La Maladie Thrombo-Embolique Veineuse (MTEV) est une maladie dont les facteurs de risque sont à la fois environnementaux et génétiques. Les facteurs de risque génétiques bien établis sont les déficits en anti-thrombine, en protéine S, en protéine C, la mutation du Facteur V de Leiden (FVL), la mutation du Facteur (F) II G20210A, ainsi que le gène ABO dont les allèles A1 et B augmentent le risque de MTEV par rapport aux allèles A2 et O. Alors qu’une part importante de l’héritabilité de la MTEV reste inexpliquée, les études contemporaines se heurtent à un manque de puissance pour découvrir de nouveaux facteurs génétiques dont les effets sont de plus en plus faibles. En vue d’augmenter la puissance de détection de nouveaux gènes de susceptibilité à la MTEV, j’ai recherché les déterminismes génétiques de deux de ses phénotypes intermédiaires : les taux d’activité plasmatique du FVIII et les taux d’antigénémie de sa protéine de transport, le Facteur de von Willebrand (vWF). Dans un premier temps, j’ai réalisé une analyse de liaison des taux de FVIII et de vWF à partir d’un échantillon de cinq grandes familles franco-canadiennes (totalisant 255 personnes) recrutées via un cas de MTEV avec mutation FVL. Quatre régions liées aux taux de FVIII et/ou vWF ont été identifiées. L’une de ces régions correspondait au locus du gène ABO déjà connu pour influencer les taux de FVIII et vWF. La recherche de gènes candidats au sein des autres signaux de liaison s’est effectuée par l’étude in silico d’une analyse d’association pangénomique de la MTEV incluant 419 cas et 1228 témoins. Deux gènes candidats ont été identifiés : STAB2 et BAI3. J’ai ensuite réalisé des études d’associations de cinq polymorphismes de BAI3. L’un d’entre eux était d’une part associé à une élévation des taux de vWF (résultat obtenu dans un échantillon de 108 familles nucléaires en bonne santé et reproduit dans un échantillon de 916 patients non apparentés atteints de MTEV), et d’autre part associé au risque de survenue de MTEV parmi les sujets non porteurs de mutations FVL et FII de deux échantillons cas-témoins (respectivement 916 cas et 801 témoins, et 250 cas et 607 témoins). Quant à STAB2, durant le courant de ma thèse, deux de ces polymorphismes ont été décrits comme associés aux taux de FVIII et vWF au cours d’une vaste étude d’association pangénomique (GWAS) menée par le consortium CHARGE rassemblant 23 600 personnes. Dans un second temps, j’ai réalisé une méta-analyse de trois GWAS des taux de FVIII et vWF. Ces analyses avaient été conduites avec l’échantillon des cinq grandes familles franco-canadiennes et deux échantillons de 972 et 570 patients atteints de MTEV. Elles étaient ajustées sur les polymorphismes du gène ABO permettant de distinguer les allèles A1, A2, B et O, dans l’optique d’augmenter la puissance des analyses en diminuant la variance résiduelle des phénotypes. Aucun polymorphisme n’était associé ni aux taux de vWF ni à ceux de FVIII après prise en compte de la correction de Bonferroni pour tests multiples (p<10-7). Cependant, parmi les onze gènes qui présentaient des polymorphismes associés aux taux de vWF ou de FVIII avec une significativité p<10-5, de manière intéressante se trouvait STAB2. Cette étude a de plus permis de confirmer les associations nouvellement découvertes de polymorphismes situés dans les gènes VWF, STXBP5 et STX2. / The Venous Thromboembolism (VTE) risk factors are environmental and genetic. The well established risk factors are anti-thrombin, protein C, protein S deficiency, Factor V Leiden and factor II mutation and ABO gene, with A1 and B allele increasing the risk of VTE. While an important part of VTE heritability remains unexplained, contemporary studies fail to discover new susceptibility genes with weaker effects. In order to increase the discovery power, I searched for genetic geterminism of two intermediary phenotypes of VTE : Factor VIII plasmatic activity (FVIII) and von Willebrand factor antigenemia (vWF)First, I performed a linkage study of FVIII and vWF from a sample of 5 large pedigrees (N=255). Four loci have been identified. One included ABO gene. I searched for candidate genes located in the others loci by studying in silico results from o Genome Wide Association Study (GWAS) of the VTE including 419 cases and and 1228 controls. témoins. Two candidate genes were identified : STAB2 et BAI3. Then I performed association studies of five SNPs in BAI3 with FVIII and vWF. One of them was associated to vWF (in a sample of 108 nuclear families and 916 VTE patients), and associated to VTE in two case-controls samples (respectively 916 cases and 801 controls, and 250 cases et 607 controls).Second, I performed a meta-analysis of three GWAS of FVIII and vWF from the same 5 pedigrees and two samples of VTE (N=972 and 570) adjusted on ABO blood group. No polymorphisms were significant after Bonferoni correction (p<10-7). Nevertheless, among 11 genes carrying polymorphisms with a p<10-5, interestingly was STAB2. Futhermore, this study allowed to confirm newly discoverd association with VWF, STXBP5 et STX2.

