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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
41

Impact of autocrine factors on physiology and productivity in Trichoplusia ni serum-free cultures

Eriksson, Ulrika January 2005 (has links)
The aim of this study was to increase the understanding of the mechanisms regulating cell proliferation and recombinant protein production in serum-free cultures of Trichoplusia ni (T. ni) insect cells. Conditioned medium (CM) was shown to contain both stimulatory and inhibitory factors (CM factors) influencing cell growth. Metalloproteinase (MP) activity was the major factor responsible for the growth stimulating effect of CM as shown by using the specific MP inhibitor DL-thiorphan. MPs may exist in several different molecular mass forms due to autoproteolysis. Although the main band of the MP was determined to be around 48 kDa, precursor forms above 48 kDa as well as autocatalytic degradation products below the main band could be observed. It is not clear whether all forms of the MP or just the main band is involved in the growth regulation. Further, a proteinase inhibitor could be identified in the inhibitory fraction. Thus, we speculate that the proteinase inhibitor may be part of an autocrine system regulating cell proliferation. Analysis of the cell cycle phase distribution revealed a high proportion of cells in the G1 (80-90 %) and a low proportion of cells in the S and G2/M phases (10-20 %) during the whole culture, indicating that S and G2/M are short relative to G1. After inoculation, a drastic decrease in the S phase population together with a simultaneous increase of cells in G1 and G2/M could be observed as a lagphase on the growth curve and this may be interpreted as a temporary replication stop. When the cells were released from the initial arrest, the S phase population gradually increased again. This was initiated earlier in CM-supplemented cultures, and agrees with the earlier increase in cell concentration. Thus, these data suggests a correlation between CM factors and the cell cycle dynamics. In cultures supplied with CM, a clear positive effect on specific productivity was observed, with a 30 % increase in per cell productivity. The specific productivity was also maintained at a high level much longer time than in fresh-medium cultures. The positive effect observed after 20 h coincided with the time a stimulatory effect on cell growth first was seen. Thus, the productivity may be determined by the proliferation potential of the culture. A consequence of this would be that the secreted MP indirectly affects productivity. Finally, the yeast extract from Express Five SFM contains factors up to 35 kDa which are essential for T. ni cell growth. The optimal concentration was determined to be 2.5-fold that in normal medium, while higher concentrations were inhibitory. However although vital, they were not solely responsible for the growth-enhancing effect, as some other, more general, component present in yeast extract was needed for proliferation as well. / <p>QC 20101129</p>
42

Optimisation du traitement du cancer du sein Triple-Négatif : développement des modèles de culture cellulaire en trois dimensions, efficacité de l'Olaparib (anti-PARP1) en combinaison avec la radiothérapie et chimiorésistance instaurée par les protéines Multi Drug Résistance / Optimization of triple-negative breast cancer treatment : development of three-dimensional cell culture models, efficacy of Olaparib (anti-PARP1) in combination with radiotherapy and chemoresistance introduced by "Multi Drug Resistance" proteins

