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Characterisation of the central region of the sheep major histocompatibility complexQin, Jinyi January 2008 (has links)
The major histocompatibility complex (MHC) is a chromosomal region encoding molecules controlling adaptive immune response in vertebrates. In farm animals, many associations between MHC loci and productivity traits including disease susceptibility have been described. However, current knowledge about the structure and function of the MHC in domestic animals, especially sheep, is very limited. Characterization of the sheep MHC may potentially facilitate breeding for enhanced disease-resistant animals through use of marker assisted selection. The main aim of this project has been to provide insights into the organization of the genomic content of the central region of the sheep MHC. The work described herein has utilized subcloning of a sheep BAC genomic library in conjunction with DNA sequencing to generate a map of the central region of the sheep MHC covering ≈700 kbp. Within this map the relative order and identity of twenty five recognized loci were established. For some loci the intergenic distances were also determined. The final map is the most accurate map of this region reported to date and shows a high degree of similarity to the analogous region of the human MHC. This work has been published and a copy of the paper is included in Appendix 1. During the course of this work detailed genomic sequences were obtained for several sheep central region loci. Complete nucleotide sequences were generated for the complement factor B locus (CFB) and the TNFα locus and a comparative analysis of these sequences confirmed their homology with other vertebrate orthologues. Extensive partial sequences for complement components C2 and C4 were also obtained and reported to GenBank. / In addition, a previously identified short tandem repeat locus designated BfMs believed to be in the CFB locus was mapped to an intron within the adjacent SKI2VL locus. Single nucleotide polymorphisms (SNPs) were identified by analysing homologous sequences from a minimum of five individual sheep. In total 33 SNPs were discovered distributed over eleven distinct loci. Allele frequencies for SNPs from ten of these loci were determined and reported for a panel of 71 sheep comprising 58 unrelated sheep from the Rylington Merino flock plus a further 13 unrelated parental animals from a three generation half sibling sheep pedigree. The availability of an independently confirmed pedigree constructed from a three generation half sibling sheep family permitted the identification by deduction of central region MHC haplotypes based on a panel of SNPs derived from 10 loci. This is the first reporting of haplotypes covering this region of the sheep MHC. Analysis of SNP panel genotypes in the cohort of 71 unrelated sheep using the expectation maximization algorithm permitted the prediction of a group of approximately 20 haplotypes, which accounted for more than 90% of the expected haplotype distribution. Four of these predicted haplotypes were also present in the known haplotype cohort deduced from the sheep pedigree. Analysis of pairwise linkage disequilibrium between SNP loci in the cohort of 71 unrelated sheep showed a centre-most region displaying relatively high levels of linkage disequilibrium which was bounded by two regions displaying more variable linkage disequilibrium. / It is hypothesised that this mid region of the central region of the sheep MHC may be a block like structure characterized by low recombination similar to those that have been widely described in the human and mouse genomes. The discoveries reported in this thesis provide a more accurate and detailed description of the central region of the sheep MHC together with a panel of SNPs, which reflect the diversity of this important genomic region which is known to be associated with immune responsiveness. The description, for the first time, of central region haplotypes provides a practical means of seeking candidate loci associated with disease resistance and productivity traits. The application of molecular techniques will enhance the rate at which the genomic composition of this region is elucidated and the work described in this thesis will contribute to final characterization of this important complex in health and disease.
