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

Recombinant synthesis of elastin-mimetic proteins has been employed for several decades, however, long-term biocompatibility and biostability of such proteins was not fully defined. We present virtually crosslinked elastin-mimetic proteins which exhibit exceptional biocompatibility and long-term biostability over a period of at least seven months. This report is the first evidence of a non-chemically or ionically crosslinked system that exhibits long-term in vivo stability. Although, physically crosslinked protein-based materials possess a number of advantages over their chemically crosslinked counterparts, physical crosslinks and the related domains so formed may be deformed or damaged at applied stresses lower than those required to disrupt covalent crosslinks. In this regard, we have synthesized a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. We have demonstrated that chemical crosslinking provides an independent mechanism for control of protein mechanical responses. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. A number of reports have described the design of synthetic genes, which encode elastin-like proteins for bacterial expression in Escherichia coli. Although advantages with this expression system exist, significant limitations including the lack of eukaryotic post-translational systems, the tendency to sequester mammalian proteins into inclusion bodies, difficult purification protocols, and endotoxin contamination have been noted. We demonstrate the expression of a recombinant elastin-mimetic protein from P. pastoris. A novel synthetic strategy, monomer library concatamerization, was utilized in designing non-repetitive elastin genes for highly repetitive protein sequences. It is likely that this strategy will be useful for creating large, repetitive genes for a variety of expression systems in order to more closely approach the genetic diversity inherent to native DNA sequences. All told, elastin-based protein polymers are a promising class of material characterized by high degree of biocompatibility, excellent biostability, and a tunable range of mechanical properties from plastic to elastic. A variety of options facilitate the processing of these biopolymers into chemically crosslinked or non-crosslinked gels, films, or nanofibers for any of a number of implant applications including structural components of artificial organs and engineered living tissues, carriers for controlled drug release, or biocompatible surface coatings.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Le Gallid herpesvirus 2 (GaHV-2), responsable de lymphomes T du poulet, est contrôlé par le vaccin CVI988/Rispens. Mes travaux de thèse ont montré que le vaccin était composé, au contraire des souches virulentes, d’une population dynamique de variants viraux majoritairement délétés de la région promotrice et d’une partie variable de l’extrémité 5’ du gène LAT codant des microARN et associé à la latence virale. Dans une approche vaccinale, un virus recombinant correspondant à l’un des variants majoritaires du vaccin CVI988/Rispens a été généré à partir d’une souche GaHV-2 hypervirulente, clonée en bacmide. Nous avons montré que ce recombinant, présentant une perte de pathogénicité presque totale, était capable de protéger significativement les poulets lors d’une épreuve avec des souches GaHV-2 hypervirulentes. Ces travaux posent les bases du développement de nouveaux vaccins à partir de souches hypervirulentes émergentes. / Gallid herpesvirus 2 (GaHV-2), responsible for T-cell lymphomas chicken, is controlled by the vaccine CVI988/Rispens. My work has shown that the vaccine contains, unlike virulent strain, a viral variants population mostly deleted from the promoter region and a variable portion of the 5' end of the gene LAT encoding microRNA and associated with viral latency. In a vaccine approach, a recombinant virus corresponding to a majority variant of the CVI988/Rispens vaccine was generated from a hypervirulent strain GaHV-2, cloned as bacmid. We showed that recombinant, with an almost total loss of pathogenicity, was able to significantly protect chickens against challenge with virulent strains GaHV-2. This work lays the basis for the development of new vaccines from emerging virulent strains.

