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Assessing the functional asymmetry of the Bacillus subtilis MutL homodimerLiu, Linda January 2017 (has links)
DNA mismatch repair corrects base-base mismatches and small insertion/deletion loops generated during normal DNA replication. If left unrepaired, these errors become permanent mutations and can lead to increased susceptibility to cancer. In most prokaryotes and all eukaryotes, the mismatch repair protein MutL is a sequence-unspecific endonuclease that plays an essential role in the strand discrimination step of this pathway. Prokaryotic MutL forms homodimers with two endonuclease sites, whereas eukaryotic MutL homologs form heterodimers with a single active site. To elucidate the mechanistic differences between prokaryotic and eukaryotic MutL, we tested whether both endonuclease sites are necessary for prokaryotic MutL nicking activity. MutL interaction with the processivity clamp is required to stimulate endonuclease activity. Therefore, we also tested whether both subunits of the MutL dimer needed to interact with the processivity clamp. To this end, we engineered a system to independently manipulate each protomer of the homodimer. We demonstrated that prokaryotic MutL is regulated by the processivity clamp to act in a similar manner to eukaryotic MutL with only one functional site contributing to the endonuclease activity. We also devised a strategy to stabilize the transient interactions between MutL, the β-clamp, and DNA through disulfide bridge crosslinking and heterobifunctional crosslinking. Stabilizing transient protein-protein and protein-DNA interactions will help optimize future structural studies in obtaining the ternary complex for mechanistic insights to the MutL endonuclease activity and regulation imposed by the β-clamp. / Thesis / Master of Science (MSc)
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Organic Synthesis using Bimetallic CatalysisEnce, Chloe Christine 23 April 2020 (has links)
Bimetallic Catalysis is an emerging field of study that uses two metals to cooperatively perform organic transformations. These metals can serve to activate or bind substrates in order to increase the rate and selectivity of reactions. This work first describes the synthesis and utilization of six new chiral, titanium-containing phosphinoamide ligands. These Lewis acidic ligands withdraw electron density from an active palladium center to induce chirality and increase the rate of allylic amination of hindered, secondary N-alkyl amines. X-ray quality crystals were grown for each ligand and completed the allylic amination of hindered secondary amines in minutes whereas other non-titanium-containing ligands produced trace product. Although enantioselectivity was low initially, through a dynamic kinetic resolution enantioselectivity was increased over time, reaching 53% enantioselectivity. The second type of bimetallic catalysis discussed is dinuclear Pd(II) and Pd(I) catalysis. These dimers were built on a 2-phosphinoamide ligand scaffold and present interesting molecular structure and unique reactivity. These dimers were found to perform tandem arylketone coupling to produce disubstituted naphthalene products under oxidative conditions. It is proposed that the Pd(II) dimer undergoes oxidative addition to produce a Pd(III) dimer which subsequently produces an aryl-ketone intermediate. This process is made possible by the cooperativity of the two palladium centers which enable the formation of a Pd(III) dimer, circumventing the need for the high energy Pd(IV) oxidation state. Oxidative conditions then allows coupling and cyclization of a second ketone to form the naphthalene product.
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L'homodimérisation du CD40 et son implication indirecte dans l'asthme allergiqueEl Salti, Saly 01 1900 (has links)
No description available.
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Computational Prediction of the Agregated Structure of Denatured LysozymeChotikasemsri, Pongsathorn 01 December 2009 (has links)
Mis-folded proteins and their associated aggregates are a contributing factor in some human diseases. In this study we used the protein lysozyme as a model to define aggregation structures under denaturing conditions. Sasahara et al. (2007), Frare et al. (2009, 2006), and Rubin et al. (2008) observed conditions where heat denatured lysozyme formed fibril structures that were observed to be 8-17 nanometers in diameter under the electron microscope. Even though the crystal structure of lysozyme is known, the denatured form of this protein is still unknown. Therefore, we used Rosetta++ protein folding and blind docking software to create in silico models of the protein at denaturing temperatures and subsequently docked them into aggregates. Here we compare those structures and select forms consistent with the fibril structure from the previous papers. The next step is to be able to use the predicted models of the fibrilar forms of denatured lysozyme to help us understand the exact conformation of fibril structures. This will let us confirm the docking interactions during the fibril aggregation process. The ultimate goal is to use the validated denatured structures to model interactions with heat shock proteins during the dis-aggregation process.
