Les microtubules sont des éléments dynamiques du cytosquelette qui contrôlent à la fois l’organisation du cytoplasme, la polarité, la migration et la division cellulaire. Notre laboratoire a précédemment montré que la kinase de stress JNK (c-Jun NH2-terminal Kinase) régule la dynamique des microtubules dans les cellules épithéliales de mammifères, en augmentant les vitesses de polymérisation, ainsi que les fréquences de sauvetage (transition vers une phase de repolymérisation). Alors que certaines protéines neuronales capables de réguler la dynamique des microtubules ont été identifiées comme des substrats de JNK, leurs équivalents dans les cellules épithéliales sont largement méconnus. Dans le but de comprendre comment JNK module la dynamique des microtubules dans les cellules épithéliales de mammifère, nous avons étudié deux substrats potentiels de JNK : la -tubuline et le facteur de sauvetage CLIP-170. Nous avons bien mis en évidence in vitro, une phosphorylation de la -tubuline par JNK sur une thréonine non-consensus, mais cette phosphorylation n’a pas été retrouvée dans les cellules HeLa, suggérant que la -tubuline n’est pas un substrat naturel de JNK in vivo. Nous avons mis en évidence par ailleurs que CLIP-170 est un nouveau substrat de JNK. Dans les cellules épithéliales, JNK activée phosphoryle trois résidus (Thr25, Thr45 et Ser147) situés dans la partie N-terminale de CLIP-170 de part et d’autre du premier domaine CAP-Gly qui est nécessaire pour l’interaction avec les microtubules. Ces acides aminés présentent des différences aussi bien dans leur phosphorylation basale que dans leurs cinétiques de phosphorylation par JNK sous divers stress. De plus, nous avons trouvé que dans différentes cellules épithéliales, la phosphorylation de ces sites est conservée. In vitro, ces résidus sont directement phosphorylés par JNK, préférentiellement quand le domaine N-terminal de CLIP-170 lie la tubuline. De plus, l’expression de mutants de CLIP-170 phospho-mimétiques et non-phosphorylables a montré que la phosphorylation de chaque site augmente la fréquence des sauvetages microtubulaires. Cette modulation n’est pas corrélée à une augmentation de la capacité de CLIP-170 à former des comètes aux extrémités plus en croissance ou à être retenue aux croissements microtubulaires, qui sont des sites de sauvetage potentiels.Ce travail a permis de décrire les premières phosphorylations de CLIP-170 qui stimulent sa fonction de sauvetage in vivo. Il souligne par ailleurs la complexité des mécanismes de sauvetage, qui demeurent un aspect encore énigmatique de l’instabilité dynamique des microtubules. L’activité de JNK sur CLIP-170 ne permet d’expliquer qu’une partie des effets de la kinase sur la dynamique des microtubules, aussi la recherche d’autres protéines cibles de JNK pouvant réguler notamment leur vitesse de polymérisation, reste à entreprendre. / Microtubules are dynamic cytoskeleton elements, which control cytoplasm organization, cell polarity, migration and division. Our laboratory has previously shown that the stress kinase JNK (c-Jun NH2-terminal Kinase) regulates microtubule dynamics in mammalian epithelial cells, by increasing their growth rates, and their rescue frequencies (transition towards phases of repolymerization). While several neuronal proteins regulating microtubule dynamics have been identified as JNK substrates, their counterparts in epithelial cells are largely unknown. With the aim to understand how JNK modulates microtubule dynamics in mammalian epithelial cells, we studied two putative substrates of JNK: -tubulin and the rescue factor CLIP-170. Regarding -tubulin, using an in vitro kinase assay, we found that a non-consensus threonine is actually phosphorylated by JNK, but we were not able to find this phosphorylation in HeLa cells, suggesting that -tubulin is not a natural JNK substrate. In parallel, we found that CLIP-170 is a new substrate of JNK in epithelial cells. Activated JNK phosphorylates three residues (Thr25, Thr45 and Ser147) located in the N-terminal part of CLIP-170, on each side of the first CAP-Gly domain, which is required for CLIP-170 interaction with microtubules. These residues exhibit differences in their level of basal phosphorylation and their kinetics of phosphorylation by JNK under various stresses. Moreover, we found that in different epithelial cells, the phosphorylation of these sites is conserved. Using an in vitro kinase assay, we found that all these residues are directly phosphorylated by JNK, preferentially when the N-terminal domain of CLIP-170 binds tubulin. Furthermore, using phospho-mimetic and non-phosphorylatable CLIP-170 mutants in epithelial cells, we revealed that the phosphorylation of each site increases microtubule rescues. Such modulation operates without increasing CLIP-170 capability to form comets at the microtubule growing plus ends or to accumulate at microtubule crossings, which are potential rescue sites.This work described the first phosphorylations that enhance CLIP-170 rescue factor function in vivo. It also points out to which extent rescue mechanisms are complex and remain an elusive aspect of dynamic instability. JNK-mediated phosphorylation of CLIP-170 only partly explains the kinase effects on microtubule dynamics. Therefore, identifying other JNK targets that may regulate microtubule polymerization rate, remains to be addressed.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13378号 / 論医博第2212号 / 新制||医||1047(附属図書館) / (主査)教授 井上 治久, 教授 髙橋 良輔, 教授 渡邉 大 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

