohistochemistry the presence of anti-TRPV4 immunoreactivity in the endothelium of rat thoracic aorta. In rat thoracic aortic rings pre-constricted with Phe (0.1 µM) the addition of different concentrations of GSK (either single or cumulative concentrations) caused concentration-dependent relaxations (EC50=0.5 nM, 95%CI=0.35-0.72 nM, n=7); 4PDD (in single concentrations) also caused relaxations of rat thoracic aortic rings. Similar results were observed for GSK in thoracic aortic rings from rabbit (EC50=4.3 nM, 95%CI=3.58-5.14 nM, n=5), mouse (EC50=1.4 nM, 95%CI=0.85-2.24 nM, n=3) and guinea-pig (EC50=0.2 nM, 95%CI=0.12-0.22 nM, n=4). GSK also produced relaxations of rings from rabbit abdominal aorta (EC50=6.5 nM, 95%CI=3.71-11.3 nM, n=3) and femoral artery (EC50=17 nM, 95%CI=16.8-18.7 nM, n=4). Relaxations caused by both GSK and 4PDD started 1-2 min after their addition and reached a steady-state in 5-8min; they were reversible after washing-out and did not exhibit tachyphylaxis. In all the studied arteries GSK or 4PDD induced- relaxations were strictly endothelium- and extracellular calcium- dependent. Pre-incubation of rat thoracic aortic rings with HC (1 µM for 5min) abolished the effect of GSK but did not affect relaxations elicited by Ach (1 µM). In all the arterial rings HC (1-3 µM) also completely reverted the relaxations caused by GSK or 4PDD; in rabbit and rat thoracic aortic rings ruthenium red (1 µM) also completely reverted the relaxations caused by GSK or 4PDD. The present findings showing that TRPV4 channels are present in endothelial cells and that their activation results in the production and release of relaxing factors constitute an indication that TRPV4 channels could be involved in the regulation of endothelial cell functions under physiological or patho-physiological conditions.

Nas últimas décadas, têm aumentado substancialmente a quantidade de pesquisas focando no desenvolvimento e caracterização de materiais biodegradáveis, particularmente, filmes comestíveis. O uso de polímeros de fontes renováveis, preparados a partir de produtos vegetais, vem ganhando importância nessa abordagem. O concentrado de proteína de soja e amido de mandioca podem ser considerados uma alternativa aos polímeros petroquímicos. O processamento pela radiação ionizante pode ser empregado para a modificação de polímeros e macromoléculas, resultando em novos materiais com grandes perspectivas de utilização industrial. A indústria de alimentos, uma das indústrias tradicionalmente mais inovadoras, exige o desenvolvimento constante de novos produtos. A capacidade de proteínas e polissacarídeos de formar filmes, amplamente conhecida, é um ponto de partida para o desenvolvimento de novos materiais que atendam os variados requerimentos dessa pungente indústria. Neste trabalho elaboraram-se filmes a base de fécula de mandioca e proteína isolada de soja em duas proporções diferentes e posteriormente irradiados e analisados quanto às suas propriedades mecânicas, cor, absorção de água, permeabilidade ao vapor de agua, análise térmica TGA e DSC entre outros. Os filmes tornaram-se aparentemente mais solúveis e menos resistentes a perfuração com o aumento da dose de radiação aplicada. Com relação às propriedades térmicas observou-se que os filmes com maior proporção de proteína são mais resistentes. Os filmes apresentaram-se menos permeáveis na dose de 40 kGy, e, com relação a absorção de água, esta foi reduzida em função da dose de radiação. Filmes com boa resistência ao vapor de água e com reduzida absorção podem ser considerados adequados para embalagens de alimentos. A radiação mostrou ser uma ferramenta conveniente na modificação de materiais poliméricos principalmente para elaboração de filmes solúveis onde esta é uma nova tendência para embalagens bioativas. / In recent decades, there has been a substantial increase in the amount of research focusing on the development and characterization of biodegradable materials, particularly edible films. The use of polymers from renewable sources, prepared from plant products, has gained importance in this approach. Soy protein concentrate and cassava starch may be considered an alternative to petrochemical polymers. Processing by ionizing radiation can be used for the modification of polymers and macromolecules, resulting in new materials with great prospects of industrial use. The food industry, one of the traditionally most innovative industries, requires the constant development of new products. The widely known ability of film forming proteins and polysaccharides is a starting point for the development of new materials that meet the varying requirements of this pungent industry. In this work, films based on manioc starch and isolated soy protein were prepared in two different proportions and later irradiated and analyzed for their mechanical properties, color, water absorption, water vapor permeability, TGA and DSC thermal analysis between others. The films became apparently more soluble and less resistant to drilling with the increased radiation dose applied. Regarding the thermal properties, it was observed that the films with greater protein orientation are more resistant. Properties such as water vapor permeability and water absorption, the films were less permeable at the 40 kGy dose. With regard to water absorption, it was reduced as a function of the radiation dose. Films with good resistance to water vapor and with low absorption are considered efficient for food packaging. Radiation has proven to be a convenient tool in the modification of polymeric materials mainly for the production of soluble films where it is a new trend for bioactive packaging.

