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L’objectif de notre travail était de déterminer le rôle joué par l’érythropoïétine et la<p>thrombopoïétine, respectivement, dans l’anémie et la thrombocytopénie observées<p>chez des enfants souffrant d’une hémopathie maligne.<p>Par le dosage simultané de la forme soluble du récepteur de la transferrine et de<p>l’érythropoïétine dans le sérum nous avons montré que l’anémie observée chez ces<p>patients est bien la conséquence d’une réduction du nombre de progéniteurs<p>érythropoïétiques (atteinte médullaire centrale) mais que celle-ci n’est pas la<p>conséquence d’une production insuffisante d’érythropoïétine. Nous avons fait la<p>même observation chez des enfants souffrant d’une tumeur solide non<p>hématologique et chez des patients en cours de traitement par chimiothérapie.<p>Chez ces derniers patients, en appliquant un modèle de culture de moelle à long<p>terme, nous avons pu démontrer l’existence d’une altération du microenvironnement<p>médullaire, probablement induite par la chimiothérapie, se<p>traduisant par une réduction de son aptitude à supporter le développement de la<p>lignée érythroïde. Ceci expliquant au moins partiellement l’inadéquation de la<p>réponse érythropoïétique observée chez ces patients en réponse à l’anémie.<p>Dans la dernière partie du travail, nous avons montré que la thrombocytopénie très<p>fréquemment observée chez les patients leucémiques s’accompagne dans la<p>majorité des cas d’une élévation exponentielle de la concentration de<p>thrombopoïétine, excepté dans les cas de leucémies de la lignée myéloïde. Chez ces<p>derniers la concentration de thrombopoïétine est proche des valeurs observées chez<p>des sujets normaux alors qu’elle devrait être 10 à 100 fois plus élevée compte tenu<p>du nombre de plaquettes extrêmement bas. Nous avons pu montrer que ces taux<p>très bas sont la conséquence de la liaison de la thrombopoïétine à un récepteur<p>spécifique et fonctionnel présent à la surface des cellules leucémiques myéloïdes<p>qui, en l’utilisant comme facteur de croissance, (stimulant leur prolifération et<p>retardant leur mort cellulaire) « consomment » la thrombopoïétine présente dans le<p>sérum. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Erythropoiesis maintains stable tissue oxygenation in the basal state, while accelerating red cell production in anemia, blood loss or high altitude. The principal regulator of erythropoiesis is the hormone erythropoietin (Epo). In response to hypoxic stress, Epo can increase a 1000-fold, driving erythropoietic rate by up to 10-fold. It’s been suggested that survival pathways activated by the Epo receptor (EpoR) underlie its regulation of erythropoietic rate. A number of apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here I assessed the roles of three survival pathways activated by EpoR in erythroblasts in-vivo: the suppression of cell-surface Fas and FasL, the suppression of the pro-apoptotic regulator Bim, and the induction of the anti-apoptotic regulator Bcl-xL. I used the novel CD71/Ter119 flow-cytometric method of identifying erythroblast maturation stages in vivo to measure these apoptotic pathways in fetal liver and adult erythropoietic tissues. I found that these pathways differ markedly in their regulation of erythropoietic rate. Using mouse genetic models, I found that apoptosis mediated by interaction between erythroblasts that co-express cell-surface Fas and FasL plays a key autoregulatory role in stabilizing the size of the erythroblast pool in the basal state. Further, mice mutant for Fas or FasL showed a delayed erythropoietic response to hypoxia or high Epo. This suggests that Fas and FasL accelerate the stress response by providing an apoptotic ‘cell reserve’ that can be rescued by Epo in stress. I also examined the in-vivo behavior of two cell-intrinsic apoptotic regulators, Bcl-xL and Bim, previously unexamined in stress. The induction of Bcl-xL was rapid but transient, whilst the suppression of Bim was slower but persistent. My data suggest that Bcl-xL is a key mediator of EpoR’s anti-apoptotic signal very early in the stress response, before Bim and Fas are suppressed. Bcl-xL adaptation to high Epo occurs through inhibition of Stat5 activation, and resets it for the next acute stress. My findings suggest that in vivo, Epo regulates erythropoietic rate through erythroblast apoptosis, and that various apoptotic regulators play distinct and unique roles in this process. My work provides new molecular insights into erythropoiesis that are relevant to cytokine biology and to clinical approaches of disease treatment.