CHARLES UNIVERSITY FACULTY OF SOCIAL SCIENCES Institute of Sociological Studies Department of Sociology Analysis of the political discourse on migration in the Chamber of Deputies of the Czech Republic: restrictive or liberal approach? Abstract of the Master's thesis Author: Ilia Kovalev Study programme: Sociology Supervisor: doc. PhDr. Zdeněk Uherek, CSc. Year of the defence: 2021 References KOVALEV, Ilia. Analysis of the political discourse on migration in the Chamber of Deputies of the Czech Republic: restrictive or liberal approach? Prague, 2021. 80 pages. Master's thesis (Mgr.). Charles University, Faculty of Social Sciences, Institute of Sociological Studies. Department of Sociology. Supervisor doc. PhDr. Zdeněk Uherek, CSc. Length of the thesis: 120256 characters Abstract Most of the previous research that has examined migration in the Czech Republic has focused primarily on the development of migration flows and changes in government strategies in this area, not on how we think about this issue and how we talk about it. Therefore, the aim of this Thesis is to analyze the political discourse on migration in the Chamber of Deputies of the Parliament of the Czech Republic. The analysis is based on the hypothesis of the existence of two consistent discourse attitudes, which represent restrictive and...

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / Angiotensinogen (ANG) is a single peptide glycoprotein that contains 452 amino acids in human (453 amino acids in rodents). ANG releases a 10-amino acids peptide known as angiotensin l (Ang I) from it's N-terminal when acted by renin, a acidic protease. Ang I can be fiirther converted into angiotensin II (Ang II) by angiotensin converting enzyme (ACE). Ang II is one of the most potent vasoconstrictors known. Ang II also acts on the brain to mcrease blood pressure. Ang II acts on the adrenal cortex to increase the secretion of aldosterone. The levels of ANG in plasma or local tissues diïectly contribute to the levels ofAng II. Therefore the studies of regulation ofANG gene expression is important to understand the molecular mechanisms of some related diseases, such as hypertension. Previous studies m which the transfection of the fusion genes that were generated with various lengths of 5'-flanking region of the rat ANG gene linked to a bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter into mouse hepatoma (Hepa 1- 6) ceUs and opossum kidney (OK) cells, identified a putative cyclic AMP responsive element (CRE) in the rat ANG gene 5'-regulatory region (N-806/-779). Compared with palindromic CRE octamer (TGACGTCA), the putative CRE of the ANG gene (ANG-CRE) is almost identical except the last two bases were in reversed order (TGACGTAC). In the present study, we isolated a fuU-length cDNA of 1345 base pairs from mouse liver cDNA library, which encodes a nuclear DNA-binding protein consisting of 436 amino acids with an apparent molecular weight of 52 kilodalton (kDa). This protein binds to the ANG-CRE, and was designated as 52-kDa protein. Southwestern blot revealed that this 52 kDa protein was present in the following tissues, such as liver, kidney, testis, brain, but not in spleen. Analysis of the deduced amino acid sequence shows no apparent basic regionleucine zipper (bZIP) stmcture, indicatmg ANG-CREB is structurally distinct fi-om bZIP family members. The antiserum against 43-kDa-CREB or ATF-2 can not interact with this 52-kDa protein, further supporting that the 52-kDa protem is immunologically different fi-om the bZIP family. The 52-kDa protein may represent a new group of CRE-binding proteins. Competitive gel mobility shift assays and Southwestern blot analysis revealed that the ANG-CREB binds to ANG-CRE, and this bindmg is not displaceable by excess amounts of CREs fi-om somatostatin (SOM), phosphoenopymvate carboxyl kmase PEPCK), and tyrosine ammo transferase (TAT) genes in vitro. These data suggest that the 52-kDa protein binds specifically to ANG-CRE and may regulate the ANG gene expression. The biological function of the 52-kDa protein is not known at present. The primary data from transient gene transfection assays showed that this protein has represser activity on the ANG gene promoter when ANG gene was stimulated by isoproterenol, but showed no effect on the ANG basal level (without any stimulation). Further experiments will be needed to study the biological function of the 52-kDa protein. / L'angiotensinogène (ANG) est une glycoprotéine fonnée de 452 acide aminés chez l'humain (453 acide aminés chez les rongeurs). Une protéase acide, la rénine, clive l'ANG de son côté N-tenninal en libérant un peptide de 10 acide aminés connu sous le nom de l'angiotensine I (Angl). Ce dernier, peut être converti en angiotensine II (AngII) sous Faction d'une enzyme de conversion dite ACE. L'Ang II est un puissant vasocoiisticteur. Il agit au niveau du cerveau en augmentant la pression sanguine et au niveau du cortex rénal en augmentant la sécrétion de l'aldostérone. Les niveaux de l'ANG daiis le plasma ou dans les tissus locaux sont directement proportionnels aux niveaux de Ang II. Ainsi les études de regulation de l'expression du gène d'Ang II sont importantes pour comprendre les mécanismes moléculaires de certames maladies, comme l'hypertension. Des études antérieiu-es par transfection en utilisant des gènes de fusion générés avec différentes longueurs de région flanquées en 5' du gène ANG de rat lié au gène rapporteur de bactérie, le chloramphénicol acetyl transférase (CAT), dans les ceUules hépatome (Hepa 1-6) de souris et dans les ceUules rénales de sarigue, ont permis d'identifier un présumé élément réponse (CRE) d'AMP cyclique dans la région régulatrice en 5' du gène ANG de rat (N- 806/-779). En comparant l'octamère palindromique de CRE (TGACGTCA) avec le présumé CRE du gène ANG (ANG-CRE), il semble qu'ils sont identiques à l'exception des deux dernières bases qui sont dans un ordre inversé (TGACGTAC). Dans cette étude, nous avons isolé à partir d'une banque de cDNA du foie de souris, une cDNA de 1345 paire de bases qui code pour une protéine de liaison à l'ADN nucléaire. Cette protéine est fonnée de 436 acide aminés avec un poids moléculaire apparent de 52 kilodalton (kDa). En se liant à ANG-CER, cette protéme est désignée comme la 52-kDa protéine. Le "Southwestern blot" a révélé que la 52-kDa protéine de 52 kDa est présente dans les tissus suivants: foie, rein, testicule et cerveau; mais absente dans la rate. L'analyse de cette séquence déduite d'acide aminés ne montre pas du stmcture apparent à la région basique de la fermeture éclaire à leucine, bzip (basic region-leucme zipper), indiquant que la 52-kDa protéine est structurellement distincte des membres de la famille bzip. L'antisérum dérigé contre le 43- kDa-CREB ou contre ATF-2 n'intéragit pas avec la 52-kDa protéme confermant amsi que la 52-kDa protéine est immunologiquenient dififérente des membres de famille bzip. La 52-kDa protéine représente donc, un nouveau groupe des protéines de liaison à CRE. Les essais sur gel à mobilité compétitive et les analyses de "southwestern blot" ont révélé que la 52-kDa protéine lié ANG-CRE et que cette liaison n'était pas déplaçable par un excès du gène CRE de somatostatin (SOM), phosphoenopymvate carboxy kmase (PEPCK) et tyrosme ammo transférase (TAT) m vitro, suggérant que la 52-kDa protéine se lié spésifiquement à ANGCRE et qu'il peut réguler l'expression du gène ANG. Les fonctioiis biologiques de la 52- kDa protéine ne sont pas encore connus. Les premiers résultats obtenus à partir des essais de transfections des gènes transitoirs montrent que cette protéine a une activité represseur sur le promoteur du gène d'ANG quand ce dernier est stimulé par l'isoprotérénol, et que, sans stimulation, elle a aucun effet sur le niveau basai d'ANG. Les expériences futures seront nécessaires poiir étudier les fonctions biologiques d'AND.