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Unravelling major histocompatibility complex diversity in the Soay sheep of St KildaDicks, Kara Leanne January 2018 (has links)
The major histocompatibility complex (MHC) is one of the most variable regions in the vertebrate genome. Many genes within the MHC play important roles in the development of an immune response, including the response to pathogens, by presenting pathogen fragments to T cells. Pathogen-mediated balancing selection is thought to be important in maintaining the high levels of allelic variation at these loci, though the precise mechanism remains unclear. The number of studies of MHC diversity in non-model organisms has increased dramatically in recent years as genotype data have become cheaper and easier to generate; however, key limitations in many such studies remain a lack of high quality MHC genotypes and associated phenotype data. Many studies focus on a single MHC locus, assuming that one locus will represent the full range of variation within each MHC haplotype. Alternatively, the products of different loci may co-amplify, preventing locus-specific genotypes and hence heterozygosity being accurately determined. Non-model systems are also often limited by small sample sizes and limited recording of associated host and pathogen measures, which, combined with high levels of allelic variation at MHC loci, can limit statistical power. Finally, few MHC studies control for the general effect of relatedness in explaining host traits before testing for MHC effects. With so many methodological impediments, it is challenging to identify robust associations between MHC variation and host phenotypes, such as parasite burden or fitness, and to draw conclusions about the mechanisms underpinning the maintenance of diversity at MHC loci. In this thesis, I address these problems by developing a SNP-based haplotyping system for a population of unmanaged Soay sheep (Ovis aries) on Hirta, St. Kilda, for which data is available on pedigree, phenotypic traits and fitness and its components over a 30- year study period. The ovine MHC consists of four classes of loci, within which loci are tightly clustered and show reduced recombination rates compared to the genome average. Although the mammalian MHC is usually highly variable, one would expect that the number of haplotypes within an MHC class in an island population of sheep with no immigration to be limited. The class IIa region of the ovine MHC includes the classical class II loci which are typically thought to be involved in the presentation of peptides derived from extracellular pathogens, including gastrointestinal helminths, in sheep and other mammals. In chapters 2 to 4, I describe the characterisation of class IIa haplotypic diversity in the Soay sheep using direct Sanger sequencing of PCR amplified fragments, which, in combination with cloning, revealed eight distinct haplotypes. With this knowledge of haplotypic diversity, and genotypes for a sample of Soay sheep typed on the Ovine Infinium HD chip (approximately 600K SNPs), I developed a panel of 13 SNPs which could be used to impute the class IIa haplotypes. This panel was genotyped by KASP (Kompetitive Allele Specific PCR) in 6034 samples and used to impute the class IIa haplotypes. After quality control measures, class IIa haplotypes were successfully imputed for 5349 individuals. Evidence of balancing selection was identified using the Ewens-Watterson test at different life history stages and within the standing population each year between 1985 and 2012, showing that allele frequencies were more even than would be expected under neutrality. However, there was no evidence of deviation from Hardy-Weinberg equilibrium identified at different life stages or in the standing population in any year. In chapter 5, I investigate associations between the MHC class IIa haplotypes and individual-level data on host phenotypes - body weight, plasma immunoglobulin levels (measured as anti-Teladorsagia circumcincta third larval stage IgA, IgE and IgG) and strongyle faecal egg counts (FEC). Associations were tested within mixed effects models which were used to account for repeated measures and control for fixed effects known to affect the response variables, as well as within an animal model framework to account for relatedness between individuals. Haplotype heterozygosity was unrelated to any of the traits investigated, suggesting a general heterozygote advantage is unlikely to be operating within the Soay sheep. Six of the eight class IIa haplotypes were associated with multiple traits in different age-sex classes, although many of these associations were removed after inclusion within animal models. The evidence of balancing selection and associations between class IIa haplotypes and phenotypes related to health offers a promising glimpse into the evolutionary mechanisms which may be operating to maintain diversity within this region.
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Genetic Diversities among Founder Populations of the Endangered Avian Species, the Japanese Crested Ibis and the Oriental Stork in Japan / 希少鳥類トキおよびコウノトリの国内始祖集団における遺伝的多様性に関する研究Taniguchi, Yukio 25 January 2016 (has links)
京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12986号 / 論農博第2826号 / 新制||農||1038(附属図書館) / 学位論文||H28||N4961(農学部図書室) / 32456 / 名古屋大学大学院農学研究科生化学制御専攻 / (主査)教授 祝前 博明, 教授 今井 裕, 教授 廣岡 博之 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM
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Rôle de l'ubiquitination dans le trafic cellulaire des molécules de présentation antigénique. / Role of the ubiquitination in the intracellular trafficking of antigen presenting moleculesDe Angelis Rigotti, Francesca 12 April 2011 (has links)
L’ubiquitinylation a été largement étudiée comme étant un mécanisme impliqué dans la régulation du trafic intracellulaire de nombreuses protéines membranaires. Mon travail a permis d’identifier MARCH-IX, une ubiquitine ligase exprimées dans les cellules de mammifères, comme un acteur important du trafic intracellulaire des molécules de présentation antigénique CD1a et CMH-I. En condition d'over-expression, MARCH-IX ubiquitinyle spécifiquement CD1a et CMH-I. Par ailleurs, en utilisant la technique d’ARN interférence, nous avons mis en évidence que l’ubiquitination des CMH I dépendante de MARCH IX facilite l’export des CMH I néosynthétisés du TGN vers la membrane plasmique et permet leur accès à des compartiments endosomaux. Notamment l’expression de MARCH-IX est régulée au niveau transcriptionnel pendant la maturation de DCs humaine; son expression est largement diminuée suite à l’activation des DCs plasmacytoïdes (pDCs), alors qu’elle augmente dans des DCs dérivées de monocytes (MoDCs) stimulées par du LPS. Ces résultats laissent envisager que MARCH IX puisse avoir un rôle important dans le contrôle de la présentation antigénique médiée par les CMH I dans les DCs humaines. Enfin, l’adressage intracellulaire des molécules de CD1a dans les MoDCs apparait également comme un processus régulé au cours de la maturation. Si CD1a est localisé à la membrane plasmique et dans des compartiments endosomaux précoce dans des cellules immatures, cette molécule n’apparaît plus qu’à la surface des cellules matures. Nous postulons donc que la régulation de MARCH-IX durant la maturation des MoDCs puisse être directement liée à la modification du trafic intracellulaire de CD1a. / Ubiquitination has been largely studied as regulator of the intracellular trafficking of several membrane proteins, inducing their internalization or their sorting from TGN to endosomes. Interestingly, pathogens adopted this mechanism to evade the immune response. For example, Kaposi’s sarcoma herpesvirus synthesizes two ubiquitin ligases, MIR1 and MIR2, which target the antigen presenting molecule, MHC class I, inducing its internalization. We identified the mammalian ubiquitin ligase MARCH-IX as important factor in the intracellular trafficking of antigen presenting molecules, CD1a and MHC-I. In conditions of MARCH-IX over-expression, CD1a and MHC-I are ubiquitinated and they accumulated in early endosomes. In MARCH-IX silenced cells, the arrival of MHC-I at the plasma membrane appear to be delayed and MHC-I accumulates in the TGN. During dendritic cell maturation, MARCH-IX expression and CD1a intracellular localization showed a correlation, which is compatible with a role of the ubiquitin ligase in the export pathway of CD1a. We concluded that MARCH-IX acts on neo-synthesized molecules, facilitating their sorting from the TGN. In addition to the function analysis of MARCH-IX, we also investigated its ability to conjugate ubiquitin on non-conventional residues. Our results demonstrated that, differently from viral ubiquitin ligases, MARCH-IX could target MHC class I and CD1a only in presence of lysine residues on their cytoplasmic tail, suggesting a stronger restriction in the control of the ubiquitination mechanism on mammals.
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Minderung der allogenen Immunogenität künstlicher Gewebe am Modell Epithelien durch Suppression der MHC-I-OberflächenexpressionBusch, Annette 07 November 2000 (has links)
Die Expression von Haupthistokompatibilitätsantigenen auf der Zelloberfläche kernhaltiger Zellen ist die Hauptursache für die Detektion durch das Immunsystem und die Rejektion allogener Transplantate. Eine vielversprechende Strategie, die MHC-I-Expression auf der Zelloberfläche zu senken, ist der Einsatz intrazellulär lokalisierter Antikörpern, sogenannter Intrabodies. In der vorliegenden Arbeit wurde erstmals versucht, eine die Expression an MHC-I-Molekülen auf der Zelloberfläche durch den Einsatz von anti-MHC-I-Intrabodies zu verhindern. Ein Hauptproblem in der Transplantation ist der Mangel an geeignetem Spendermaterial. Um diesem Zustand entgegenzuwirken, wird versucht, adäquaten Organersatz durch Tissue Engineering bereitzustellen. Die in vitro-Züchtung autologer epithelialer Zellen zur Generierung transplantierbarer Hautstücke spielt heute bereits eine große Rolle bei der Transplantation artifizieller Gewebe. Ferner werden diverse vollsynthetische Materialien für diesen Zweck hergestellt. Eine Verbesserung in diesen Bereich würde die Generierung von nicht-immunogenen allogenen Keratinozyten darstellen. Im Rahmen dieser Arbeit wurden primäre Rattenkeratinozyten mit anti-MHC-I-Intrabodies transfiziert. Diese Zellen zeigten einen MHC-I-"knock-out"-Phänotyp - eine starke Expression der Intrabodies war jedoch essentiell für die vollständige Zurückhaltung aller MHC-I-Moleküle in der Zelle. Neben der Applikation von Intrabodies in Rattenkeratinozyten konnte die MHC-I-Expression auf der Zelloberfläche von 293-Zellen und