The focus of this research is to describe the production and characterization of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in insect cells, using Autographa californica buclear polyhedrosis virus (AcNPV) as an expression vector. All three forms of biological activity of hGM-CSF. Following N-glycanase treatment, the two glycosylated hGM-CSF proteins (15.5 and 16.5 KDa) which bound to Concanavalin A affinity column ran as a 14.5-15.5 KDa band on SDS-PAGE. Western blot analysis of expression in Sf9 cells treated with tunicamycin revealed only the presence of the 14.5 KDa species. The N-terminal amino acid sequence of the recombinant hGM-CSF was identical to that of natural hGM-CSF deduced from cDNA. These results demonstrate that baculovirus-produced hGM-CSF could be N-glycosylated in Sf9 cells, the signal peptide of recombinant hGM-CSF could be recognized and cleaved by infected insect cells and the resultant molecule secreted into the medium.

No description available.

Recombinant tags are used extensively in protein expression systems to allow purification through IMAC (Immobilized Metal Affinity Chromatography), identification through Western blot, and to facilitate crystal formation for structural analysis. While widely used, their role in enzyme characterization has raised concerns with respect to potential impact on activity. In this study, a flavonol-specific 3-O glucosyltransferase (Cp3GT) from grapefruit (Citrus paradisi) was expressed in Pichia pastoris, and was assayed in its untagged form and with a C-terminal c-myc/6x His tag under various conditions to determine the effect of tags. Prior characterization of pH optima for Cp3GT obtained through expression in Escherichia coli, containing an N-terminal thioredoxin/6x His tag, indicated an optimal pH of 7–7.5, which is indicative of a normal physiological pH and agrees with other glucosyltransferase (GT) pH optima. However, characterization of Cp3GT expressed using P. pastoris with a C-terminal c-myc-6x His tag showed a higher optimal pH of 8.5–9. This suggests a possible tag effect or an effect related to physiological differences between the cell expression systems. Results testing recombinant Cp3GT expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima.

Résumé confidentiel / Résumé confidentiel

Neste trabalho foram estudados diversos métodos cromatográficos para a purificação de fatores recombinantes de coagulação VII (FVIIr) e VIII (FVIIIr) derivados de linhagens celulares humanas SK-Hep. O FVIIIr é utilizado para o tratamento da Hemofilia A, enquanto o FVIIr é utilizado para o tratamento da Hemofilia B e também a Hemofilia A. Produzir estes fatores em linhagens celulares humanas faz com os padrões de glicosilação, sulfatação e enovelamento destas proteínas sejam extremamente parecidos com os fatores endógenos produzidos no organismo humano. A purificação do FVIIIr através de técnicas de cromatografia multimodais usando a resina CaptoMMC, afinidade usando a resina FVIIISelect e troca iônica (SP-Sepharose) permitiu obter um produto bastante homogêneo e com perfil de banda (por SDS-PAGE) bem definido que demonstrou a presença esperada das cadeias leve e pesada (o Westen-Blott indicou que os anticorpos comerciais reconheceram a cadeia pesada da molécula estudada). As técnicas permitiram uma alta reprodutibilidade do processo onde sequencias de purificação indicaram o mesmo comportamento de perfis cromatográficos e o processo eliminou 99.5% ± 0,5% de proteínas inespecíficas, recuperando até 64% de FVIIIr. O FVIIr foi purificado com apenas uma única técnica cromatográfica usando a resina FVIISelect que isolou a proteína de interesse eliminando cerca de 99% de impurezas, recuperando praticamente todo o produto. O eluido da cromatografia de afinidade foi dialisado em membranas de 5 kDa o que resultou no processo de auto ativação da molécula de FVIIr, resultando em um aumento de sinal de até 5x em relação a quantidade inicial. O gel de SDS-PAGE e o Westen-Blott comprovaram o processo de auto-ativação no qual uma migração de banda de 50 kDa para 30kDa foi observada e os anticorpos comerciais contra FVII foram capazes de detecta-la. O método de purificação também foi bastante reproduzível e o perfil de banda muito semelhante se comparado ao produto comercial existente no mercado. Sendo assim, foi possível obter plataformas de purificação para as proteínas FVIIr e FVIIIr. / In this work, several chromatographic methods were studied for the purification of recombinant clotting factors VII (FVIIr) and VIII (FVIIIr) derived from human SK-Hep cell lines. The FVIIIr is used for the treatment of Hemophilia A, while the FVIIr is used for the treatment of Hemophilia B and Hemophilia A. Producing these factors in human cell lines results in glycosylation, sulphation and folding patterns similar to the endogenous factors produced in the human organism. Purification of FVIIIr by multimodal chromatography techniques using CaptoMMC resin, affinity using FVIIISelect resin and ion exchange (SP-Sepharose) yielded a fairly homogeneous and well-defined band profile (by SDS-PAGE) which demonstrated the expected presence of the light and heavy chains, Westen-Blott indicated that commercial antibodies recognized the heavy chain of the studied molecule. The techniques allowed a high reproducibility of the process where purification sequences indicated the same behavior of chromatographic profiles and the process eliminated 99.5% ± 0.5% nonspecific proteins and recovering up to 64% FVIIIr. FVIIr was purified with only a single chromatographic technique using the FVIISelect resin which isolated the protein by removing about 99% impurities and recovering virtually the entire product. The affinity chromatography eluate was dialyzed on 5 kDa membranes which resulted in the autoactivation process of the FVIIr molecule resulting in a signal increase of up to 5 fold over the initial amount. The SDS-PAGE gel and Westen-Blott demonstrated the auto-activation process where a migration of 50 kDa to 30 kDa band was observed and the commercial antibodies against FVII were able to detect the band. The purification method was also quite reproducible and the band profile very similar compared to the commercial products. Thus, it was possible to obtain purification platforms for the FVIIr and FVIIIr proteins.