In sheep, large ovarian antral follicles grow in waves with a periodicity of every 4 to 5 days; each wave is initiated by a peak in serum concentrations of follicle stimulating hormone (FSH). In the present thesis, follicular data and hormone estimations acquired from daily ultrasonography and blood samples, respectively, were used to study mechanisms regulating the number of follicular waves per estrous cycle. Using additional approaches such as implants releasing estradiol-17â and or progesterone, immunization against gonadotropin releasing hormone (GnRH), and injections of GnRH, the role of pulsed luteinizing hormone (LH) secretion and FSH peaks in follicular wave emergence and growth and the dependency of FSH peaks on pulsed GnRH secretion, were studied in sheep. The viability of aged follicles was also addressed.<p> The results of the present studies showed that ewes with three or four waves per cycle had cycles of the same length. The inter-wave interval was longer for the first and the last or ovulatory wave of the cycle in three compared to four wave cycles. The length of the lifespan and regression phase of the largest follicle of a wave declined across the cycle as FSH peak concentration and amplitude decreased. The maximum follicular diameter of the largest follicle growing in the first wave and the last or ovulatory wave of the cycle was greater compared to other waves of the cycle. Treatment of anestrous ewes with estradiol releasing implants alone completely abolished pulsed LH secretion and suppressed follicular wave development; however, FSH secretion was only minimally affected and the pool of small follicles was not affected. When pulsed secretion of LH was restored by frequent injections of GnRH, follicular waves were re-established. Treatment of anestrous ewes with implants releasing estradiol and progesterone, decreased FSH peak amplitude and abolished LH pulses and follicular waves; the size of the pool of small follicles increased. Immunization against GnRH in anestrous ewes abolished pulsatile LH secretion and suppressed follicular wave emergence; however, FSH peaks continued to occur for several weeks. In cyclic ewes, creating an LH pulse frequency typical of the follicular phase, during the luteal phase of the cycle by giving GnRH, increased maximum diameter of the largest follicle in a wave and serum concentrations of estradiol and progesterone. The enhanced growth of follicles in a wave blocked the next expected FSH peak and its associated follicular wave. Decreasing LH pulse frequencies lower than the minimal frequency seen in the luteal phase, by implants releasing progesterone, did not affect the growth of follicular waves.<p> It was previously demonstrated that treatment of non-prolific WWF ewes with Prostaglandin F2á (PGF2á) and medroxy progesterone acetate (MPA) increased the ovulation rate by adding ovulations from the penultimate wave in addition to the final wave of the cycle; however, fertility was not improved. In the last study of my thesis, we collected follicles, with an extended lifespan, from the penultimate wave of the cycle in ewes given the PGF2á and MPA treatment. We compared their quality with follicles from the final wave of the cycle by looking at the expression of markers of follicular development. The results showed that theca cells of follicles from the final wave had significantly higher mRNA expression for vascular endothelial growth factor (VEGF) compared to follicles from the penultimate wave. Granulosa cells of follicles from the final wave had significantly higher mRNA expression for connexion 43 (Cx43) compared to follicles from the penultimate wave. Protein expression for Cx43, proliferating cell nuclear antigen (PCNA) and Factor VIII was greater in follicles from the final compared to the penultimate wave.<p> We concluded from the present studies that: 1) the mechanism that makes a three wave or four wave cycle is unclear; 2) some level of pulsatile LH secretion is required for an FSH peak to trigger emergence of follicular waves in anestrous ewes; 3) progesterone enhances the inhibitory effects of estradiol on FSH secretion in anestrous ewes, suppressing specifically FSH peak amplitude; 4) an endogenous rhythm may exist that drives the peaks in FSH secretion independent of secretory products from the follicles growing in a wave and pulsed GnRH secretion; 5) follicular waves in ewes, when exposed to an LH pulse frequency similar to the follicular phase, during the luteal phase of the cycle, when serum progesterone concentrations are high, can grow and function like ovulatory follicles growing in the follicular phase of the cycle; 6) expression of some markers of vascularization/ angiogenesis, gap-junctional communication and cell proliferation, appeared to be decreased in follicles from the penultimate compared to the final wave of an estrous cycle, when the lifespan of follicles from the penultimate wave was extended such that they were present in the ovary with follicles from the final wave of the cycle.