Dubois, Clémence 21 December 2018 (has links)
Le cancer du sein est une maladie complexe et difficile à caractériser. Parmi les différents sous-types moléculaires, les tumeurs du sein Triple-Négatives (TN) sont particulièrement agressives et de mauvais pronostic. Elles sont caractérisées par une absence d’expression des récepteurs aux œstrogènes (ER), à la progestérone (PR), l’absence de surexpression du récepteur Human Epidermal growth factor 2 (HER2) et de fréquentes mutations sur les gènes BRCA1/2 (profil « BRCAness »). En absence de thérapies ciblées efficaces, de nombreux traitements ciblés notamment les inhibiteurs de poly-ADP-ribose polymérases (anti-PARPs) sont actuellement en cours de développement, en recherche préclinique et clinique. Basés sur le principe de létalité synthétique, les anti-PARPs ciblent les propriétés BRCAness des tumeurs TN. Dans ce contexte, ces travaux de recherche ont été orientés sur le développement d’outils diagnostics afin d’optimiser l’efficacité des anti-PARPs sur des tumeurs TN. Pour ce faire, dans un premier temps, des cultures cellulaires en 3D via la technique Liquid Overlay ainsi que des tests de cytotoxicités associés ont été développés, à partir des lignées cellulaires MDA-MB-231 et SUM1315 de phénotype TN. Ces deux modèles de sphéroïdes ont ensuite été optimisés/normalisés dans un milieu de culture synthétique intitulé OPTIPASS (BIOPASS). Dans un deuxième temps, l’efficacité d’un co-traitement combinant l’anti-PARP1 Olaparib à faibles et à fortes doses et la radiothérapie fractionnée (5x2 Gy) a été modélisée sur les deux lignées MDA-MB-231 et SUM1315, en conditions 2D et 3D. Ces expériences ont clairement mis en évidence un effet potentialisateur de l’Olaparib sur la radiothérapie (i) en présence de faibles doses de cet anti-PARP (5 µM ou inférieur) (ii) à long terme et (iii) en présence d’un fractionnement maximum (5x2 Gy). De plus, les lignées tumorales TN étudiées présentaient des différences de sensibilité vis-à-vis du co-traitement. Ainsi, une analyse transcriptomique in silico a mis en évidence des profils très différents de ces lignées hautement métastatiques et très agressives. Notamment, la lignée SUM1315 semblait présenter un engagement neuronal, suggérant son origine métastatique cérébrale. Ces résultats encourageants pourraient ouvrir de nouvelles perspectives pour le traitement des métastases cérébrales de tumeurs mammaires TN, très fréquentes chez ce sous-type. Dans un troisième temps, afin de mieux caractériser le mode d’action de l’Olaparib sur ces modèles de sphéroïdes, un dérivé fluorescent de l’Olaparib, l’Ola-FL, a été synthétisé et caractérisé. L’analyse de la pénétration et de la distribution de l’Ola-FL au sein des sphéroïdes MDA-MB-231 et SUM1315 a mis en évidence une distribution rapide et homogène du composé ainsi que sa persistance après 3h d’incubation, dans toute la profondeur des sphéroïdes et notamment dans les zones hypoxiques centrales. Enfin, l’analyse de la co-expression de deux pompes Multidrug Resistance (MDR) majeures, la MRP7 et la P-gp après le traitement des deux lignées TN avec l’Olaparib, a mis en évidence sur les cultures 2D, une expression de type relai de la MRP7 et la P-gp. Sur les sphéroïdes traités avec une faible dose d’Olaparib à long terme, une expression basale de la MRP7 et une surexpression de la P-gp ont été détectées, au sein des cellules résiduelles périphériques des sphéroïdes. Ces résultats mettent clairement en évidence l’implication des pompes d’efflux dans les mécanismes de résistances à l’Olaparib, dans ces tumeurs agressives. L’ensemble des résultats issus de la modélisation de l’action de l’Olaparib sur des sphéroïdes MDA-MB-231 et SUM1315 laissent supposer sa plus grande efficacité à faible dose et à long-terme, notamment dans les zones hypoxiques des sphéroïdes, probablement aussi à l’origine de son effet potentialisateur avec la radiothérapie. / Breast cancer is a very complex and heterogeneous disease. Among the different molecular subtypes, Triple-Negative (TN) breast cancers are particularly aggressive and of poor prognosis. TN tumours are characterized by a lack of estrogen receptors expression (ER), progesterone receptors expression (PR), the absence of Human Epidermal growth factor receptor 2 overexpression (HER2) of the frequent mutations on BRCA1 / 2 genes ("BRCAness" phenotype). In the absence of effective targeted therapies, many targeted therapies including poly-ADP-ribose polymerase inhibitors (anti-PARPs) are currently under development in preclinical and clinical studies. Based on the synthetic lethality concept, the anti-PARPs specifically target the BRCAness properties of TN tumors. In this context, these works were focused on the development of diagnostic tools for the optimization of TN tumours treatment with anti-PARPs. For this, firstly, 3D cell cultures formed with the Liquid Overlay technique as well as associated cytotoxicity tests were developed, from the TN breast cancer cell lines MDA-MB-231 and SUM1315. These two spheroid models were then optimized and standardized in a synthetic culture medium called OPTIPASS (BIOPASS). Secondly, the efficacy of a co-treatment combining anti-PARP1 Olaparib at low and high doses and fractioned radiotherapy (5x2 Gy) was analyzed on the two cell lines MDA-MB-231 and SUM1315 cultured in 2D and 3D conditions. These experiments clearly demonstrated a potentiating effect of Olaparib on radiotherapy (i) in presence of low doses of this anti-PARP (5 μM or inferior) (ii) at long term and (iii) in presence of the maximum fractionation (5x2 Gy). In addition, these two TN cell lines showed a heterogeneous sensitivity to the co-treatment. Thus, an in silico transcriptomic analysis revealed very different profiles of these highly metastatic and highly aggressive cell lines. Notably, the SUM1315 cell line presented a neuronal commitment, suggesting its cerebral metastatic origin. These promising results could open up new perspectives for the treatment of TN tumours brain metastases, which are very common in this subtype. Thirdly, in order to better characterize the mode of action of Olaparib on these spheroid models, a fluorescent derivative of Olaparib, Ola-FL, was synthesized and characterized. The analysis of Ola-FL penetration and distribution in MDA-MB-231 and SUM1315 spheroids showed a rapid and homogeneous distribution of the compound as well as its persistence after 3h of incubation, in all the depth of the spheroids and especially in the central hypoxic zones. Finally, the analysis of the co-expression of two major Multidrug Resistance (MDR) pumps, MRP7 and P-gp after the treatment of the two TN lines with Olaparib, revealed on 2D cultures, a relay type expression of the MRP7 and the P-gp. On spheroids treated with a low dose of Olaparib art long term (10 days), a basal expression of MRP7 and an overexpression of P-gp were detected in the peripheral residual cells of the spheroids. These results clearly highlighted the involvement of these efflux pumps in Olaparib resistance mechanisms, in these aggressive tumors. All the results resulting from the modeling of the action of Olaparib on MDA-MB-231 and SUM1315 spheroids suggest its greater efficacy at low dose and at long-term, especially in the hypoxic zones of the spheroids. This parameter might be probably at the origin of its potentiating effect with radiotherapy.
43

Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino. / Production of recombinant proteins in BHK-21 cells cultured in serum free media.

Patiño, Sandra Fernanda Suárez 06 May 2016 (has links)
Células eucariotas usadas como plataforma de expressão de proteínas recombinantes são geralmente cultivadas com soro fetal bovino (SFB), porém, abordagens biotecnológicas atuais sobre cultura de células devem evitar o uso deste suplemento, devido a problemas de custo, variações entre os lotes e risco de contaminação. Assim, nosso objetivo foi expressar as proteínas recombinantes: GFP (proteína verde fluorescente), NS3 (proteína não estrutural 3 do vírus da hepatite C) e RVGP (glicoproteína do vírus da raiva) em células BHK-21 adaptadas em meios livres de soro fetal bovino (SFM) usando o sistema de expressão baseado no Semliki Forest Virus (SFV). Os resultados do presente trabalho mostraram que células adaptadas em SFM cresceram de forma eficiente, produziram mais partículas virais recombinantes de SFV do que células suplementadas com soro, sendo que estas partículas virais podem ser usadas diretamente para imunização, pois garantiram uma amplificação e expressão eficiente das diferentes proteínas dentro da célula hospedeira. / Eukaryotic cells are cultured with serum, however current biotechnological approaches of cell culture need to avoid using of this supplement, due to the high costs, lot-to-lot variation and risk of contamination. Thus, our aim was to express the recombinant protein: GFP (green fluorescent protein); NS3 (Hepatitis C virus non-structural protein 3) and RVGP (rabies virus glycoprotein) in BHK-21 cells cultured in serum free culture based on Semliki Forest Virus system. The results of this work showed that cells cultured in serum-free media (SFM) were grown efficiently, they were produce more recombinant viral particles when compared with cells supplemented with SFB. These viral particles can be used directly for immunization, since generated amplification and expression efficient of different proteins within the host cell.
44

Adaptção de linhagens celulares humanas para crescimento em suspensão e meios de cultura livres de soro fetal bovino / Serum-free suspension adaptation of human cell lines