The ship emission already occupy the eighth position in the world biggest emitters ranking. This happens because the ship operations have a huge demand variation therefore in order to reduce the ship emissions is required an efficient operation of the generators. This work aims at integrating advanced storage systems into the operation of diesel generators. The variation of the operation point has a direct interference on the emissions and on the diesel consumption, this variation is allowed through the frequency and voltage control. The use of lithium batteries for various operation points of the generators is analyzed. The use of an energy storage system allowed the operation of the generators in a better operation point therefore there was a reduction in diesel consumption and in CO2 emissions when the diesel generators. The main result of this work could also shed light in the operation of isolated power systems equipped with advanced storage systems and diesel generators. / As emissões dos navios já ocupam a oitava posição entre os países com maior emissão no mundo. Isso pode ser explicado pelo fato de que as operações dos navios têm uma grande variação de demanda de potência, com isso a operação inteligente dos geradores a diesel é fundamental para a redução das emissões. A abordagem desenvolvida nesse trabalho integra o uso de sistemas de armazenamento avançados na operação dos geradores a diesel. A variação do ponto de operação dos geradores a diesel interfere diretamente no consumo e nas emissões, essa variação só é possível por meio do controle de frequência e tensão providos pelo sistema de armazenamento de energia. Nesse trabalho foram analisados o uso de baterias de lítio para diferentes pontos de operação do gerador a diesel. O uso das baterias possibilitou a operação dos geradores num melhor ponto de carga com isso houve uma redução das emissões e do consumo de combustível. Os resultados encontrados nesse trabalho podem ser extrapolados qualitativamente para outros sistemas de potência offshore, como plataformas de petróleo e de perfuração, que operem com sistemas de baterias avançadas e geradores a diesel.

O imprinting genômico é um processo regulado epigeneticamente que faz com que os alelos sejam expressos de acordo com a sua origem parental. No cromossomo 11 (11p15.5), existem duas regiões controladoras de imprinting (ICR1 e ICR2), que controlam a expressão de genes marcados (imprinted). Os padrões de metilação dessas regiões podem ser alterados pela dissomia uniparental (DUP), que ocorre quando parte de ou um cromossomo inteiro do mesmo par de homólogos é herdado de somente um genitor. Erros mitóticos podem gerar mosaicismo com uma linhagem de células com DUP e a outra biparental. As síndromes de Silver-Russell (SSR) e Beckwith-Wiedemann (SBW) são doenças de alterações do imprinting genômico, envolvendo os cromossomos 7 (SSR) e 11 (SSR e SBW). A Hemihiperplasia Isolada (HHI) parece corresponder a uma forma mais leve da SBW.. No presente trabalho, foi realizada uma varredura in silico para busca de novos microssatélites nos cromossomos 7 e 11, e selecionados seis do tipo tetra ou pentanucleotídeos, no cromossomo 7, e 12, no cromossomo 11. O perfil de metilação nas ICRs foi verificado por três técnicas distintas: MS-MLPA, DESM-RT e por uma nova estratégia desenvolvida neste trabalho denominada DESM-QFPCR. Foram avaliados 32 pacientes com SBW, 16 HHI, 20 com SSR e seus pais, quando disponíveis, além de um paciente com fenótipo aparentemente normal com cariótipo 46,XX/46,XY e cuja placenta apresentou displasia mesenquimal placentária (DMP) a qual está associada à SBW. Os novos marcadores apresentaram alta taxa de heterozigose (média de 70%), e ausência das características indesejáveis dos dinucleotídeos predominantemente utilizados para detecção de DUP. Seis marcadores estão entre genes controlados pelas ICRs 1 e 2. A DUP paterna do cromossomo 11 (DUPpat Cr11), sempre restrita a 11p15.5, foi responsável por 13% dos casos de HHI e 19% dos de SBW. As alterações estruturais foram confirmadas por minissequenciamento quantitativo de SNPs e por MS-MLPA. Um paciente apresentou duplicação paterna abrangendo ambas as ICRs. Uma deleção não descrita anteriormente no gene