In sheep, large ovarian antral follicles grow in waves with a periodicity of every 4 to 5 days; each wave is initiated by a peak in serum concentrations of follicle stimulating hormone (FSH). In the present thesis, follicular data and hormone estimations acquired from daily ultrasonography and blood samples, respectively, were used to study mechanisms regulating the number of follicular waves per estrous cycle. Using additional approaches such as implants releasing estradiol-17â and or progesterone, immunization against gonadotropin releasing hormone (GnRH), and injections of GnRH, the role of pulsed luteinizing hormone (LH) secretion and FSH peaks in follicular wave emergence and growth and the dependency of FSH peaks on pulsed GnRH secretion, were studied in sheep. The viability of aged follicles was also addressed.<p> The results of the present studies showed that ewes with three or four waves per cycle had cycles of the same length. The inter-wave interval was longer for the first and the last or ovulatory wave of the cycle in three compared to four wave cycles. The length of the lifespan and regression phase of the largest follicle of a wave declined across the cycle as FSH peak concentration and amplitude decreased. The maximum follicular diameter of the largest follicle growing in the first wave and the last or ovulatory wave of the cycle was greater compared to other waves of the cycle. Treatment of anestrous ewes with estradiol releasing implants alone completely abolished pulsed LH secretion and suppressed follicular wave development; however, FSH secretion was only minimally affected and the pool of small follicles was not affected. When pulsed secretion of LH was restored by frequent injections of GnRH, follicular waves were re-established. Treatment of anestrous ewes with implants releasing estradiol and progesterone, decreased FSH peak amplitude and abolished LH pulses and follicular waves; the size of the pool of small follicles increased. Immunization against GnRH in anestrous ewes abolished pulsatile LH secretion and suppressed follicular wave emergence; however, FSH peaks continued to occur for several weeks. In cyclic ewes, creating an LH pulse frequency typical of the follicular phase, during the luteal phase of the cycle by giving GnRH, increased maximum diameter of the largest follicle in a wave and serum concentrations of estradiol and progesterone. The enhanced growth of follicles in a wave blocked the next expected FSH peak and its associated follicular wave. Decreasing LH pulse frequencies lower than the minimal frequency seen in the luteal phase, by implants releasing progesterone, did not affect the growth of follicular waves.<p> It was previously demonstrated that treatment of non-prolific WWF ewes with Prostaglandin F2á (PGF2á) and medroxy progesterone acetate (MPA) increased the ovulation rate by adding ovulations from the penultimate wave in addition to the final wave of the cycle; however, fertility was not improved. In the last study of my thesis, we collected follicles, with an extended lifespan, from the penultimate wave of the cycle in ewes given the PGF2á and MPA treatment. We compared their quality with follicles from the final wave of the cycle by looking at the expression of markers of follicular development. The results showed that theca cells of follicles from the final wave had significantly higher mRNA expression for vascular endothelial growth factor (VEGF) compared to follicles from the penultimate wave. Granulosa cells of follicles from the final wave had significantly higher mRNA expression for connexion 43 (Cx43) compared to follicles from the penultimate wave. Protein expression for Cx43, proliferating cell nuclear antigen (PCNA) and Factor VIII was greater in follicles from the final compared to the penultimate wave.<p> We concluded from the present studies that: 1) the mechanism that makes a three wave or four wave cycle is unclear; 2) some level of pulsatile LH secretion is required for an FSH peak to trigger emergence of follicular waves in anestrous ewes; 3) progesterone enhances the inhibitory effects of estradiol on FSH secretion in anestrous ewes, suppressing specifically FSH peak amplitude; 4) an endogenous rhythm may exist that drives the peaks in FSH secretion independent of secretory products from the follicles growing in a wave and pulsed GnRH secretion; 5) follicular waves in ewes, when exposed to an LH pulse frequency similar to the follicular phase, during the luteal phase of the cycle, when serum progesterone concentrations are high, can grow and function like ovulatory follicles growing in the follicular phase of the cycle; 6) expression of some markers of vascularization/ angiogenesis, gap-junctional communication and cell proliferation, appeared to be decreased in follicles from the penultimate compared to the final wave of an estrous cycle, when the lifespan of follicles from the penultimate wave was extended such that they were present in the ovary with follicles from the final wave of the cycle.

Pacientes envenenados pela serpente Bothrops jararaca manifestam uma tendência hemorrágica em que a plaquetopenia é um achado consistente. Manifestações clínicas sistêmicas, como sangramento de mucosas e microangiopatia trombótica em alguns pacientes, apresentam similaridades com sinais clínicos de doença de von Willebrand e púrpura trombocitopênica trombótica. Algumas proteínas do veneno - como a botrocetina (uma proteína relacionada às lectinas do tipo C) e as metaloproteinases do veneno (SVMP) - interferem direta ou indiretamente na interação entre plaquetas e o fator de von Willebrand (vWF) in vitro e in vivo. E dessa forma, podem contribuir para os sangramentos induzidos pelo envenenamento devido à importância que o vWF tem para a hemostasia primária. Pensando em compreender a participação do vWF do organismo e a botrocetina e as SVMP do veneno bruto de B. jararaca (BjV) na plaquetopenia induzida pelo envenenamento, utilizamos dois modelos experimentais: ratos Wistar heterogênicos e camundongos nocautes do gene Vwf (Vwf-/-). No modelo em ratos, o BjV foi pré-incubado com salina (controle positivo), um inibidor de metaloproteinases (Na2-EDTA), anticorpos policlonais anti-botrocetina, glicerol (veículo dos anticorpos), ou a combinação do Na2-EDTA e anticorpos anti-botrocetina; o grupo controle