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Evaluation of Stallion Sperm Membrane Integrity Using Varied Flow Cytometer-Based MethodologiesStump, Karen Elizabeth 03 October 2013 (has links)
Artificial insemination using cooled, transported semen has become a popular practice in the equine industry. However, equine sperm are assumed to show a decline in their fertilizing ability after 24 to 48 hours of cooled storage. Two measures that are commonly used to estimate the fertility of an ejaculate are sperm motility and sperm membrane integrity (SMI). Recently, it has been suggested that SMI may have a better correlation with fertility of an inseminate than sperm motility. The effect of cooled-storage on sperm quality over an extended time period was evaluated to illustrate changes in sperm characteristics that might be related to an ejaculate’s fertility.
Semen was stored at 4°C in INRA 96 extender containing 10% seminal plasma for a period of 10 days. Data were collected daily on sperm motion characteristics, SMI, mitochondrial membrane potential, and DNA quality. To measure daily changes in SMI in stallion sperm, two fluorescent vital-staining protocols used in flow cytometric analysis were compared – a combination of SNARF-1, Yo-Pro-1, and Ethidium Homodimer 1 (SYE) and a combination of lectin from Pisum sativum and propidium iodide (PSA/PI). We hypothesized that the SYE protocol adapted for use with stallion sperm could detect more subtle, and perhaps earlier, damage to the sperm plasma membrane than the PSA/PI protocol. A combination of SYBR 14, propidium iodide, and JC-1 (SYPIJC) was used to measure mitochondrial membrane potential, as well as SMI. A computer-assisted sperm motion analysis (CASA) instrument was used to evaluate sperm motion characteristics; the sperm chromatin structure assay (SCSA) was used to measure the degree of DNA fragmentation.
In this study, with the exception of sperm motility, the measures of sperm quality retained values consistent with “viability” after 10 days of cooled-storage. This suggests that the fertility of some stallions may last considerably longer than previously assumed, which could ultimately alter the time-table used for artificial insemination using cooled, transported semen.
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Měření viability buněk / Cell Viability MeasurementProkopyeva, Elena January 2012 (has links)
This master's thesis deals with the ways of cell viability determination and its measuring. The first part of this thesis has a brief introduction into the theory of cell viability, also the methods of cell viability measurement and different types of fluorescent probes are described. The thesis further includes a brief description of the image processing methods, as well as the fluorescent microscope and applied filters are described. In the practical part the system implemented at a graphical programming environment LabVIEW used for the cell viability evaluation is described, functions of applied units are explained. The program is checked on model data.
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Protein NMR studies of two systems involved in bacterial pathogenicity / Untersuchungen mittels Protein NMR an zwei Systemen mit Einfluss auf bakterielle PathogenitätRumpel, Sigrun 01 November 2006 (has links)
No description available.
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Le rôle du CD40 homodimère dans la réponse immunitaireJundi, Malek 06 1900 (has links)
Le CD40 est une glycoprotéine transmembranaire de type I, appartenant à la famille des TNFRs, exprimée à la surface des cellules immunitaires, hématopoïétiques, vasculaires, épithéliales, et d’autres types de cellules, y compris les cellules tumorales. Le CD40 ne possédant pas de domaine kinase, pour induire un signal il interagit directement ou indirectement avec des protéines adaptatrices telles que les TRAFs et les JAKs. L’interaction du CD40 avec son principal ligand, le CD154, joue un rôle primordial dans la régulation de la réponse immunitaire et le maintien de l’homéostasie. L’activation du CD40 à la surface des cellules B augmente leur capacité de présentation d’antigène, en plus d’induire la prolifération, la commutation isotypique et l’apoptose. Les patients souffrant de mutations au niveau du gène codant pour