京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20793号 / 医博第4293号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 鈴木 茂彦, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22366号 / 医博第4607号 / 新制||医||1043(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 岩田 想, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...

The immune system is essential for keeping the integrity of multicellular organisms. We were able to make a step forward in studying the complex immune reactions in mammals in vivo and/ or in situ using the major histocompatibility complex (MHC) class II/ enhanced green fluorescent protein (EGFP) knock-in mouse model. Due to the EGFP visualization of MHC II expressing cells we were able to observe antigen presenting cells, which are essential for the onset of immune responses, in their natural environment. Thus, we report some original features of the immune system. We have identified MHC II+ cell clusters with unknown, probably unique function, in the intestine. We have also described MHC II+ cell migration to the lactating mammary gland and tested few hypotheses about the role of this phenomenon for the development of the mammary gland, milk secretion or infant immune system establishment. Lastly, we observed residential macrophages in the cornea. The presence of APCs in the cornea is a very contradictory issue due to the fact that cornea is an immunologically privileged tissue and therefore harbors special immune features. key words: antigen presenting cells (APC), major histocompatibility complex class II (MHC II), enhanced green fluorescent protein (EGFP), immune system, knock-in mouse model

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No description available.

Värmechockprotein 90 kDa (HSP90) bildar ett anmärkningsvärt komplicerat nätverk med en mängd olika cochaperones. Komplexet av FK506-bindande protein 51 kDa (FKBP51) och HSP90 förmedlar proteinveckning och funktion, främjar tau aggregation vid Alzheimers sjukdom och påverkar stressrelaterade störningar, fetma, typ två-diabetes, etc. I samarbete med den molekylära chaperonen HSP90, FKBP51 har nyligen föreslagits som ett lovande terapeutiskt mål för Alzheimers sjukdom (AD). Således skapades knock-in-musen med punktmutationer i tetratricopeptide repeat (TPR) domänen av FKBP51, vilket gör den oförmögen att interagera med HSP90, för att undersöka de potentiella terapeutiska målen för behandling av dessa sjukdomar. Glukokortikoidreceptorn (GR) fungerade traditionellt som utgångspunkten för de initiala studierna av FKBP51-funktion och mekanism som kan stimuleras av den syntetiska glukokortikoiden dexametason (Dexa). Det primära målet med projektet är att förstå den biologiska betydelsen av FKBP51-HSP90 interaktioner. Det är oklart hur FKBP51-mutation påverkar protein-protein-interaktionen och glukokortikoidsignalering. Här analyserades embryonala fibroblaster (MEF) isolerade från vildtyp och FKBP51 mutant mus med avseende på proteinlokalisering, proteinuttryck och genuttryck. Även om ingen säker skillnad mellan vildtyp och mutantmöss sågs i Dexa-medierad glukokortikoidsignalering, förekommer de posttranslationella modifieringarna (PTM) vid exponering för Dexa-behandling av FKBP51 i vildtypmöss i en signifikant högre utsträckning än i Fkbp51mute-möss.Fosforyleringsmodifieringen av FKBP51 antogs initialt och bekräftades av fosforyleringsanrikningsstrategier. Bekräftelse har dock ännu inte erhållits. / Heat shock protein 90 kDa (HSP90) forms a remarkably complicated network with a variety of cochaperones. The complex of FK506-binding protein 51 kDa (FKBP51) and HSP90 mediates protein folding and function, promoting tau aggregation in Alzheimer's disease and influencing stress-related disorders, obesity, type two diabetes, etc. In collaboration with the molecular chaperone HSP90, FKBP51 has recently been proposed as a promising therapeutic target for Alzheimer's disease (AD). Thus, the knock-in mouse harboring point mutations in the tetratricopeptide repeat (TPR) domain of FKBP51 rendering it unable to interact with HSP90 were created to investigate the potential therapeutic targets for the treatment of these diseases. Glucocorticoid receptor (GR) traditionally served as the starting point for the initial studies of FKBP51 function and mechanism which can be stimulated by the synthetic glucocorticoid, dexamethasone (Dexa). The primary goal of the project is to comprehend the biological significance of FKBP51-HSP90 interactions. It is unclear how FKBP51 mutation affects the protein-protein interaction and glucocorticoid signaling. Here, embryonic fibroblasts (MEFs) isolated from wildtype and FKBP51 mutant mouse were analyzed with respect to protein localization, protein expression, and gene expression. Although no certain difference between wildtype and mutant mice was seen in Dexa-mediated glucocorticoid signaling, the post-translational modifications (PTMs) in exposure to Dexa treatment of FKBP51 occur in wildtype mice to a significantly higher extent than in Fkbp51mute mice. The phosphorylation modification of FKBP51 was initially hypothesized and confirmed by phosphorylation enrichment strategies. However, confirmation has not yet been obtained.