primären humanen Keratinozyten ebenfalls durch die Expression von anti-MHC-I-Intrabodies vermindert werden. Im Vergleich mit dem ebenfalls MHC-I-bindenden adenoviralen Protein p19 bewirkten die eingesetzten anti-MHC-I-Intrabodies bei gleich starker Expression eine wesentlich stärkere "Downregulation" der MHC-I-Oberflächenexpression von 293-Zellen. Intrabody-exprimierende Zellen zeigten keine signifikanten morphologischen oder physiologischen Veränderungen gegenüber untransfizierten Zellen: Wachstum, die Expression anderer Oberflächenmoleküle und morphologische Erscheinung waren unverändert. Es konnte lediglich eine verstärkte intrazelluläre Akkumulation von MHC-I-Molekülen detektiert werden. Die funktionelle Bedeutung der MHC-I-"Downregulation" durch Intrabodies konnte durch die stark verminderte zytolytische Aktivität zytotoxischer T-Zellen gegenüber Intrabody-exprimierender Rattenkeratinozyten im Vergleich zu unmodifizierten Zellen gezeigt werden. In der vorliegenden Arbeit ist es erstmals gelungen, Zellen mit einem vollständigen MHC-I-"knock-out"-Phänotyp zu erzeugen - bisherige Modifikationen der MHC-I-Oberflächenexpression durch die Generierung _2-Mikroglobulin- oder TAP-defizienter Mäuse führte zu keinem restlosen Verlust der Klasse-I-Moleküle auf der Zelloberfläche. MHC-I-"downregulierte" Keratinozyten könnten in der Hauttransplantation anstelle der limitiert zur Verfügung stehenden autologen Zellen eingesetzt werden oder in Kombination mit synthetischen Materialien verwendet werden, wo sie durch die Sekretion von heilungsfördernden Faktoren eine Verbesserung des Wundheilungsprozesses bewirken würden. / The expression of major histocompatibility antigens on the surface of eucaryotic cells is the predominant reason for immunologic detection and the rejection of allogeneic transplants. A promising strategy to lower the MHC I expression on the cell surface is the use of intracellular localized antibodies, termed intrabodies. In this work it has been tried for the first time to prevent the expression of MHC class I molecules on the cell surface by intrabody expression. A major problem in transplantation is the shortage of suitable donor material. To overcome this situation tissue engineering has and will continue to enlarge the scope of organ grafting. Today, the in vitro culture of autologous epithelial cells to generate transplantable skin sheets plays an important role in the transplantation of artificial tissues. Furthermore various fully synthetic materials are produced for transplantation. An improvement in this field could be the generation of non-immunogeneic allogeneic keratinocytes. Within this work primary rat keratinocytes have been transfected with anti-MHC-I-Intrabodies. These cells show a MHC I "knockout" phenotype - yet a strong intrabody expression was essential for the complete retention of all MHC I molecules inside the cell. Besides the application of intrabodies in keratinocytes the MHC I expression on the surface of 293 cells and human primary keratinocytes could be reduced by anti-MHC I intrabodies as well. In comparison with the also employed adenoviral protein p19 the applied intrabodies generated at the same expression level a much stronger down-regulation of the MHC I surface expression of 293 cells. Intrabody expressing cells did not show any significant morphologic or physiologic alterations compared to untransfected cells: growth, the expression of other surface molecules and the morphological appearance were unaltered. Merely an enhanced intracellular accumulation of MHC I molecules could be detected. The functional relevance of the MHC I down-regulation by intrabodies could be shown by the strong diminished cytolytic activity of cytotoxic T cells to intrabody expressing rat keratinocytes in comparison to unmodified cells. In this work cells with a completely MHC I knock-out phenotype has been successfully generated for the first time - modifications of the MHC I surface expression by generation of _2-microglobulin or TAP deficient mice did not lead to a complete loss of all MHC I molecules on the cell surface so far. MHC I down-regulated keratinocytes could be employed in skin transplantation instead of the limited available autologous cells or utilized in combination with synthetic materials where they would induce an improvement of the healing process by the secretion of cytokines.
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IFN-Gamma-Mediated Immunoevasive Strategies in Multiple MyelomaCiarlariello, Paul David 08 August 2016 (has links)
No description available.
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Infection of Human Cell Lines by Japanese Encephalitis Virus : Increased Expression and Release of HLA-E, a Non-classical HLA MoleculeShwetank, * January 2013 (has links) (PDF)
Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain.
Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules.
Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses.
In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs.
Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process.
Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA-
E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells.
HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL.
The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells.
In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.