le CD40 ou de son ligand sont immunosupprimés et sensibles à des infections opportunistes. Des études ont montré que le CD40 comme d’autres membres de la famille des TNFRs est capable de former des homodimères. Plus récemment, on a montré que la formation du CD40 homodimère est le résultat de son engagement sur les cellules B. En plus, cette homodimérisation du CD40 est importante pour la phosphorylation de l’Akt. L’interaction CD40/CD154 peut avoir un rôle direct dans l’immunothérapie par l’induction de l’apoptose de certaines cellules cancéreuses ou un rôle indirect en activant les cellules présentatrices d’antigènes (CPA) afin d'augmenter l’efficacité de l’activation des cellules T cytotoxiques. Nos résultats montrent que l’induction de la mort cellulaire par le CD40 requiert la perméabilisation du lysosome, la libération de la cathepsine B, la présence de ROS et une interaction avec le TRAF6, cette mort cellulaire programmée est plus importante en présence de la forme monomérique du CD40, muté au niveau de la cystéine 238. Par ailleurs, l’homodimérisation du CD40 requerrait sa translocation vers les radeaux lipidiques et nécessiterait la présence des ROS. Cette homodimérisation du CD40 semble être importante pour l’activation des cellules B par le biais de l’induction de l’expression du CD23, CD69 et CD80. De plus, nos résultats montrent pour la première fois une implication du CD40 homodimère dans l’induction du CD23 par le biais du TLR4. Nos résultats soulignent l’importance du CD40 homodimère dans certaines voies de signalisation. Ainsi, ils mettent en évidence le rôle de la Cys-238 dans la coopération entre des récepteurs de la réponse immunitaire innée et adaptative. Toutes ces données permettraient une meilleure compréhension de certaines voies de signalisation impliquées dans plusieurs maladies auto-immunes et faisant objet de plusieurs essais thérapeutiques. / CD40 is a type I transmembrane glycoprotein belonging to the TNFRs family, which is expressed on the surface of immune, hematopoietic cells, vascular, epithelial, and other cell types, including a wide range of tumour cells. CD40 does not have a kinase domain. Thus, to induce a signal, CD40 interacts directly or indirectly with adapter proteins such as TRAFs and Jaks. The interaction of CD40 with its main ligand, CD154, plays an important role in regulating the immune response and homeostasis. The activation of CD40 on the surface of B cells increases its ability to promote antigen presentation, in addition to inducing proliferation, isotype switching, and apoptosis. Patients affected by mutations in the gene encoding the CD40 or its ligand are immunosuppressed and susceptible to opportunistic infections. Studies have shown that CD40, as other members of the family of TNFRs is capable of forming homodimers. More recently, it was shown that the formation of the CD40 homodimer is the result of the engagement of CD40 on B cells by CD154. In addition, the homodimerization of CD40 is important for the phosphorylation of Akt. The CD40/CD154 interaction can have a direct role in immunotherapy by inducing apoptosis of some cancer cells or an indirect role in activating antigen-presenting cells (APCs), thereby increasing the effectiveness of activation of cytotoxic T cells. Our results show that the induction of cell death by CD40 requires permeabilization of the lysosome, the release of cathepsin B, the presence of ROS and interaction with TRAF6, this programmed cell death is greater in the presence of the monomeric form of CD40, due to a mutation at the level of the cysteine 238. Moreover, the homodimerization of CD40 requires its translocation to lipid rafts and the presence of ROS. This homodimerization is necessary for the CD40 B-cell activation via the induction of expression of CD23, CD69 and CD80. In addition, our results show for the first time the involvement of the CD40 homodimer in the induction of CD23 expression via TLR4. Our results emphasize the importance of CD40 homodimer in signaling pathways and highlight the role of Cys-238 in the cooperation between receptors of the innate and adaptive immune response. All together our results will allow a better understanding of CD40 signaling pathways involved in several autoimmune diseases, which give a rise to a better therapeutic trial design.
