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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.
61

The Histidine-rich Glycoprotein in Reproduction

Lindgren, Karin E January 2016 (has links)
Infertility affects 15% of reproductive-aged couples. The milieu surrounding the growing embryo is of outmost importance, and should be optimised during in vitro fertilisation (IVF). Many biological processes, such as angiogenesis, coagulation, and immune processes need to be well regulated for a pregnancy to occur and progress normally. Histidine-rich glycoprotein (HRG) is a plasma protein that regulates components of these systems by building complexes with various ligands. A single nucleotide polymorphism (SNP) in HRG, denoted HRG C633T, seem to be of importance for IVF treatment outcomes. The aim of this thesis was to further investigate the proposed human fertility effects of the HRG C633T SNP. According to the findings of this thesis, the HRG C633T genotype is associated with primary recurrent miscarriage. Male HRG C633T genotype is associated with semen characteristics in infertile men, and pregnancy rates following IVF. However, the distribution of the HRG C633T SNP does not differ between infertile and fertile couples. We further examined the role of the region surrounding the HRG C633T SNP for regulation of endometrial angiogenesis and human embryo development. The region affects primary endometrial endothelial cell migration, proliferation and tube-formation in vitro but does not appear to affect human embryo development. No effect of the HRG peptide was noted on the secretome of human embryos. However, early embryos secrete proteins into the surrounding culture media and the level of secretion of VEGF-A, IL-6, EMMPRIN and PlGF is greater in embryos of higher developmental stages. In conclusion, the HRG C633T genotype appears to play a role only if infertility is established. The region surrounding HRG C633T SNP is of relevance in vitro for regulation of human endometrial endothelial cell angiogenesis. To predict which embryos to transfer in IVF, we have highlighted a number of proteins of interest for further investigation.
62

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.
63

Diagnostic rapide de la tuberculose par culture / Rapid diagnosis of tuberculosis by culture

Asmar, Shady 30 September 2015 (has links)
L'isolement de Mycobacterium tuberculosis par culture est la méthode de référence pour le diagnostic de la tuberculose. Le but de notre travail était d'améliorer et de faciliter le diagnostic par culture de la tuberculose. Dans un premier temps, nous avons produit une revue bibliographique en comparant les différentes techniques ou protocoles utilisés pour le diagnostic de la tuberculose. Ce travail nous a permis d'actualiser notre protocole de diagnostic, avec la mise en place d’un "kit-tuberculose" contenant des containers imprégnés de chlorhexidine pour la récupération et la décontamination directe d’échantillons cliniques non- invasifs, suivi par la culture sur un milieu solide à base d'oeuf, et détection des colonies par microscope inversé ou par un système d'imagerie en temps réel. Nous avons mis en place une méthode de décontamination par 0,7%-chlorhexidine et avons montré que cette méthode était plus efficace que la méthode de référence NALC-NaOH. Ensuite, nous avons développé un milieu de culture à base de sérum animal, le MOD9 dont nous avons montré par une étude comparative qu'il était supérieur au milieu solide LJ de référence. Une deuxième étude comparant un protocole de décontamination par la chlorhexidine et culture sur milieu MOD9 au protocole standard, NALC-NaOH/Bactec960 a montré une supériorité par rapport au protocole standard. Enfin, la mise en place d'un système de détection des micro-colonies de M. tuberculosis sur MOD9 par imagerie en temps réel Advencis-Biosystem a permis de réduire le temps de détection de M. tuberculosis à 3,2 jours avec le protocole chlorhexidine/MOD9/Advencis, avec un record mondial de détection en 25h. / Isolation of Mycobacterium tuberculosis by culture is the gold standard for the diagnosis of tuberculosis. The aim of my thesis work was to simplify and improve the culture diagnosis of tuberculosis. At first we started with a bibliographic study, comparing step by step the different techniques and protocols that have been used for the diagnosis of tuberculosis. This work has allowed us to update our tuberculosis diagnosis protocol, starting with the implementation of a "Tuberculosis-kit" consisting of chlorhexidine containing containers for the recovery and decontamination of non-invasive specimens, followed by culture on an egg-based medium, a micro- colonies detection using an inverted microscope or an automated real-time imaging incubator system and finally an identification using mass spectrometry. We established a new chlorhexidine- based decontamination method that we showed to be more efficient for the recovery and isolation of M. tuberculosis than the standard NALC-NaOH method. Than we developed a new serum-based culture medium, the MOD9 that we showed in a comparative study to be superior to the reference LJ medium for the recovery of M. tuberculosis. In a second study we proved that our chlorhexidine/MOD9 protocol was superior to the standard NALC-NaOH/Bactec 960 MGIT protocol for the isolation of M. tuberculosis. And finally the implementation of a real time imaging system for the detection of M. tuberculosis micro-colonies on MOD9 permits us to dramatically reduce the detection time from 15 days with the standard NALC-NaOH/Bactec 960 MGIT protocol to 3.2 days with our 0.7%-chlorhexidine/MOD9/Advencis-Biosystem protocol with a world record detection time of 25h.
64

Electrochemical behaviors of micro-arc oxidation coated magnesium alloy

Liu, Jiayang January 2014 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / In recent years, magnesium alloys, due to their high strength and biocompatibility, have attracted significant interest in medical applications, such as cardiovascular stents, orthopedic implants, and devices. To overcome the high corrosion rate of magnesium alloys, coatings have been developed on the alloy surface. Most coating methods, such as anodic oxidation, polymer coating and chemical conversion coating, cannot produce satisfactory coating to be used in human body environment. Recent studies demonstrate that micro-arc oxidation (MAO) technique can produce hard, dense, wear-resistant and well-adherent oxide coatings for light metals such as aluminum, magnesium, and titanium. Though there are many previous studies, the understanding of processing conditions on coating performance remains elusive. Moreover, previous tests were done in simulated body fluid. No test has been done in a cell culture medium, which is much closer to human body environment than simulated body fluid. In this study, the effect of MAO processing time (1 minute, 5 minutes, 15 minutes, and 20 minutes) on the electrochemical behaviors of the coating in both conventional simulated body fluid and a cell culture medium has been investigated. Additionally a new electrolyte (12 g/L Na2SiO3, 4 g/L NaF and 4 ml/L C3H8O3) has been used in the MAO coating process. Electrochemical behaviors were measured by performing potentiodynamic polarization and electrochemical impedance spectroscopy tests. In addition to the tests in simulated body fluid, the MAO-coated and uncoated samples were immersed in a cell culture medium to investigate the corrosion behaviors and compare the difference in these two kinds of media. The results show that in the immersion tests in conventional simulated body fluid, the 20-minute MAO coated sample has the best resistance to corrosion due to the largest coating thickness. In contrast, in the cell culture medium, all MAO coated samples demonstrate a similar high corrosion resistance behavior, independent of MAO processing time. This is probably due to the organic passive layers formed on the coating surfaces. Additionally, a preliminary finite element model has been developed to simulate the immersion test of magnesium alloy in simulated body fluid. Comparison between the predicted corrosion current density and experimental data is discussed.

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