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Regulace genové exprese HSP70 genů a její závislost na genotypu HSP70 genů. / Regulation of gene expression of HSP70 genes and its dependence on the genotype of HSP70 genes.Ambrož, Antonín January 2011 (has links)
The topic of the presented thesis is the regulation of gene expression level of the three HSP70 genes in mononuclear cells. We investigated the dependence of expression regulation (induction) abiliy on selected point mutations, so-called SNPs (single nucleotide polymorphism) in the observed genes. The mononuclear cells were obtained from peripheral blood samples of healthy individuals. In order to analyze their gene expression, we selected individuals who were homozygous for at least one of the monitored point mutations. Taking into account the chosen criteria for healthy individuals we based on interviews with these individuals and their personal history. We determined the polymorphisms observed in two cell stress-inducible HSP70-1 (HSPA1A) and HSP70-2 (HSPA1B) genes and in one constitutively expressed gene HSP70-Hom (HSPA1L). Further, we have analyzed HSP70s gene expression regulation and the relation between the expression regulation and studied polymorphisms. We determined the degree of regulation of a gene expression in the studied genes in relation to two SNPs -110A/C (rs1008438), +190G /C (rs1043618) gene HSP70-1, and two SNPs +1267A/G (rs1061581), +2074G /C (rs539689 ) of the HSP70-2 gene, and the mutation of one five-nucleotide (rs9281590) HSP70-2 gene, and one SNP +2437T/C (rs2227956) of...
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Développement d’approches de contrôle de qualité pour la caractérisation de l’immunopeptidome de cellules infectées par les coronavirusDespault-Duquette, Jérôme 12 1900 (has links)
La présentation de l’antigène est un mécanisme par lequel les cellules nucléées présentent un court peptide sur la molécule de classe 1 du Complexe Majeur d’Histocompatibilité (CMH-1) codée par les gènes « antigènes d’histocompatibilité humains ». Le terme “immunopeptidomique” est utilisé pour décrire l’ensemble des peptides associés aux molécules du CMH-1. Les cellules T CD8+ patrouillent l’organisme, s’attachent à la molécule CMH-1 par leur récepteur T et détruisent les cellules affichant un peptide atypique. Ce domaine présente un grand intérêt au niveau du traitement des infections virales et dans la conception de vaccins.
Compte tenu que les coronavirus ont été à l’origine de trois épidémies durant les 20 dernières années, et que de multiples souches circulent chez l’humain ainsi que dans le règne animal, il est impératif de développer des vaccins universels qui pourrait prévenir de futurs événement épidémiologiques mondiaux reliés aux coronavirus.
L’immunopeptidomique souffre d'un manque de protocoles normalisés et de contrôle et d’assurance de la qualité des échantillons afin de libérer tout son potentiel dans la recherche biomédicale. Dans le cadre de cette étude, la spectrométrie de masse, la cytométrie de flux et des approches bio-informatiques ont été utilisées pour développer des protocoles de contrôle de qualité pour la caractérisation de l’immunopeptidome de cellules infectées par les coronavirus. Nous avons isolé et analysé l’immunopeptidome de cellules MRC-5 avant et après infection par le coronavirus humain OC-43. En plus d’observer une forte baisse de l’abondance des molécules HLA et de la variété des peptides présentés après l’infection, 9 peptides viraux ont été isolés à partir des molécules du CMH-1. Ces peptides pourraient être utilisés afin de contribuer à formuler un vaccin pan-coronavirus qui élicite une réponse balancée entre la réponse humorale et la réponse cytotoxique. / Antigen presentation is a mechanism by which nucleated cells present a short
peptide on the Major Histocompatibility Complex (MHC-1) class 1 molecule encoded by
the “human histocompatibility antigen” genes. The term "immunopeptidomics" is used
to describe the set of peptides associated with MHC-1 molecules. CD8+ T cells patrol the
body, attach to the MHC-1 molecule through their T receptor and destroy cells displaying
an atypical peptide. This field is of great interest in the treatment of viral infections and
in vaccine design.
Given that coronaviruses have been responsible for three epidemics in the last 20
years, and that multiple strains circulate in humans and animals, it is imperative to
develop universal vaccines that could prevent future global epidemiological events
related to coronaviruses.
Immunopeptidomics suffers from a lack of standardized protocols and sample quality
control and assurance to unleash its full potential in biomedical research. In this study,
mass spectrometry, flow cytometry, and bioinformatics approaches were used to develop
quality control protocols for characterizing the immunopeptidome of coronavirusinfected
cells. We isolated and analyzed the immunopeptidome of MRC-5 cells before and
after infection with human coronavirus OC-43. In addition to observing a strong decrease
in the abundance of HLA molecules and in the variety of peptides presented after
infection, 9 viral peptides were isolated from MHC-1 molecules.
These peptides could be used to help formulate a pan-coronavirus vaccine that
elicits a balanced response between humoral and cytotoxic responses.
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