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Le rôle de l'inflammation dans le développement et la progression des maladies rénales chroniques (MRC) chez le chat a été peu étudié. L'hepcidine est une protéine de la phase aigue (PPA) de l'inflammation qui contribue au développement des anémies lors de MRC chez l'homme. Les objectifs de cette étude sont de comparer les concentrations en PPAs, en erythropoietine (EPO) ainsi que le statut en fer entre un groupe de chats sains et en MRC. 18 chats sains et 38 chats en MRC ont été recrutés de façon prospective. Les examens réalisés incluaient hématologie, biochimie, analyse d'urine, Serum amyloid A (SAA), haptoglobine (HAP), EPO, hepcidine,fer, TIBC et ferritinne. Nous avons observé une augmentation significative des concentrations en SAA et en hepcidine ainsi qu'une diminution significative du fer et du TIBC dans le groupe MRC (P < .05). Une corrélation positive entre la créatinine et certaines PPAs (SAA and hepcidin; P < .05) était présente. L'augmentation de SAA et hepcidine était significativement associé avec une diminution du TIBC et de l'hématocrite dans le groupe MRC. Les 14 (37%) chats anémiques du groupe MRC avaient une concentration significativement plus basse en fer et en TIBC (P < .05), changements compatibles avec une déficience fonctionelle en fer. Aucun chat n'avait un panel de fer compatible avec une carence en fer absolue. En conclusion, les résultats de cette étude suggèrent que les MRC chez le chat sont des conditions pro-inflammatoires, ayant un impact sur le métabolisme du fer. / The role of inflammation in the development and progression of feline chronic kidney disease (CKD) is not well characterized. Hepcidin, a recently discovered acute phase protein (APP) contributes to the development of anemia in human patients with CKD. The objectives of our study was to compare plasma APP including hepcidin, iron status, and erythropoietin (EPO) concentrations between healthy and cats with naturally occurring CKD. Eighteen healthy control cats and 38 cats with CKD were prospectively recruited. Complete physical examination along with hematology, biochemistry, plasma amyloid A (SAA), haptoglobine (HAP), EPO, iron, TIBC and ferritin were performed using routine laboratory analyses and commercially available feline ELISA-assays. Hepcidin-25 concentration was assessed with a human ELISA kit (DRG® Diagnostics). We found that mean SAA and hepcidin concentration were significantly higher and mean total iron and TIBC were significantly lower in the CKD group (P < .05). There was a significant positive correlation between creatinine and APPs (SAA and hepcidin; P < .05). Increase in SAA and hepcidin was also significantly associated with decrease of TIBC and PCV in the CKD group. The 14 (37%) anaemic cats with CKD had significantly and lower iron, TIBC, consistent with functional iron deficiency (P < .05). There were no patients with an iron profile suggestive of a true iron deficiency. There was no association with survival. In conclusion, our data suggest that feline CKD is a pro-inflammatory state, having significant impact on iron metabolism. With further validation, hepcidin may help better characterize these interactions.

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite &beta;-lactoglobulina, &alpha;-caseína, &beta;-caseína e &kappa;-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de &beta;-caseína e &beta;-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou &kappa;-caseína além da &beta;-caseína e &beta;-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de &beta;-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P&lt;0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P&gt;0,05), não sendo observada diferença com a LND tratada com SOL (P&gt;0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax&reg;) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da &beta;-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as &beta;-lactoglobulin, &alpha;-casein, &beta;-casein and &kappa;-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel&reg;. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed &beta;-lactoglobulin and &beta;-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed &beta;-lactoglobulin, &beta;-casein and &kappa;-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of &beta;-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P&lt;0,05). However, the medium did not have effect on growth speed of LD (P&gt;0,05) and no difference was observed at the NDL treated with LOS (P&gt;0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax&reg;) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of &beta;-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