CDKN1C foi observada em uma paciente e sua mãe. Para os pacientes com DUPpat Cr11, foram investigados microssatélites em 13 autossomos e nos cromossomos sexuais para detecção de mosaicismo global. Apenas o paciente com DMP apresentou mosaicismo [células androgenéticas (25-30%) e biparentais], sugerindo evento de dupla fertilização. Nos pacientes com SSR, foi observada hipometilação na ICR1 em 25% dos casos. Para a SBW, foi observada hipermetilação na ICR1 e hipometilação na ICR2 em 6% e 42% dos casos, respectivamente. Os casos com DUPpat Cr11 apresentaram alteração de metilação em ambas as ICRs. As frequências de alterações (epi) genéticas encontradas foram semelhantes às previamente descritas na literatura para as SBW, SSR e HHI. Neste trabalho, foi desenvolvida uma nova técnica para estudo de metilação do DNA de ICRs e testados marcadores microssatélites inéditos na região 11p15, que quando comparados com metodologias mais tradicionais de avaliação, como DESM-RT e MS-MLPA, mostraram elevada correlação dos resultados. Os achados mostram a complexidade da etiologia das doenças estudadas no presente trabalho e os dados moleculares serão imprescindíveis para o aconselhamento genético adequado para cada caso em particular e suas famílias. / Genomic imprinting is a epigenetically regulated process where the alleles are expressed in terms of their parental origin. On chromosome 11 (11p15.5) there are two regions controlling imprinting (ICR1 and ICR2), which control imprinted gene expression. The methylation patterns in these regions may be altered by uniparental disomy (UPD), which occurs when part or whole chromose is inherited from only one parent. Mitotic errors can lead to mosaicism with a cell line with DUP and other, biparental. The Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) are diseases of abnormal genomic imprinting, involving chromosomes 7 (SSR) and 11 (SRS and BWS). The Isolated Hemihiperplasia (IHH) seems to correspond to a milder form of the SBW. In the present study, we performed an in silico scan to search for new microsatellites on chromosomes 7 and 11, and selected six tetra- and/or pentanucleotides on chromosome 7, and 12 on chromosome 11. The pattern of methylation in ICRs was verified by three different techniques: MS-MLPA, DESM-RT and a new strategy developed in this work called DESM-QFPCR. We evaluated 32 patients with BWS, HHI 16, with 20 SSR and their parents, when available, and one patient with apparently normal phenotype with karyotype 46, XX/46, XY and whose placenta showed placental mesenchymal dysplasia (PMD) which is associated with SBW. The new markers showed a high heterozygosity rate (average 70%), and absence of undesirable characteristics of dinucleotides, predominantly used for detection of DUP. Six markers spans genes controlled by the ICRs 1 and 2. The paternal UPD for chromosome 11 (UPDpat Cr11), all restricted to 11p15.5, was responsible for 13% of cases of HHI and 19% of the SBW. Structural changes were confirmed by quantitative SNaPshot sequencing of SNPs and MS-MLPA. One patient had paternal duplication encompassing both ICRs. A not previously described deletion in the gene CDKN1C was observed in one patient and her mother. For patients with DUPpat Cr11, microsatellites were investigated in 13 autosomes and sex chromosomes to detect wide mosaicism. Only patients with DMP showed mosaicism [androgenetic cells (25-30%) and biparental], suggesting double fertilization. In patients with SRS, ICR1 hypomethylation was observed in 25% of cases. For BWS, ICR1 hypermethylation and in ICR2 hypomethylation were observed 6% and 42% of cases, respectively. All cases with UPDpat Cr11 presented abnormal methylation in both ICRs. The (epi) genetic change frequencies were similar to those previously described in the literature for BWS, SRR andIHH. In the present work, we developed a new technique to study DNA methylation of ICRs and tested novel microsatellite markers in the 11p15 region, which showed high correlation of results, when compared with more traditional methods such as RT-DESM and MS-MLPA. The results show the complex etiology of these diseases and the molecular data are essential for appropriate patient and families genetic counseling.