negativo foi injetado somente com salina. Após a administração subcutânea (s.c.) dos venenos tratados (1,6 mg/kg), amostras de sangue foram coletadas após 3, 6 ou 24 h, e analisaram-se a contagem de plaquetas, quantificação antigênica (vWF:Ag) e da atividade de ligação do vWF ao colágeno (vWF:CB), a atividade de ADAMTS13, a distribuição multimérica de vWF, e a atividade coagulante de fator VIII (FVIII). Para explorar a participação do vWF na plaquetopenia, camundongos nocautes de vWF (Vwf-/-) e camundongos controles (C57BL/6) foram injetados s.c. com BjV incubado com salina (grupo positivo do envenenamento) ou apenas salina (grupo controle negativo). As injeções dos tratamentos, bem como os períodos analisados foram idênticos aos dos ratos. Em nossos resultados, todos os ratos injetados com algum tratamento de BjV, inclusive nos animais que receberam veneno pré-tratado com anticorpo anti-botrocetina e/ou Na2-EDTA, apresentaram plaquetopenia, com maior intensidade em 6 h. Na avaliação do vWF foi encontrada uma grande variação individual nos grupos de tratamentos, porém ainda assim houve uma tendência a redução nos níveis de vWF:Ag em 3 e 6 h nos ratos que receberam BjV sem inibidores. A administração de BjV tratado somente com anticorpo anti-botrocetina promoveu uma maior redução nos níveis de vWF:Ag em 3 h, com retorno aos níveis semelhantes aos de controle negativo em 6 h e 24 h. A inibição sozinha das metaloproteinases não promoveu efeito importante, porém em 6 h, potencializou a ação do anticorpo anti-botrocetina na inibição conjunta do decréscimo de vWF:Ag e vWF:CB. A análise dos multímeros do vWF mostrou perfis bastante variáveis individualmente, porém os multímeros de alto peso molecular e intermediário tenderam a diminuir e os de baixo peso a aumentar nos animais que receberam algum tratamento com BjV, especialmente em 24 h. Na dosagem de FVIII, houve redução em 3 e 6 h em todos os ratos que receberam qualquer tratamento de BjV, sem grandes variações entre esses grupos. A atividade de ADAMTS13 apresentou uma redução dos valores em 3 e 6 h, que foi revertida pela inibição das metaloproteinases do veneno. Já nos camundongos, a plaquetopenia esteve presente em todos os animais nocautes e controles que receberam BjV, mostrando ser independente da presença de vWF. Nos camundongos controles (C57BL/6), não houve alterações evidentes em vWF:Ag durante o envenenamento, porém em 3 h houve uma tendência a sua diminuição. Em conjunto, nossos resultados mostram que a presença da botrocetina no veneno bruto não afeta a plaquetopenia desencadeada pelo envenenamento, porém influencia o vWF plasmático quantitativa e funcionalmente. As metaloproteinases do veneno têm forte efeito sobre a enzima fisiológica reguladora da atividade biológica do vWF, a ADAMTS13, que indiretamente pode afetar os níveis de vWF. Ademais, a intensidade da plaquetopenia durante o envenenamento de B. jararaca não depende da presença de vWF, e tendo em conta o caráter multifatorial do consumo plaquetário durante o envenenamento, sugerimos que outros mecanismos possam ser responsáveis pela plaquetopenia induzida pelo BjV. Com isso, concluímos que o consumo de vWF no envenenamento por B. jararaca é um fator contribuinte, porém não determinante, para as alterações da contagem plaquetária / Patients bitten by Bothrops snakes manifest a bleeding tendency in which thrombocytopenia is consistently observed. Systemic clinical manifestations, such as mucous bleeding and thrombotic microangiopathy in some patients, share similarities with symptoms of von Willebrand disease and thrombotic thrombocytopenic purpura. Some venom proteins - e.g. botrocetin (a C-type lectin-related protein) and snake venom metalloproteinases (SVMP) - disturbs, direct or indirectly, the interaction between platelets and von Willebrand factor (vWF) in vitro and in vivo, and may contribute thereby to snakebite-induced bleedings, once vWF is required for primary hemostasis. To better understand the relation between plasma vWF, and botrocetin and SVMPs from B. jararaca crude venom (BjV) in the thrombocytopenia induced by envenomation, we used two experimental models: Wistar heterogenic rats and vWF knockout mice (Vwf-/-). In the rat model, BjV was pre-incubated with saline (positive control), metalloproteinase inhibitor (Na2-EDTA), polyclonal anti-botrocetin antibodies, glycerol (antibody vehicle), or the combination of Na2-EDTA and anti-botrocetin antibodies; the negative control group was injected with saline only. After subcutaneous injection (s.c.) of treated venom (1.6 mg/kg), blood samples were collected after 3, 6 or 24 h, and platelet count, vWF antigen (vWF:Ag) and collagenbinding activity (vWF:CB), ADAMTS13 activity, vWF multimer distribution, and factor VIII (FVIII) coagulant activity were analyzed. To investigate the participation of vWF in thrombocytopenia, vWF knockout mice (Vwf-/-) and control mice (C57BL/6) were injected s.c. with saline only (negative control group) or BjV pre-incubated with saline (positive control group). The same protocols used for rats were accomplished in mice. Our results showed that all rats injected with any BjV treatment, including animals which received anti-botrocetin antibodies and/or Na2-EDTA-treated BjV, showed thrombocytopenia, with the nadir at 6h. vWF analysis exhibited a large individual variation among treatment groups, but there was a tendency to reduce vWF:Ag levels at 3 and 6 h in rats that received BjV pre-incubated with saline (without any inhibitor). Administration of BjV pre-incubated only with anti-botrocetin antibodies evoked a large reduction in vWF:Ag levels at 3 h, which returned to levels similar to those of the negative control group at 6 and 24 h. SVMP inhibition alone did not induce an important effect, but potentialized the activity of anti-botrocetin antibodies to inhibit the fall in both vWF:Ag and vWF:CB levels at 6 h. VWF multimer analysis had a large individual profile variation, although animals that received any BjV treatment tended to decrease the high and intermediate molecular weight multimers and to increase the low ones, especially at 24 h. FVIII showed diminished levels in all rats that received any BjV treatment at 3 and 6 h, without important variations among groups. Decreased levels of ADAMTS13 activity were noticed at 3 and 6 h, which were reverted