Alterações macro e microvasculares ocorrem no Diabetes Mellitus (DM). A Retinopatia Diabética (RD) é uma complicação bem prevalente do DM e resulta de microangiopatia generalizada no tecido retiniano que, em diferentes estágios da doença, se observa como edema local, exsudatos, formação de neovasos e hemorragias, sendo a principal causa de cegueira na faixa etária de 20 a 74 anos. Alguns fatores angiogênicos são apontados como possíveis mediadores no desenvolvimento das alterações microvasculares no DM. Dentre eles, a Eritropoietina (EPO), hormônio glicoproteico, possui expressão mediada por alelos específicos do gene EPO. Determinados perfis genéticos para três SNPs-single nucleotide polymorphisms do gene EPO (rs1617640, rs507392, rs551238) encontram-se associados com maiores concentrações de EPO no humor vítreo de pacientes diabéticos com RD, o que pode ser indicativo da relação entre determinado genótipo de risco (TTA) e o desenvolvimento de tal comorbidade. O presente trabalho pretendeu investigar a associação entre o genótipo considerado de risco para o desenvolvimento de RD e alterações funcionais na visão de cores de pessoas com diagnóstico de DM. Tal estudo contou com uma amostra de 95 diabéticos (49 mulheres e 46 homens, média de idade: 48,33 anos; DP: 16,90), com um total de 31 DM tipo 1 e 64 tipo 2 e 114 controles (73 mulheres e 41 homens, média de idade: 38,38 anos; DP: 12,81). Foi realizada avaliação através de teste psicofísico (Cambridge Colour Test CCT), que visou medir o limiar de discriminação nos três eixos de confusão de cores e sequenciamento genético, a partir de amostras de sangue. Para os diabéticos, no eixo de confusão protan, os resultados mostraram uma piora de desempenho para o genótipo TTA/GCC (p= 0,048), com relação aos controles. No eixo de confusão deutan, não houve diferença para qualquer dos genótipos (p= 0,0207), enquanto que para o eixo tritan, o genótipo TTA/GCC esteve associado a uma piora de desempenho (p=0,014). Para os controles, não houve diferença entre os genótipos. Assim, os resultados mostraram uma piora na visão de cores de pacientes DM, associada ao haplótipo TTA/GCC / Diabetic Retinopathy (DR), a prevalent complication of diabetes, is the leading cause of blindness among those aged 20-74 years. Micro and macrovascular changes occur in Diabetes Mellitus (DM) with DR being the most common among these vascular complications, resulting in generalized retinal microgangiopathy. At different stages of the disease, localized edema, exudates and hemorrhages occur. Several pro-angiogenic factors have been suggested as possible mediators in the development of these microvascular changes. Among them is erythropoietin (EPO), a glycoprotein hormone whose expression is mediated by specific alleles of the EPO gene. Specific genetic profiles for three single nucleotide polymorphisms (SNPs) in the EPO gene (rs1617640, rs507392, rs551238) are associated with larger EPO concentrations in the vitreous humor of diabetic patients with RD. This association indicates a possible relationship between the identified genotypes (TTA) and the comorbidity´s development. This work aimed to investigate the association between genotypes considered at risk for the development of DR and functional changes in color vision among individuals diagnosed with DM. Thus, this study included a sample of 95 diabetic patients (49 women and 46 men, average age: 48.33 years; SD: 16.90), with a total of 31 DM type 1 and 64 type 2 and 114 controls (73 women and 41 men, average age: 38.38 years; SD: 12.81). Evaluation was performed using a psychophysical test (Cambridge Colour Test -CCT), to measure the discrimination threshold in three-color confusion axes in addition to genetic sequencing. In the protan axis of confusion color, performance among diabetics with the TTA/GCC genotype was significantly lower (p=0.048) than those in the control group. The results showed a poorer performance among those with the TTA / GCC genotype (p = 0.048), compared to controls. In deutan axis confusion, there was no difference among any of the genotypes (p = 0.0207), while for the tritan axis, the TTA / GCC genotype was associated with a poorer performance (p = 0.014). For the controls, there was no difference between genotypes. Thus, the results showed a worsening in color vision among DM patients associated with TTA / GCC haplotype

Alterações macro e microvasculares ocorrem no Diabetes Mellitus (DM). A Retinopatia Diabética (RD) é uma complicação bem prevalente do DM e resulta de microangiopatia generalizada no tecido retiniano que, em diferentes estágios da doença, se observa como edema local, exsudatos, formação de neovasos e hemorragias, sendo a principal causa de cegueira na faixa etária de 20 a 74 anos. Alguns fatores angiogênicos são apontados como possíveis mediadores no desenvolvimento das alterações microvasculares no DM. Dentre eles, a Eritropoietina (EPO), hormônio glicoproteico, possui expressão mediada por alelos específicos do gene EPO. Determinados perfis genéticos para três SNPs-single nucleotide polymorphisms do gene EPO (rs1617640, rs507392, rs551238) encontram-se associados com maiores concentrações de EPO no humor vítreo de pacientes diabéticos com RD, o que pode ser indicativo da relação entre determinado genótipo de risco (TTA) e o desenvolvimento de tal comorbidade. O presente trabalho pretendeu investigar a associação entre o genótipo considerado de risco para o desenvolvimento de RD e