Nesta tese abordaremos dois tipos de problemas relacionados aos célebres Teorema da Fibração de Milnor e Teorema da Fibração de Milnor-Lê para o caso real com valores críticos não isolados. Primeiramente, asseguramos fibrações do tipo Milnor-Lê para F : (Xm, 0) &rarr; (Yn, 0), germe de aplicação subanalítico com X e Y espaços subanalíticos sobre C \\ {0} uma curva subanalítica conexa em Y e sobre um subespaço analítico suave W &sub; Y de dimensão p, n &ge; p &ge; 2, sob algumas condições. Em particular, mostramos a existência das fibrações sobre o discriminantes de germe de aplicações subanalíticos, caso esse ainda não estudado na literatura, normalmente o conjunto dos valores críticos são desconsiderados. Finalizando nossa análise da categoria subanalítica, certificamos que existe a fibração de Milnor-Lê para f : (X, 0) &rarr;(Rp, 0), com dimensão de X maior que p &ge; 2, subanalítica e X subanalítico com valores críticos não isolados, definindo d-regularidade. Abordamos estes problemas utilizando resultados de campos de vetores rugosos. Em uma segunda etapa apresentamos um novo critério necessário e suficiente para verificar a importante propriedade de transversalidade de um germe de aplicação real f de classe Cl, l &ge; 1. Fazendo uso também de uma recente ferramenta desenvolvida, a D-regularidade, verificamos condições para a existência das fibrações do germe de aplicação &Psi; F, X : (Cn, 0) &rarr; (C, 0) não holomorfo, dado por &Psi; (z, z&#772;) = &Sigma;nj=1 kjtjzj a<sub<jzj bj, aj, bj &ge; 0 com aj = bj para pelo menos um j e aj &ne; bj para ao menos um j, com j = 1, ... , n. Observamos que &Psi;F, X são polinômios homogêneos pesados mistos com R+ -ação. Consideramos &Psi;F, X : (R2n ,0) &rarr; (R2, 0) germe de aplicação analítico real. Estudamos a topologia dessas fibrações nos reais, constatando que o discriminante tem dimensão 1 e por isso tem ambas as fibrações conhecidas. Por fim exibimos um homeomorfismo entre as fibras dos valores regulares e dos valores críticos. / In this thesis two types of problems related to the famous Milnor Fibration Theorem and Milnor-Lê Fibration Theorem for the real case with non-isolated critical values will be addressed. Primarily, we assure the fibrations of type Milnor-Lê for the germ F : (X, 0) &rarr; (Y, 0) subanalytic with X and Y subanalytic spaces on C \\ {0} a subanalytic connected curve in Y and over a smooth analytical subspace W &sub; Y of dimension p, n &ge p &ge; 2, under some conditions. In particular, we show the existence of the fibrations about the discriminants of subanalytical map-germ, if this not been studied in the literature, usually the set of critical values are disregarded. Finalizing our analysis of this subanalytic category, we certify that there exist the fibrations of type Milnor-Lê to f : (X, 0) &rarr; (Rp, 0), with dimension of X greater than p &ge; 2, subanalytic and X subanalytic with non-isolated critical values, setting d -regularity. We address these problems using results of the rugose vector fields. In a second part, we present a new necessary and sufficient criterion to verify the important transversality property of a real map-germ f of class Cl, l &ge; 1. Using a recent developed tool, D-regularity, we verify conditions for the existence of the fibrations of map-germ &Psi; F, X : (Cn, 0) &rarr; (C, 0) non holomorphic, given by &Psi; (z, z&#772;) = &Sigma;nj=1 kjtjzj ajzb<sup<j, aj, bj &ge; 0 with aj = bj for at least one j and aj &ne; bj for at leeast one j = 1, ..., n. We note that &Psi; F, X are mixed weighted homogeneous polynomials with R+-action. We consider &Psi;F, X : (R2n, 0) &rarr; (R2, 0) real analytic map-germ. We studied the topology of these fibrations, noting that the discriminant has dimension 1 and therefore has both the fibrations known. Lastly we show a homeomorphism between the fibers of the regular values and the critical values for a case special this family.