O objetivo deste estudo foi a avaliação histopatológica e radiográfica da resposta pulpar e periapical de dentes de cães após pulpotomia e utilização da Proteína Morfogenética Óssea Recombinante Humana 7. Foram utilizados 60 dentes (120 raízes) de 6 cães, divididos em 8 grupos, nos períodos experimentais de 7 dias (Grupos I, II, III, IV) e 70 dias (Grupos V, VI, VII, VIII). Após a pulpotomia, o remanescente pulpar foi recoberto com os seguintes materiais: Grupos I e V - Proteína Óssea Morfogenética Recombinante Humana 7 (rHuBMP-7) associada ao Colágeno Recombinante Humano (rHuCollagen); Grupos II e VI - Colágeno Recombinante Humano (rHuCollagen); Grupos III e VII (Controle Negativo) - Hidróxido de Cálcio p.a. e soro fisiológico e Grupos IV e VIII (Controle Positivo) - Óxido de Zinco e Eugenol. Decorridos os períodos experimentais, os animais foram mortos, as peças removidas e submetidas ao processamento histológico. A avaliação histopatológica foi realizada subjetivamente em microscópio óptico. A avaliação radiográfica foi realizada considerando-se a integridade da lâmina dura, presença de áreas de rarefação óssea periapical, de reabsorções radiculares (interna e externa) e de ponte de dentina, sendo os resultados submetidos à análise estatística utilizando-se o teste exato de Fisher. Nos espécimes que apresentavam áreas de rarefação periapical, as medidas radiográficas das lesões foram comparadas entre os grupos por meio do teste de Kruskall-Wallis. Os achados histopatológicos evidenciaram que no período de 7 dias, nos Grupos I e II havia um infiltrado inflamatório severo e intensa proliferação vascular no tecido pulpar, no Grupo IV um infiltrado inflamatório moderado enquanto no Grupo III foi observado um infiltrado inflamatório leve, estando o tecido pulpar íntegro. Em todos os grupos não havia formação de ponte de dentina e a região periapical apresentava aspectos de normalidade. No período de 70 dias, nos Grupos V, VI e VIII não houve formação de ponte de dentina, o tecido pulpar apresentava áreas de necrose com presença de células inflamatórias na região periapical e reabsorção cementária e óssea. Por outro lado, no Grupo VII, foi observada presença de ponte de dentina, ausência de processo inflamatório e ausência de reabsorção dos tecidos mineralizados. Com relação aos achados radiográficos, no período de 7 dias, todos os espécimes dos Grupos I, II, III e IV apresentavam integridade da lâmina dura, ausência de rarefação óssea periapical, ausência de reabsorção radicular (interna e externa) e ausência de ponte de dentina. No período de 70 dias, nos Grupos V, VI e VIII não houve formação de ponte de dentina em nenhum espécime sendo observadas áreas de rarefação óssea periapical em 100% das raízes do Grupo VI, 60% das raízes do Grupo VIII e 40% das raízes do Grupo V, sendo as maiores lesões encontradas no Grupo VI, seguida pelos Grupos V e VIII (p<0,05). No grupo VII, foi observada presença de ponte de dentina em 60% dos casos, integridade da lâmina e ausência de rarefação óssea periapical em 100% dos casos. Pode-se concluir que a Proteína Óssea Morfogenética Recombinante Humana 7 quando associada ao Colágeno Recombinante Humano não apresentou resultados