by SVMP inhibition. In mice, thrombocytopenia was present in all control and knockout mice that received BjV, demonstrating independence of vWF presence. In control mice (C57BL/6), there were no relevant alterations in vWF:Ag during envenomation, although at 3 h there was a tendency to decrease it. Al together, our results showed that botrocetin present in crude venom does not affect thrombocytopenia induced by envenomation, but it changes the levels and function of plasma vWF. SVMP had a marked effect in ADAMTS13, the physiological enzyme that regulates vWF biological activity, which may affect vWF levels indirectly. In addition, thrombocytopenia during B. jararaca envenomation is independent of vWF, and considering the multifactorial features of platelet consumption during envenomation, we suggest that other mechanisms might account for BjV-induced thrombocytopenia. Therefore, we conclude that vWF consumption during B. jararaca envenomation is an ancillary mechanism, but not the main one to decrease platelet counts

A hemofilia A é uma doença ligada ao cromossomo X causada pela deficiência do fator VIII da coagulação sanguínea (FVIII). O tratamento disponível consiste na terapia de reposição da proteína do fator VIII derivada do plasma (FVIIIdp) ou recombinante (FVIIIr). Atualmente, dos 7 produtos recombinantes disponíveis no mercado, 6 são produzidos em linhagens celulares de roedores. A expressão dessa proteína em sistemas celulares não-humanos pode gerar uma molécula com perfil de modificações diferente do endógeno, podendo levar a reações imunogênicas e geração de inibidores anti-FVIIIr. Em função disso, novas estratégias de produção têm sido avaliadas, como a utilização de células hospedeiras mais eficientes no que diz respeito ao potencial de expressão da proteína de interesse. Dentre as linhagens de interesse, a linhagem hepática SK-HEP-1 tem se destacado por apresentar altos níveis de expressão do FVIIIr e potencial para o cultivo em suspensão em meios livres de soro fetal bovino (SFB). Dessa forma, o objetivo deste trabalho foi avaliar a produção de FVIIIr na linhagem celular humana SK-Hep-1 comparando duas estratégias para o estabelecimento de processos de produção em condições livres de soro e em suspensão: Estratégia 1 - adaptação para tais condições da linhagem já modificada geneticamente e Estratégia 2 - modificação gênica para a expressão da proteína já em células previamente adaptadas à tais condições. Para a estratégia 1, foram geradas duas linhagens recombinantes produtoras de FVIIIr, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1 aderentes e em cultivos suplementados com SFB. Na caracterização da cinética e produção do FVIIIr as linhagens apresentaram taxas específicas máxima de crescimento (?max) de 0,064 e 0,0031h-1 produzindo 1,0 e 0,78UI/mL de FVIIIr, respectivamente. Diversos protocolos de adaptação foram empregados, entretanto, não foi possível obter sucesso na adaptação das linhagens recombinantes para condições livres de soro e em suspensão. Para a estratégia 2, as células SK-HEP-1 selvagens adaptadas ao meio de cultura livre de SFB SFMII apresentaram um valor de ?max de 0,0186h-1 e Xmax de 1,9x106cels/mL. Para as etapas de modificação gênica da linhagem selvagem foram utilizados os mesmos vetores lentivirais empregados para a geração das células recombinantes aderentes, pLVmpsvFVIII?B-Neo e pLVCMVFVIII?B-GFP. Para o primeiro, não foi possível gerar uma linhagem produtora do FVIIIr. Para o segundo, foi possível obter duas linhagens produtoras do FVIIIr com 0.14 e 0.12IU/mL de atividade pelo ensaio cromogênico. O presente trabalho mostrou que a linhagem humana Sk-Hep-1 é apropriada para a produção de altos níveis de FVIIIr. No entanto, maiores esforços devem ser voltados ao desenvolvimento de meios de cultura livres de soro específicos para a linhagem para possibilitar a produção eficiente do FVIIIr em suspensão em meios livre de soro. / Hemophilia A is a genetic X-linked disorder caused by the coagulation factor VIII (FVIII) deficiency. The current treatment is the replacement therapy with plasma derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) products. Nowadays, of the seven products available in the market, six are produced in rodent expression systems, which can result in a rFVIII molecule with different post-translational modifications and may lead to immune responses to non-human epitopes. Therefore, new production strategies have been evaluated, as the use of more efficient hosts in terms of protein expression potential. Among potential cell lines, the hepatic SK-HEP-1 cell line features high levels of rFVIII production and potential for serum-free suspension culture. In face of the exposed above, the goal of this study was to evaluate rFVIII production in the SK-HEP-1 human cell line comparing two strategies for the establishment of production process in a suspension serum-free condition: strategy 1 - adaptation to these conditions of a genetic modified cell line; strategy 2 - genetic modification of an already adapted cell line to rFVIII protein expression. For strategy 1, two adherent rFVIII producer cell lines were established in serum containing medium, SK-HEP-F8/Neo-E1 e SK-HEP-F8/GFP-E1. Characterization of cell growth and rFVIII production showed a maximum specific growth rate (?max) of 0.064 and 0.00311h-1 with rFVIII production of 1.0 and 0.78UI/mL, respectively. Different adaptation protocols were used; however, it was not possible to adapt the recombinant cell lines to growth in suspension serum-free conditions. For strategy 2, the wildtype SK-HEP-1 cell line adapted growth in SFMII BSF medium, showed a ?max of 0.0186h-1 and a maximum cell concentration (Xmax) of 1.9x106cells/mL. For the genetic modification, it were employed the same lentiviral vectors used for the recombinant adherent cells generation, pLVmpsvFVIII?B-Neo and pLVCMVFVIII?B-GFP. For the first, no attempts were successful. For the second, it was possible to generate two rFVIII producer populations with 0.14 and 0.12IU/mL activity, measured by chromogenic assay. These results demonstrate that the SK-HEP-1 cell line is appropriate for the production of high levels of rFVIII. Nevertheless, efforts should be made in developing specific medium to support efficient rFVIII production in suspension and suspension serum-free conditions.