La cryoconservation de cortex ovarien est la seule technique envisageable pour les patientes pré-pubères et les femmes dont la pathologie nécessite l’administration d’un traitement hautement gonadotoxique dont l’initiation ne peut être différée. L’autotransplantation est jusqu’à présent la seule méthode disponible de réutilisation du tissu ovarien cryoconservé, et a permis d’obtenir plus de 130 naissances dans le monde, dont trois au CHRU de Besançon. Toutefois, en cas de pathologie néoplasique à risque de localisation métastatique ovarienne, cette technique peut présenter un risque de réintroduction de cellules malignes susceptibles d’être présentes dans le greffon. Des méthodes alternatives à l’autogreffe de tissu ovarien cryopréservé fondées sur l’utilisation de follicules ovariens isolés sont actuellement en développement. L’objectif de cette thèse a été de développer un protocole permettant l’isolement et la qualification de follicules ovariens qui pourront être utilisés en thérapie cellulaire à usage humain. Dans un premier temps, une validation de la technique d’isolement de follicules ovariens a été réalisée à partir de la dissociation de fragments de cortex ovarien, issus de patientes ayant subit une résection percœlioscopique, à l’aide d’une collagénase NB6 produite selon les bonnes pratiques de fabrication. Les follicules ainsi obtenus ont été analysés en termes de viabilité (immédiate et après culture in vitro), rendement, morphologie et état prolifératif. Dans un deuxième temps, la sécurité carcinologique des suspensions folliculaires obtenues après isolement a été évaluée par cytométrie en flux multicouleurs à l’aide d’une modélisation ayant consisté en la contamination de suspensions folliculaires avec des cellules leucémiques issues de patients souffrant de leucémies aigües myéloïdes (LAM) ou lymphoblastiques (LAL) d’immunophénotype connu. La collagénase NB6 a permis l’isolement d’un grand nombre de follicules vivants, principalement au stade primordial, et dont la majorité était non activée, même après trois jours de culture in vitro dans un gel de fibrine. La technique d’isolement suivie de trois lavages a permis d’éliminer les cellules leucémiques préalablement ajoutées aux suspensions folliculaires dans 23 cas sur 24, sans endommager les follicules isolés. La cytométrie en flux multicouleurs est une technique d’analyse efficace pour évaluer la contamination, par des cellules leucémiques, de suspensions contenant des follicules ovariens isolés. Le protocole d’isolement de follicules ovariens humains, ayant été réalisé avec la collagénase NB6 de grade clinique, peut être envisagé pour une reconstruction ovarienne à visée thérapeutique humaine. / The cryopreservation of ovarian cortex is the only technique available for prepubertal girls and women when their pathology requires the administration of a highly gonadotoxic treatment whose initiation cannot be delayed. Autotransplantation has so far been the only available method to re-use cryopreserved ovarian tissue, and has resulted in more than 130 births worldwide, including three at the Besançon Hospital. However, in cases of neoplastic disease with a risk of ovarian metastatic localization, this technique may present a risk of reintroducing malignant cells likely to be present in the graft. Alternative methods to cryopreserved ovarian tissue autograft based on the use of isolated ovarian follicles are currently under development. The aim of this thesis was to develop a protocol allowing the isolation and the qualification of ovarian follicles that can be used in cell therapy for human purposes. In a first step, a validation of the technique to isolate ovarian follicles was carried out from the dissociation of cortical ovarian fragments, taken from patients undergoing a laparoscopic ovarian drilling, using collagenase NB6 which is produced according to good manufacturing practices. Follicles thus obtained were analyzed in terms of viability (before and after in vitro culture), yield, morphology and proliferative state. In a second step, the carcinologic safety of follicular suspensions obtained after isolation was evaluated by multicolor flow cytometry using a model involving the contamination of follicular suspensions with leukemic cells from patients suffering from acute myeloid leukemia (AML) or acute lymphoblastic leukemia (LAL) with known immunophenotype. Collagenase NB6 has allowed the isolation of a large number of viable follicles, mostly at the primordial stage and the majority of which were unactivated even after three days of in vitro culture in a fibrin matrice. Our isolation technique followed by three washes has allowed the elimination of leukemic cells previously added to follicular suspensions, in 23 out of 24 cases, without damaging isolated follicles. Multicolor flow cytometry is an effective analytical technique for assessing leukemic cell contamination of suspensions containing isolated ovarian follicles. The protocol to isolate human ovarian follicles, performed with the clinical grade collagenase NB6, may be considered for an ovarian reconstruction to human therapeutic purposes.