Ahnak ist ein ubiquitäres Protein, das an einer Vielzahl biologischer Prozesse beteiligt ist. In der Herzmuskelzelle ist Ahnak überwiegend am Sarkolemma lokalisiert und bindet an Aktin und an die regulatorischen Beta2-Untereinheit des L-Typ-Kalzium-Kanals. Das Ziel dieser Arbeit war die Funktion von Ahnak im Herzen mit Hilfe eines Knock-out-Maus-Modells und in Bindungsstudien zu untersuchen. Morphologische Untersuchungen zeigten, dass das Längenwachstum adulter Kardiomyozyten bei Ahnakdefizienz signifikant reduziert war. Die Kontraktionseigenschaften adulter isolierter Ahnak-defizienter Kardio-myozyten (im Alter von 6 Monaten) waren ebenfalls verändert. Die Kontraktions- und Relaxaktionsgeschwindigkeiten waren erhöht. Eine Erhöhung des diastolischen Kalzium-Spiegels zeigten die Kardiomyozyten schon im Alter von 3 Monaten. Diese beobachteten phänotypischen Veränderungen lassen vermuten, dass die Aktivität des L-Typ-Kalzium-Kanals erhöht ist. In dieser Arbeit konnte das PXXP-Motiv, in der C-terminalen Ahnak-Domäne, als die hochaffine Beta2-Bindungsstelle (KD ~ 60 nM) identifiziert werden. Substitution von Prolin gegen Alanin verringerte zwar die Bindung zur Beta2-Untereinheit dramatisch (KD ~ 1 µM), hob sie aber nicht auf. In weiteren Bindungsstudien zeigte sich, dass die natürlich vorkommende Missensmutation I5236T die Bindung zur regulatorischen Beta2-Untereinheit verstärkte, dagegen verminderte die PKA-abhängige Phosphorylierung der beiden Proteinpartner die Bindung. Experimente am ganzen isoliert perfundierten Herzen zeigten, dass Ahnak-Knock-Out-Herzen geringer Beta-adrenerg stimulierbar waren. Ahnak scheint wie eine physiologische Bremse des kardialen Kalzium-Kanals zu wirken. / Ahnak is an ubiquitous protein with in unique structure, which has been implicated in cell type specific functions. In cardiomyocytes, ahnak is predominantly localized at the sarcolemma and is associated with actin and with the regulatory beta2 subunit of the L-type calcium-channel. The aim of this work was to unravel the function of ahnak in the heart, using a knock-out-mouse model and binding studies. Morphological studies showed a significant decrease in the cell-length of ahnak deficient cardiomyocytes. The contractile parameters of isolated adult ahnak deficient cardiomyocytes (in the age of 6 month) were altered. The development of tension and relaxation were increased. An increase of diastolic calcium was already observed at the age of 3 month. In general the observed phenotypic changes suggested an increased activity of the L-type calcium-channel. In this study, a PXXP-motif, which locates in ahnaks C-terminus, was identified as the high affinity beta2 subunit binding site (KD ~ 60 nM). Substitution of both proline residues by alanine reduced, but did not abolish the binding (KD ~ 1 µM). Further binding studies revealed that the natural occurring ahnak missense mutation I5236T increases the binding affinity to the regulatory beta2 subunit. By contrast PKA dependant phosphorylation of both protein partners decreases the interaction. In studies with isolated perfused working heart preparations, the ahnak deficient hearts were less beta-adrenergic stimulated than hearts from wild type. Taken together ahnak seems to be a physiological brake of the cardiac calcium-channel.