Biaggio, Rafael Tagé 28 March 2014 (has links)
Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8,6x106 e 1,9x106 células/mL, respectivamente) e apresentou crescimento celular durante 18 dias em cultura. A velocidade específica de crescimento máxima (?max) foi semelhante nas duas células (0,0159 h-1 para a HKB-11 e 0,0186 h-1 para SK-Hep-1). A limitação do crescimento das células adaptadas não parece estar associada à exaustão de glicose e glutamina, tampouco à formação de lactato em concentrações inibitórias. Todavia, para ambos os casos, foi observada produção de amônia em concentrações consideradas inibitórias (2 - 5 mM). De maneira geral, foi possível estabelecer culturas celulares em condições compatíveis com o desenvolvimento de um bioprocesso reprodutível, seguro e em concordância com as boas práticas de fabricação. / Human cell lines have attracted great interest since they are capable of producing glycosylated proteins in a more similar way to native human proteins, reducing the potential for immune responses against non-human epitopes. However, these human cell lines have not been extensively characterized and cultured in large scale and in serum-free suspension conditions. As a result, the main objective of this work was to adapt three human cell lines: SK-Hep-1, HepG2 and HKB-11 to serum-free suspension cultures, since they are promising systems of recombinant protein expression. For this task, four commercial serum-free media were tested. Adapted cell lines in T-flasks were further adapted to suspension cultures. Results showed that both HKB-11 and SK-Hep-1 were adapted to serum-free suspension cultures in FreeStyle and SFMII, respectively and were cryopreservated in serum-free formulations. Kinetic characterization showed that HKB-11 cell concentration was four times higher than SK-Hep-1 cell (8,6x106 and 1,9x106 cells/ml, respectively) and showed cell growth in culture over 18 days. The maximum specific growth rate (?max) was similar for both cell lines (0,0159 h-1 to HKB-11 and 0,0186h-1 to SK-Hep-1). Growth limitation of adapted human cell lines does not seem to be associated with depletion of glucose and glutamine, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production achieved inhibitory concentrations (2 - 5 mM). In general, it was possible to establish human cell cultures that are compatible with reproducible and safe bioprocess conditions and in compliance with good manufacturing practices.
45

A Comparative Analysis of the Biomechanics and Biochemistry of Cell-Derived and Cell-Remodeled Matrices: Implications for Wound Healing and Regenerative Medicine

Ahlfors, Jan-Eric Wilhelm 03 May 2004 (has links)
The purpose of this research was to study the synthesis and remodeling of extracellular matrix (ECM) by fibroblasts with special emphasis on the culture environment (media composition and initial ECM composition) and the resulting mechanical integrity of the ECM. This was investigated by culturing fibroblasts for 3 weeks in a variety of culture conditions consisting of collagen gels, fibrin gels, or media permissive to the self-production of ECM (Cell-Derived Matrix), and quantifying the mechanics of the resulting ECM. The mechanical characteristics were related to the biochemistry of the resulting ECM, notably in terms of collagen accumulation and collagen fibril diameters. The ultimate tensile strength (UTS) of the collagen gels and fibrin gels at the end of the 3-week period was 168.5 ± 43.1 kPa and 133.2 ± 10.6 kPa, respectively. The ultimate tensile strength of the cell-derived matrices was 223.2 ± 9 kPa, and up to 697.1 ± 36.1 kPa when cultured in a chemically-defined medium that was developed for the rapid growth of matrix in a more defined environment. Normalizing the strength to collagen density resulted in a UTS / Collagen Density in these groups of 6.4 ± 1.9 kPa/mg/cm3, 25.9 ± 2.4 kPa/mg/cm3, 14.5 ± 1.1 kPa/mg/cm3, and 40.0 ± 1.9 kPa/mg/cm3, respectively. Cells were synthetically more active when they produced their own matrix than when they were placed within gels. The resulting matrix was also significantly stronger when it was self-produced than when the cells rearranged the matrix within gels that corresponded to a significantly larger fraction of non-acid and pepsin extractable collagen. These studies indicate that cell-derived matrices have potential both as in vitro wound healing models and as soft connective tissue substitutes.
46

Adaptção de linhagens celulares humanas para crescimento em suspensão e meios de cultura livres de soro fetal bovino / Serum-free suspension adaptation of human cell lines