O objetivo deste trabalho foi aperfeiçoar o filtro lento em escala domiciliar (FLD) à realidade das comunidades isoladas brasileiras com o uso conjunto de pré-tratamento e pós-tratamento acessíveis. As espécies cactáceas Opuntia cochenillifera e Opuntia ficus indica foram avaliadas em ensaios de tratabilidade como coagulantes naturais em pó. A melhor condição para remoção de turbidez e cor aparente foi com 30 mg.L-1 de O. cochenillifera em pH sem a necessidade de correção (± 6,8). O pré-tratamento adaptado às comunidades isoladas utilizando o coagulante natural apresentou valores compatíveis de turbidez (7,83 ± 2,32 uT) para inserção aos filtros. Os FLDs em fluxo contínuo (taxa de filtração de 1,22 m³/m².dia) e em fluxo intermitente (taxa de filtração máxima de 2,79 m³/m².dia) demonstraram redução da turbidez, da cor aparente, dos coliformes totais e da Escherichia coli e produziram água filtrada com ausência de cistos de Giardia spp. e oocistos de Cryptosporidium spp. O FLD em fluxo contínuo teve a maior eficiência em relação à turbidez (± 81,2%); enquanto que o FLD operado em fluxo intermitente apresentou melhores eficiências para a remoção da cor aparente (48,4%), dos coliformes totais (2,60log ± 0,99) e da E. coli (2,86log ± 0,79). Os principais microrganismos atuantes no schmutzdecke dos FLDs foram ciliados (como, por exemplo, a Vorticella sp.), nematódeos e rotíferos. A esponja-vegetal (Luffa cylindrica) e o quiabo (Hibiscus esculentus) foram ponderados como desinfetantes alternativos para o pós-tratamento aos FLDs. As sementes da L. cylindrica e o fruto da H. esculentus foram avaliados através do preparo do extrato e as folhas da L. cylindrica através do preparo em pó. A melhor condição para inativação de E. coli foi com 1 g.L-1 do extrato de H. esculentus em pH sem a necessidade de correção (± 6,8) durante um tempo de contato de 30 min. Essa condição forneceu 1,12log (± 0,16) para a inativação de E. coli, contudo não afetou a viabilidade dos (oo)cistos de Giardia spp. e Cryptosporidium spp. Em ensaios toxicológicos com Chironomus xanthus, a água filtrada não apresentou toxicidade. / The aim of this study was to improve the household-scale slow sand filter (HSSF) to the reality of isolated Brazilian communities with combined use of pre-treatment and post-treatment approachable. Cacti species Opuntia cochenillifera e Opuntia ficus indica were evaluated in Jar tests as powder natural coagulants. The best condition for turbidity and apparent color removal was with 30 mg.L-1 of O. cochenillifera at pH without correction (± 6,8). Pre-treatment adapted to isolated communities using natural coagulant showed compatible turbidity values (7.83 ± 2.32 uT) for insertion into the filters. The HSSFs with continuous (filtration rate of 1,22 m³/m².day) and intermittent operation (maximum filtration rate of 2,79 m³/m².day) demonstrated reductions in turbidity, apparent color, total coliforms and Escherichia coli and produced filtered water without Giardia spp. cysts and Cryptosporidium spp. oocysts. The HSSF in continuous operation had the highest turbidity efficiency (± 81.2%); while the HSSF in intermittent operation had showed better efficiencies for the apparent color (48,4%), total coliforms (2,60log ± 0,99) and E. coli (2,86log ± 0,79). The main microorganisms active in the schmutzdecke were ciliates (for example, Vorticella spp.) nematodes and rotifers. Luffa cylindrica and Hibiscus esculentus were evaluated as alternative disinfectant for post-treatment to HSSF. L. cylindrica seed and H. esculentus fruit were tested as extract and L. cylindrica leave was tested powder. The best condition for E. coli inactivation was with 1 g.L-1 of H. esculentus extract at pH without correction (± 6,8) during 30 min. This condition provided 1,12log (± 0,16) for E. coli inactivation, however did not affect Giardia spp. cysts and Cryptosporidium spp. oocysts viability. In toxicological tests with Chironomus xanthus, the filtered water showed no toxicity.