alterações funcionais na visão de cores de pessoas com diagnóstico de DM. Tal estudo contou com uma amostra de 95 diabéticos (49 mulheres e 46 homens, média de idade: 48,33 anos; DP: 16,90), com um total de 31 DM tipo 1 e 64 tipo 2 e 114 controles (73 mulheres e 41 homens, média de idade: 38,38 anos; DP: 12,81). Foi realizada avaliação através de teste psicofísico (Cambridge Colour Test CCT), que visou medir o limiar de discriminação nos três eixos de confusão de cores e sequenciamento genético, a partir de amostras de sangue. Para os diabéticos, no eixo de confusão protan, os resultados mostraram uma piora de desempenho para o genótipo TTA/GCC (p= 0,048), com relação aos controles. No eixo de confusão deutan, não houve diferença para qualquer dos genótipos (p= 0,0207), enquanto que para o eixo tritan, o genótipo TTA/GCC esteve associado a uma piora de desempenho (p=0,014). Para os controles, não houve diferença entre os genótipos. Assim, os resultados mostraram uma piora na visão de cores de pacientes DM, associada ao haplótipo TTA/GCC / Diabetic Retinopathy (DR), a prevalent complication of diabetes, is the leading cause of blindness among those aged 20-74 years. Micro and macrovascular changes occur in Diabetes Mellitus (DM) with DR being the most common among these vascular complications, resulting in generalized retinal microgangiopathy. At different stages of the disease, localized edema, exudates and hemorrhages occur. Several pro-angiogenic factors have been suggested as possible mediators in the development of these microvascular changes. Among them is erythropoietin (EPO), a glycoprotein hormone whose expression is mediated by specific alleles of the EPO gene. Specific genetic profiles for three single nucleotide polymorphisms (SNPs) in the EPO gene (rs1617640, rs507392, rs551238) are associated with larger EPO concentrations in the vitreous humor of diabetic patients with RD. This association indicates a possible relationship between the identified genotypes (TTA) and the comorbidity´s development. This work aimed to investigate the association between genotypes considered at risk for the development of DR and functional changes in color vision among individuals diagnosed with DM. Thus, this study included a sample of 95 diabetic patients (49 women and 46 men, average age: 48.33 years; SD: 16.90), with a total of 31 DM type 1 and 64 type 2 and 114 controls (73 women and 41 men, average age: 38.38 years; SD: 12.81). Evaluation was performed using a psychophysical test (Cambridge Colour Test -CCT), to measure the discrimination threshold in three-color confusion axes in addition to genetic sequencing. In the protan axis of confusion color, performance among diabetics with the TTA/GCC genotype was significantly lower (p=0.048) than those in the control group. The results showed a poorer performance among those with the TTA / GCC genotype (p = 0.048), compared to controls. In deutan axis confusion, there was no difference among any of the genotypes (p = 0.0207), while for the tritan axis, the TTA / GCC genotype was associated with a poorer performance (p = 0.014). For the controls, there was no difference between genotypes. Thus, the results showed a worsening in color vision among DM patients associated with TTA / GCC haplotype

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite &beta;-lactoglobulina, &alpha;-caseína, &beta;-caseína e &kappa;-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de &beta;-caseína e &beta;-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou &kappa;-caseína além da &beta;-caseína e &beta;-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de &beta;-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P&lt;0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P&gt;0,05), não sendo observada diferença com a LND tratada com SOL (P&gt;0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax&reg;) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da &beta;-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as &beta;-lactoglobulin, &alpha;-casein, &beta;-casein and &kappa;-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel&reg;. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed &beta;-lactoglobulin and &beta;-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed &beta;-lactoglobulin, &beta;-casein and &kappa;-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of &beta;-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P&lt;0,05). However, the medium did not have effect on growth speed of LD (P&gt;0,05) and no difference was observed at the NDL treated with LOS (P&gt;0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax&reg;) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of &beta;-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models. Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion. Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h. Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.