A Síndrome Antifosfolípide (SAF) é uma doença autoimune na qual há presença de anticorpos antifosfolípides [anticoagulante lúpico (AL), anticardiolipina (ACL), anti-beta2glicoproteína I (a-beta2GPI)]. É caracterizada por eventos trombóticos e/ou perdas gestacionais de repetição. Existe um ambiente pró-coagulante na SAF, pois os antifosfolípides (AFL) são capazes de induzir disfunção endotelial aumentando a expressão de moléculas de adesão como o fator de von Willebrand (FVW). Entre os inúmeros elementos envolvidos neste processo, o FVW e a relação com: sua principal enzima proteolítica, a ADAMTS-13, e com o FVIII são relativamente menos conhecidos. Os objetivos deste estudo foram: 1-verificar alterações no endotélio de pacientes com SAF primária (SAFP), através do marcador de lesão endotelial, o FVW, da ADAMTS-13 e do FVIII, avaliando a concentração antigênica, a atividade e a correlação entre estas proteínas; 2- Verificar se alguma destas variáveis é capaz de diferenciar os tipos de eventos trombóticos ocorridos nestes pacientes e se a presença dos AFL influenciam estas proteínas. O estudo, do tipo transversal, envolveu 39 pacientes com SAF primária, com idade mediana de 43 anos, em tratamento no ambulatório de Reumatologia do Hospital das Clínicas, e 39 controles sadios doadores da Fundação Pró-Sangue Hemocentro de São Paulo, pareados por sexo e idade com os pacientes. Os títulos de ACL e a-beta2GPI, as determinações antigênicas e de atividade das proteínas FVW, ADAMTS-13, FVIII e PF4 foram realizados por ELISA, e a atividade do FVIII foi determinada pelo método cromogênico. A análise das subunidades do FVW foi realizada por Western immunoblotting. AL foi detectado utilizando ensaios de coagulação de acordo com as recomendações da Sociedade Internacional de Trombose e Hemostasia. Os resultados foram apresentados em: 1- pacientes SAFP e controles, e 2- pacientes SAFP agrupados em relação ao tipo de evento e ao tipo de AFL presente. Os pacientes apresentaram aumento da concentração antigênica do FVW (74±6 x 69±11 UI/dL; p=0,016), ADAMTS-13 (1,3±0,34 x 0,82±0,12 Ug/mL; p < 0,0001), FVIII (106±19 x 91±15 UI/dL; p=0,0003),da ligação FVW:FVIII (144±17 x 134±20%; p=0,082) e da atividade do FVIII (117±38 x 98±30%; p=0,0021) comparado aos controles. O PF4 apresentou-se diminuído nos pacientes em relação aos controles (96±12 x 101±8 UI/mL; p=0,014). Os pacientes com trombose arterial apresentaram correlação positiva e significante entre a atividade e o antígeno do FVW (r=0,468; p=0,028) e da ADAMTS-13 (r =0,635; p=0,001); os pacientes com trombose venosa apresentaram esta correlação positiva e significante na ADAMTS-13 (r=0,635; p=0,001). Quando os pacientes foram analisados pelo tipo de antifosfolípide, não se observou diferenças nas variáveis estudadas. Os pacientes com SAFP parecem apresentar disfunção endotelial. No entanto, aparentemente existe um mecanismo de equilíbrio para evitar a formação de um novo trombo. O papel do FVW e a sua relação com a ADAMTS-13, em diferentes doenças, ainda é relativamente pouco conhecido, mas está sendo apontado como importante na patogênese de estados pró-trombóticos como os presentes em pacientes com SAF / Antiphospholipid syndrome (APS) is an autoimmune disease, characterized by vascular thrombosis and /or pregnancy morbidity, in association with antiphospholipid antibodies (aPL) (lupus anticoagulant (LA), anticardiolipin (ACL), anti-beta2glicoprotein I (a-beta2GPI). Antiphospholipid (APL) seems to induce endothelial dysfunction by increasing the expression of adhesion molecules such as von Willebrand factor (VWF). This results in a prothrombotic state in APS. Among the several elements involved in this process, some are relatively less known, such as VWF and its relationship with ADAMTS-13, its main proteolytic enzyme. The aim of this study was to evaluate endothelial dysfunctions in patients with primary APS (PAPS), by examining correlation among the soluble endothelial marker, VWF, the enzyme ADAMTS-13, and FVIII protein. The relationship of these proteins and the presence of arterial and/or venous thrombosis, and presence of APL was also evaluated. This cross-sectional study involved 39 PAPS patients, with a median age of 43 years, who have been treated in the Outpatient Clinics, Department of Rheumatology, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, and 39 healthy subjects blood donors from the Fundação Pró-Sangue Hemocentro de São Paulo, matched for sex and age with patients. Levels of APL (ACL and a-beta2GPI), concentration and activities of VWF, ADAMTS-13, FVIII and PF4 proteins were measured with ELISA. LA was detected with coagulation assays according to updated guidelines from the International Society on Thrombosis and Haemostasis, and FVIII activity was measured by chromogenic method. Analysis of VWF subunits was performed by Western immunoblotting. The results were evaluated according to:1- PAPS patients and controls, and2- PAPS patients grouped in relation to type of event and the type of APL. Patients showed higher VWF antigen concentration (74±6 x 69±11 IU/dL, p=0.016), ADAMTS-13 (1.3±0.34 x 0.82±0.12 Ug/mL, p < 0.0001), FVIII (106±19 x 91 ± 15 IU/dL, p=0.0003), VWF binding to FVIII (144±17 x 134 ± 20%, p=0.082) and activity of FVIII (117±38 x 98±30%, p=0.0021) than controls. The PF4 was decreased in patients compared to controls (96±12 vs. 101±8 IU/mL, p=0.014). VWF antigen and activity correlated well (Pearson´s r =0.468; p=0.028) as well as ADAMTS-13(Pearson´s r=0.635; p=0.001) in patients with arterial thrombosis. However, in patients with venous thrombosis only ADAMTS-13 had a good correlation (Pearson´s r =0.492; p=0.045). When patients were analyzed by the type of aPL, no differences in the studied variables were observed. Patients with PAPS seem to present endothelial dysfunction. However, apparently there is an attempt to balance mechanism to prevent a new thrombus formation. The role of VWF and its relation with ADAMTS-13 in different diseases is still relatively unknown. However it has been considered as important in the pathogenesis of prothrombotic states such as those present in patients with APS