Rafael Tagé Biaggio 28 March 2014 (has links)
Linhagens celulares humanas têm atraído grande interesse devido a sua capacidade de glicosilar proteínas de maneira mais semelhante às proteínas nativas humanas, reduzindo o potencial de respostas imunológicas contra epítopos não humanos. No entanto, por se tratar de uma aplicação recente, essas células ainda não foram extensamente caracterizadas e cultivadas em condições reprodutíveis da escala industrial, ou seja, em suspensão e em meios de cultura livres de soro fetal bovino (SFB). Em função disso, o objetivo principal deste trabalho foi estabelecer culturas livres de SFB e em suspensão para as linhagens celulares humanas SK-Hep-1, HepG2 e HKB-11, que têm despertado grande interesse devido ao potencial de produção de proteínas recombinantes. Para isso, quatro formulações comerciais livres de SFB foram avaliadas. As células que apresentaram bons resultados na adaptação aos meios realizada em garrafas estáticas foram então adaptadas para crescimento em suspensão. Foi possível realizar a adaptação satisfatória da célula HKB-11 ao meio FreeStyle e da célula SK-Hep-1 ao meio SFMII bem como a criopreservação das mesmas também em condições livres de SFB. A caracterização cinética das células adaptadas mostrou que a célula HKB-11 apresentou concentração celular quatro vezes superior a da célula SK-Hep-1 (8,6x106 e 1,9x106 células/mL, respectivamente) e apresentou crescimento celular durante 18 dias em cultura. A velocidade específica de crescimento máxima (?max) foi semelhante nas duas células (0,0159 h-1 para a HKB-11 e 0,0186 h-1 para SK-Hep-1). A limitação do crescimento das células adaptadas não parece estar associada à exaustão de glicose e glutamina, tampouco à formação de lactato em concentrações inibitórias. Todavia, para ambos os casos, foi observada produção de amônia em concentrações consideradas inibitórias (2 - 5 mM). De maneira geral, foi possível estabelecer culturas celulares em condições compatíveis com o desenvolvimento de um bioprocesso reprodutível, seguro e em concordância com as boas práticas de fabricação. / Human cell lines have attracted great interest since they are capable of producing glycosylated proteins in a more similar way to native human proteins, reducing the potential for immune responses against non-human epitopes. However, these human cell lines have not been extensively characterized and cultured in large scale and in serum-free suspension conditions. As a result, the main objective of this work was to adapt three human cell lines: SK-Hep-1, HepG2 and HKB-11 to serum-free suspension cultures, since they are promising systems of recombinant protein expression. For this task, four commercial serum-free media were tested. Adapted cell lines in T-flasks were further adapted to suspension cultures. Results showed that both HKB-11 and SK-Hep-1 were adapted to serum-free suspension cultures in FreeStyle and SFMII, respectively and were cryopreservated in serum-free formulations. Kinetic characterization showed that HKB-11 cell concentration was four times higher than SK-Hep-1 cell (8,6x106 and 1,9x106 cells/ml, respectively) and showed cell growth in culture over 18 days. The maximum specific growth rate (?max) was similar for both cell lines (0,0159 h-1 to HKB-11 and 0,0186h-1 to SK-Hep-1). Growth limitation of adapted human cell lines does not seem to be associated with depletion of glucose and glutamine, nor with the formation of lactate in inhibitory concentrations. However, in both cases, ammonia production achieved inhibitory concentrations (2 - 5 mM). In general, it was possible to establish human cell cultures that are compatible with reproducible and safe bioprocess conditions and in compliance with good manufacturing practices.
47

Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino. / Production of recombinant proteins in BHK-21 cells cultured in serum free media.

Sandra Fernanda Suárez Patiño 06 May 2016 (has links)
Células eucariotas usadas como plataforma de expressão de proteínas recombinantes são geralmente cultivadas com soro fetal bovino (SFB), porém, abordagens biotecnológicas atuais sobre cultura de células devem evitar o uso deste suplemento, devido a problemas de custo, variações entre os lotes e risco de contaminação. Assim, nosso objetivo foi expressar as proteínas recombinantes: GFP (proteína verde fluorescente), NS3 (proteína não estrutural 3 do vírus da hepatite C) e RVGP (glicoproteína do vírus da raiva) em células BHK-21 adaptadas em meios livres de soro fetal bovino (SFM) usando o sistema de expressão baseado no Semliki Forest Virus (SFV). Os resultados do presente trabalho mostraram que células adaptadas em SFM cresceram de forma eficiente, produziram mais partículas virais recombinantes de SFV do que células suplementadas com soro, sendo que estas partículas virais podem ser usadas diretamente para imunização, pois garantiram uma amplificação e expressão eficiente das diferentes proteínas dentro da célula hospedeira. / Eukaryotic cells are cultured with serum, however current biotechnological approaches of cell culture need to avoid using of this supplement, due to the high costs, lot-to-lot variation and risk of contamination. Thus, our aim was to express the recombinant protein: GFP (green fluorescent protein); NS3 (Hepatitis C virus non-structural protein 3) and RVGP (rabies virus glycoprotein) in BHK-21 cells cultured in serum free culture based on Semliki Forest Virus system. The results of this work showed that cells cultured in serum-free media (SFM) were grown efficiently, they were produce more recombinant viral particles when compared with cells supplemented with SFB. These viral particles can be used directly for immunization, since generated amplification and expression efficient of different proteins within the host cell.

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