satisfatórios. / The purpose of this study was to evaluate, both histopathologically and radiographically, the pulpal and periapical response of dogs’ teeth after pulpotomy and use of recombinant human bone morphogenetic protein-7 (rHuBMP-7). For such purpose, 60 teeth (120 roots), obtained from 6 dogs, were divided in 8 groups and evaluated in two experimental periods: 7 days (Groups I, II, III, IV) and 70 days (Groups V, VI, VII, VIII). After pulpotomy, pulp remnant was covered with the following materials: Groups I and V - recombinant human bone morphogenetic protein-7 (rHuBMP-7) associated to recombinant human like collagen (rHuCollagen); Groups II and VI - recombinant human like collagen (rHuCollagen); Groups III and VII (negative control) – calcium hydroxide and sodium chloride solution; and Groups IV and VIII (positive control) – zinc oxide and eugenol. At the established experimental periods, the animals were sacrificed and the anatomic pieces were obtained and histologically processed. The histopathologic evaluation was realized subjectively in a light microscope. The radiographic evaluation was performed considering the integrity of the lamina dura, presence of areas of periapical bone rarefaction, root resorption (internal and external) and dentin bridge formation. The results were analyzed statistically using Fisher\'s exact test. In the specimens presenting periapical bone rarefaction areas, the lesions’ radiographic measurements were compared among the groups using the Kruskall-Wallis test. The histopathologic findings in the 7-day period revealed that Groups I and II presented a severe inflammatory infiltrate and intense vascular proliferation in the pulp tissue, Group IV presented a moderate inflammatory infiltrate while Group III presented a mild inflammatory infiltrate and intact pulp tissue. In all groups, there was no dentin bridge formation and the periapical region had normal appearance. In the 70-day period, Groups V, VI and VIII showed no dentin bridge formation and pulp tissue presented necrotic areas with inflammatory cells in the periapical region as well as bone and cemental resorption. On the other hand, in Group VII, there was dentin bridge formation, absence of inflammatory process and absence of resorption of mineralized tissues. Regarding the radiographic findings, in the 7-day period, all specimens in Groups I, II, III and IV present intact lamina dura, absence of periapical bone rarefaction, absence of root resorption (internal and external) and absence of dentin bridge formation. In the 70-day period, Groups V, VI and VIII did not present dentin bridge formation in any specimen. Periapical bone rarefaction areas were observed in 100% of the roots in Group VI, 60% of the roots in Group VIII and 40% of the roots in Group V. The largest lesions were found in Group VI, followed by Groups V and VIII (p<0.05). In Group VII, there was dentin bridge formation in 60% of the cases, intact lamina dura and absence of periapical bone rarefaction in 100% of the cases. Based on these results, it may be concluded that recombinant human bone morphogenetic protein-7 associated to recombinant human like collagen did not present satisfactory results.