La drépanocytose est associée à un état d’hypercoagulabilité qui se manifeste sur le plan clinique par un risque augmenté de thromboses artérielles et veineuses. L’exploration de la coagulation chez les patients drépanocytaires montrait surtout une activation de la coagulation et des altérations des acteurs pro- et anticoagulants du système hémostatique. Les tests de coagulation globale de routine tels que l’aPTT et le PT sont peu sensibles aux états d’hypercoagulabilité. La fonction hémostatique globale des patients drépanocytaires était donc peu connue. Le test de génération de thrombine est un test de coagulation globale, sensible aux états d’hypo- et d’hypercoagulabilité, facile à réaliser de nos jours avec une bonne reproductibilité. Nous l’avons utilisé pour démontrer que la coagulation globale des enfants drépanocytaires était caractérisée par une accélération des réactions de formation de la thrombine et par une augmentation du potentiel de thrombine endogène. Nous avons par la suite montré que les taux élevés de microparticules pro-coagulantes et du facteur VIII chez les enfants drépanocytaires seraient les facteurs déterminant l’accélération des réactions de formation de thrombine tandis que la réduction de l’activité anticoagulante du système protéine C / protéine S serait le facteur déterminant l’augmentation du potentiel de thrombine endogène. Les marqueurs de l’hémolyse corrélaient significativement avec ces facteurs ainsi qu’avec les paramètres de génération de thrombine, suggérant que l’hémolyse serait le mécanisme pathologique à la base de l’augmentation du potentiel de génération de thrombine chez les enfants drépanocytaires. Les paramètres de génération de thrombine n’étaient pas significativement différents entre l’état de stabilité clinique et l’état de crise vaso-occlusive. Chez les enfants hétérozygotes composites, ces paramètres avaient des valeurs intermédiaires entre celles des enfants contrôles et celles des enfants drépanocytaires homozygotes. Près de 40 % des enfants drépanocytaires homozygotes avaient un potentiel hémostatique supérieur à la moyenne + 2DS des enfants contrôles du même âge. Ces enfants présentant une génération de thrombine élevée se distinguaient des autres par leur plus jeune âge, une plus grande intensité de l’hémolyse, une plus courte durée de traitement par l’hydroxyurée et des vélocités du flux sanguin au doppler transcrânien plus élevées. Ces données suggèrent davantage un lien entre le potentiel de génération de thrombine et la vasculopathie cérébrale chez les enfants drépanocytaires. L’analyse de 4 enfants ayant reçu une greffe de cellules souches hématopoïétiques montrait une tendance à la réduction du potentiel de génération de thrombine et des autres altérations de la coagulation trois mois après la greffe. Le test de génération de thrombine permet une meilleure exploration de la coagulation globale des enfants drépanocytaires. Sa réalisation sur sang total permettrait une analyse plus globale en intégrant la participation des éléments figurés du sang particulièrement les globules rouges./<p>Sickle cell disease is associated with a hypercoagulable state that express clinically by an increased risk of arterial and venous thrombosis. The exploration of coagulation in sickle cell patients showed mainly activation of coagulation and alterations pro-and anticoagulants actors of the hemostatic system. Routine global testing of coagulation such as the prothrombin time and the activated partial thromboplastin time are insensitive to hypercoagulable states. The overall hemostatic function in sickle cell disease was so little known. The thrombin generation test is a test of overall coagulation. It is sensitive to both hypo- and hypercoagulable states. It is easy to perform nowadays with good reproducibility. We used it to demonstrate that the overall coagulation of sickle cell disease children was characterized by an acceleration of the reactions of thrombin formation and an increase of the endogenous thrombin potential. We have subsequently shown that high levels of procoagulant microparticles and high levels of factor VIII in children with sickle cell disease are the factors determining the acceleration of reactions leading to thrombin formation. Our results also showed that the reduced activity of the protein C / S anticoagulant pathway is a determining factor of the increased endogenous thrombin potential in sickle cell children. Markers of hemolysis correlated significantly with these factors as well as the parameters of thrombin generation, suggesting that hemolysis is probably the