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Le rôle du CD40 homodimère dans la réponse immunitaireJundi, Malek 06 1900 (has links)
Le CD40 est une glycoprotéine transmembranaire de type I, appartenant à la famille des TNFRs, exprimée à la surface des cellules immunitaires, hématopoïétiques, vasculaires, épithéliales, et d’autres types de cellules, y compris les cellules tumorales. Le CD40 ne possédant pas de domaine kinase, pour induire un signal il interagit directement ou indirectement avec des protéines adaptatrices telles que les TRAFs et les JAKs. L’interaction du CD40 avec son principal ligand, le CD154, joue un rôle primordial dans la régulation de la réponse immunitaire et le maintien de l’homéostasie. L’activation du CD40 à la surface des cellules B augmente leur capacité de présentation d’antigène, en plus d’induire la prolifération, la commutation isotypique et l’apoptose. Les patients souffrant de mutations au niveau du gène codant pour le CD40 ou de son ligand sont immunosupprimés et sensibles à des infections opportunistes. Des études ont montré que le CD40 comme d’autres membres de la famille des TNFRs est capable de former des homodimères. Plus récemment, on a montré que la formation du CD40 homodimère est le résultat de son engagement sur les cellules B. En plus, cette homodimérisation du CD40 est importante pour la phosphorylation de l’Akt. L’interaction CD40/CD154 peut avoir un rôle direct dans l’immunothérapie par l’induction de l’apoptose de certaines cellules cancéreuses ou un rôle indirect en activant les cellules présentatrices d’antigènes (CPA) afin d'augmenter l’efficacité de l’activation des cellules T cytotoxiques. Nos résultats montrent que l’induction de la mort cellulaire par le CD40 requiert la perméabilisation du lysosome, la libération de la cathepsine B, la présence de ROS et une interaction avec le TRAF6, cette mort cellulaire programmée est plus importante en présence de la forme monomérique du CD40, muté au niveau de la cystéine 238. Par ailleurs, l’homodimérisation du CD40 requerrait sa translocation vers les radeaux lipidiques et nécessiterait la présence des ROS. Cette homodimérisation du CD40 semble être importante pour l’activation des cellules B par le biais de l’induction de l’expression du CD23, CD69 et CD80. De plus, nos résultats montrent pour la première fois une implication du CD40 homodimère dans l’induction du CD23 par le biais du TLR4. Nos résultats soulignent l’importance du CD40 homodimère dans certaines voies de signalisation. Ainsi, ils mettent en évidence le rôle de la Cys-238 dans la coopération entre des récepteurs de la réponse immunitaire innée et adaptative. Toutes ces données permettraient une meilleure compréhension de certaines voies de signalisation impliquées dans plusieurs maladies auto-immunes et faisant objet de plusieurs essais thérapeutiques. / CD40 is a type I transmembrane glycoprotein belonging to the TNFRs family, which is expressed on the surface of immune, hematopoietic cells, vascular, epithelial, and other cell types, including a wide range of tumour cells. CD40 does not have a kinase domain. Thus, to induce a signal, CD40 interacts directly or indirectly with adapter proteins such as TRAFs and Jaks. The interaction of CD40 with its main ligand, CD154, plays an important role in regulating the immune response and homeostasis. The activation of CD40 on the surface of B cells increases its ability to promote antigen presentation, in addition to inducing proliferation, isotype switching, and apoptosis. Patients affected by mutations in the gene encoding the CD40 or its ligand are immunosuppressed and susceptible to opportunistic infections. Studies have shown that CD40, as other members of the family of TNFRs is capable of forming homodimers. More recently, it was shown that the formation of the CD40 homodimer is the result of the engagement of CD40 on B cells by CD154. In addition, the homodimerization of CD40 is important for the phosphorylation of Akt. The CD40/CD154 interaction can have a direct role in immunotherapy by inducing apoptosis of some cancer cells or an indirect role in activating antigen-presenting cells (APCs), thereby increasing the effectiveness of activation of cytotoxic T cells. Our results show that the induction of cell death by CD40 requires permeabilization of the lysosome, the release of cathepsin B, the presence of ROS and interaction with TRAF6, this programmed cell death is greater in the presence of the monomeric form of CD40, due to a mutation at the level of the cysteine 238. Moreover, the homodimerization of CD40 requires its translocation to lipid rafts and the presence of ROS. This homodimerization is necessary for the CD40 B-cell activation via the induction of expression of CD23, CD69 and CD80. In addition, our results show for the first time the involvement of the CD40 homodimer in the induction of CD23 expression via TLR4. Our results emphasize the importance of CD40 homodimer in signaling pathways and highlight the role of Cys-238 in the cooperation between receptors of the innate and adaptive immune response. All together our results will allow a better understanding of CD40 signaling pathways involved in several autoimmune diseases, which give a rise to a better therapeutic trial design.
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Localization and dimerization of the ABC half transporter rAbcb6 as compared to rAbcb7 / Lokalisation und Dimerisation des Ratten-ABC-Halbtransporters, rAbcb6 im Vergleich zu rAbcb7Jakimenko, Ana 06 July 2006 (has links)
No description available.
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