Der renale Ischämie-/Reperfusionsschaden (IRI) stellt in der Transplantationsmedizin ein grosses Problem dar. Fucoidin, ein potenter Antagonist der Selektin-vermittelten Leukozytenaggregation, verbesserte an der Rattenleber (in einer Konzentration von 360mg/l) das Ausmass der leukozytären Gewebeinfiltration in der frühen Phase nach Ischämie und Reperfusion. In einem Modell der isoliert hämoperfundierten Schweineniere sollte die Wirkung von Fucoidin auf die postischämische Organfunktion untersucht werden. Hierzu wurden 24 Versuche durchgeführt. Dem Blut der Versuchsgruppen wurde vor Beginn der Reperfusion Fucoidin in einer Konzentration von 100 mg/l zugesetzt. Es zeigte sich unter Fucoidin ein signifikanter Abfall des renalen Blutflusses (55 ( 28 vs. 143 ( 97 ml*min-1*100g-1, p / Renal postischemic reperfusion injury constitutes a significant problem after kidney transplantation. The polysaccharide fucoidin (360 mg/l) improves postischemic function in Ratliver, presumably by blocking selectin-mediated leukocyte adhesion. Twelve pairs of ischemic pig kidneys were reperfused in an ex vivo model with autologous blood with or without fucoidin (100 mg/L). Fucoidin resulted in a significant decrease of renal blood flow (55 ( 28 vs. 143 ( 97 mL*min-1*100g-1, p < 0.001) and increased vascular resistance (2.9 ( 2.8 vs. 1.1 ( 1.5 mmHg*mL-1*min-1*100g-1, p < 0.001). Compared to untreated control kidneys significantly more interstitial and intravascular leucocytes were found in fucoidin treated kidneys. Intraglomerular fibrinogen and thrombocytic aggregates were also increased significantly. Granulocytic emboli were present in afferent glomerular arteries of 10/12 fucoidin-treated kidneys and in 2/12 controls (p < 0.001). L-selectin-dependent granulocytic aggregation under shear stress in vitro was prevented by fucoidin in a dose-dependent fashion. However similar concentrations used in reperfused kidneys caused large granulocytic aggregates. The observed formation of embolizing granulocytic aggregates indicates limited effectiveness of fucoidin as an inhibitor of selectin-mediated leukocyte adhesion.

im Postscript-Format / PostScript

112 Patienten wurden im Rahmen von 2 prospektiven multizentrischen Phase-II Studien mit einer isolierten Extremitätenperfusion mit Tumornekrosefaktor (TNF) und Melphalan behandelt. Hiervon waren 49 Patienten an einem lokoregionär metastasierten Melanom und 63 Patienten an einem lokal fortgeschrittenen Weichgewebssarkom erkrankt. Bei Patienten mit regionären Metastasen eines Melanoms fand sich in 46 % eine komplette und in 20 % eine partielle Remission. Die mittlere lokoregionäre progressionsfreie Zeit war 32,3 Monate (Median 15,6 Monate). Sie war signifikant länger bei Patienten mit einer kompletten Remission (46,0 Monate, Median 30,2 Monate) im Vergleich zu Patienten mit einer partiellen Remission (15,2 Monate, Median 16,1 Monate) oder Patienten mit unverändertem Befund (7,2 Monate, Median 6,0 Monate). Die Gesamt-Überlebenszeit der Patienten mit Melanom betrug im Mittel 42,6 Monate (Median 24,4 Monate) und die 5-Jahres-Überlebensrate 42 %. Die klinische Ansprechrate der Patienten mit Weichgewebssarkomen betrug 44,5 %. Bei kombinierter Analyse der klinischen und histologischen Remission betrug die Gesamtansprechrate in der Gruppe der Patienten mit Weichgewebssarkomen 60 % (21 % CR). Insgesamt war bei 86 % der Patienten mit Sarkomen (54/63 Patienten) ein extremitätenerhaltendes Vorgehen möglich. Die mittlere lokale progressionsfreie Zeit betrug für alle 63 Patienten mit Weichgewebssarkom 63,7 Monate und die 5-Jahres-Progessionsfreiheit 72 %. Die mittlere Überlebenszeit der Patienten mit Sarkomen betrug 61,9 Monate und die 5-Jahres-Überlebenszeit 64 %. Wichtigste Einflussfaktoren auf die Überlebenszeit waren das Tumorgrading (p=0,016) und die Tumorgrösse (p=0,046). 