pathological mechanism underlying the increased potential for thrombin generation in children with sickle cell disease. Nearly 40% of children with homozygous sickle cell disease had their hemostatic potential above the mean + 2SD that of controls children of the same age. These children with high thrombin generation differed from others by their younger age, greater intensity of hemolysis, a shorter duration of treatment with hydroxyurea. They also had higher velocity of blood flow using transcranial Doppler. These data further suggest a potential link between thrombin generation and cerebral vasculopathy in children with sickle cell disease. Analysis of four children who received hematopoietic stem cells transplantation showed a tendency towards a reversibility of thrombin generation and other alterations of coagulation three months after the transplant. Thrombin generation assay allows a better exploration of the global coagulation of sickle cell disease children. Its realization on whole blood would be a more comprehensive analysis as it would integrate the participation of blood cells particularly red blood cells. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Bakgrund - Hemofili A och B är X-kromosombundna blödarsjukdomar, som beror på genetiska avvikelser i de gener som kodar för koagulationsfaktor VIII respektive IX. I dagsläget förlitar sig hemofilipatienter på kontinuerliga intravenösa injektioner med faktorkoncentrat, för att förhindra att potentiellt livshotande blödningar uppstår. Genterapi med rekombinanta adeno-associerade virus (AAV) skulle kunna erbjuda ett kurativt behandlingsalternativ, genom införandet av friska arvsanlag i hepatocyter. Syfte - Syftet med den här litteraturstudien var att undersöka huruvida genterapi medierad av AAV-vektorer är en effektiv och säker behandling mot hemofili A och B ur ett långsiktigt perspektiv. Metod - Studien är genomförd som en systematisk litteraturstudie och är baserad på sex originalartiklar framsökta via databasen PubMed, med sökorden "hemophilia AND gene therapy". Specificerade sökkriterier användes för att underlätta relevansbedömning och valet av artiklar. Resultat - En ökad endogen koagulationsfaktorproduktion kunde påvisas hos majoriteten av studiedeltagarna efter genterapibehandlingarna. Sammantaget observerades också en väsentlig blödningsreducering och en minskad faktorkoncentratanvändning. Störst förbättring noterades i de kohorter som erhållit högre genterapidoser eller den muterade faktor IX Padua-genen. Ingen immunrespons mot transgenprodukten detekterades i någon studie. Däremot sågs ett humoralt immunsvar mot AAV-kapsiden hos samtliga studiedeltagare. En mycket stor variation i T-cellssvar mot AAV-kapsiden kunde noteras. Förhöjda nivåer av alaninaminotransferas (ALAT) var den vanligast förekommande incidenten, men samtliga fall kunde framgångsrikt behandlas med glukokortikoidpreparat. Slutsats - Genterapibehandling med rekombinanta AAV-vektorer mot hemofili A och B förefaller effektiv och säker. Förhöjda ALAT-nivåer återstår dock som en behandlingsproblematik. Längre uppföljningar av fler genterapibehandlade hemofilipatienter krävs, för att kunna dra några definitiva slutsatser, väga risker mot nytta, samt optimera och individanpassa doser. / Background - Hemophilia A and B are X-linked bleeding disorders, resulting from defects in the genes encoding coagulation factors VIII and IX respectively. The current treatment for hemophiliacs entails frequent intravenous injections of coagulation factor concentrates, to prevent potentially life-threatening hemorrhages. Gene therapy utilizing recombinant adeno-associated viruses (AAV) could offer a potentially curative treatment option through the introduction of healthy genes into hepatocytes. Aim - The aim of this literature study was to investigate the long-term efficacy and safety of AAV vector-mediated gene therapy for the treatment of hemophilia A and B. Methods - The study is conducted as a systematic literature study and is based on six original articles retrieved from the search engine PubMed, using the key words "hemophilia AND gene therapy". Specific search criteria were used to facilitate the relevance assessment and selection of articles. Results - An increased endogenous coagulation factor synthesis was noted in the majority of the study participants after the gene therapy. Overall, a significant reduction in bleeding episodes and the use of factor concentrates were observed. The greatest improvements were noted in the cohorts that received the higher gene therapy doses or the mutated factor IX Padua gene. None of the study participants had an immunologic response to the transgene product. A humoral immune response against the AAV capsid was seen in all participants though. Large differences in AAV capsid-specific T-cell activation were observed. The most common adverse event was an elevation in the alanine aminotransferase (ALT) level. However, these events could be controlled with glucocorticoids. Conclusions - AAV vector-mediated gene therapy for the treatment of hemophilia A and B had a positive efficacy and safety profile. Although increased ALT levels remain a concern. Monitoring of larger numbers of study participants for longer follow-up periods is necessary for any definite conclusions to be drawn, to weigh risks against benefits and to optimize individual dosing.