94 der 112 behandelten Patienten hatten nach der Extremitätenperfusion im Bereich der Extremität eine regionale Toxizität Grad I-II nach Wieberdink und bei 18 Patienten kam es zu einer höhergradigen Gewebeschädigung (Grad III-IV). Die postoperativen Serumkonzentrationen von Creatinkinase (CK) und Myoglobin zeigten eine signifikante Korrelation zur regionalen Toxizität (p=0,007 bzw. 0,003). An systemischen Nebenwirkungen der isolierten Extremitätenperfusion mit TNF und Melphalan fiel vor allem eine Erhöhung der Leberwerte auf, mit Anstieg der Transaminasen und des Bilirubins bis zu WHO-Schweregrad III und IV. Einschränkungen der Lebersyntheseleistungen wurden nicht beobachtet. Alle Veränderungen waren spontan rückläufig. Leuko- und Thrombopenien Grad III/IV betrafen häufig Patienten mit einer systemischen Leckrate von > 5 % (29 % bzw. 21 %). Eine Überlegenheit der isolierten Extremitätenperfusion mit TNF und Melphalan beim Melanom gegenüber der Perfusion mit Melphalan alleine kann aus den vorliegenden Daten nicht abgeleitet werden. Die von uns festgestellte klinische Remissionsrate von 66 % ist nicht höher als in der Literatur angegeben. Durch die isolierte Extremitätenperfusion mit TNF und Melphalan kann bei ca. 80 % der behandelten Patienten mit lokal fortgeschrittenen und primär nicht resektablen Weichgewebssarkomen ein Extremitätenerhalt erreicht werden. Die hohe Rate auch histologisch belegter Remissionen bei diesen Patienten untermauert den Stellenwert dieser Therapie eindrucksvoll. / 112 patients were treated by isolated limb perfusion with tumor necrosis factor (TNF) and melphalan for locoregional metastases of melanoma (49 patients) or locally advanced soft tissue sarcoma (63 patients). Patients were treated in 2 prospective multicentric phase-II trials. For patients with intransit metastases from melanoma, complete response could be reached in 46 % and partial response in 20 %. Mean time to locoregional progression was 32.3 months (median 15.6 months). This was significantly longer for patients with complete response (46.0 months, median 30.2 months) when compared to patients with a partial response (15.2 months, median 16.1 months) or patients with no change (7.2 months, median 6.0 months). The mean overall survival time for patients with melanoma was 42.6 months (median 24.4 months) and the 5-year survival rate 42 %. The clinical response rate for sarcoma patients was 44.5 %, but on combined analysis of clinical and histologically confirmed response the overall response rate was 60 % (21 % CR). Limb salvage was possible in 86 % of the sarcoma patients (54/63 patients). The mean time to local progression was 63.7 months and the 5-year progression-free rate was 72 %. Mean overall survival time in this group was 61.9 months with a 5-year survival rate of 64 %. Grading (p=0,016) and tumor size (p=0,046) were the most important factors influencing survival. Regional toxicity after isolated limb perfusion was mild (Wieberdink grade I-II) in 94 of the 112 patients, and 18 patients had a more severe reaction (grade III-IV). Postoperative serum concentrations of creatinekinase (CK) and myoglobin were significantly correlated to regional toxicity (p=0.007 and 0.003). Elevated liver enzymes and bilirubin with levels up to WHO III and IV were the most profound systemic side effect of isolated limb perfusion with TNF and melphalan. All changes resolved completely. Leucopenia and thrombopenia grade III/IV were mainly observed in patients with a systemic leakage rate greater than 5 % (29 % resp. 21 %). In melanoma patients, the observed overall response rate of 66 % after isolated limb perfusion with TNF and melphalan is not superior to literature results for limb perfusion with melphalan alone. Thus, the superiority of this treatment cannot be confirmed for this indication. In contrast, limb salvage became possible in approx. 80 % of the patients with locally advanced and unresectable sarcoma. In addition, the high rate of histologically confirmed response in these patients underlines the value of this treatment modality.