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.

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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...

This thesis deals with the increasingly developing field of biometric systems which is the identification of faces. The thesis deals with the possibilities of face localization in pictures and their normalization, which is necessary due to external influences and the influence of different scanning techniques. It describes various techniques of localization of dominant features of the face such as eyes, mouth or nose. Not least, it describes different approaches to the identification of faces. Furthermore a it deals with an implementation of the Dominant Face Features Recognition application, which demonstrates chosen methods for localization of the dominant features (Hough Transform for Circles, localization of mouth using the location of the eyes) and for identification of a face (Linear Discriminant Analysis, Kernel Discriminant Analysis). The last part of the thesis contains a summary of achieved results and a discussion.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19174号 / 医博第4016号 / 新制||医||1010(附属図書館) / 32166 / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 淳, 教授 村井 俊哉, 教授 渡邉 大 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The antimicrobial peptide (AMP) ARV-1502 was designed based on naturally occurring short proline-rich AMPs, including pyrrhocoricin and drosocin. Identification of chaperone DnaK as a therapeutic target in Escherichia coli triggered intense research on the ligand- DnaK-interactions using fluorescence polarization and X-ray crystallography to reveal the binding motif and characterize the influence of the chaperone on protein refolding activity, especially in stress situations. In continuation of this research, 182 analogs of ARV-1502 were designed by substituting residues involved in antimicrobial activity against Gramnegative pathogens. The peptides synthesized on solid-phase were examined for their binding to E. coli and S. aureus DnaK providing 15 analogs with improved binding characteristics for at least one DnaK. These 15 analogs were distinguished from the original sequence by their increased hydrophobicity parameters. Additionally, the influence of the entire DnaK chaperone system, including co-chaperones DnaJ and GrpE on refolding and ATPase activity, was investigated. The increasingly hydrophobic peptides showed a stronger inhibitory effect on the refolding activity of E. coli chaperones, reducing protein refolding by up to 64%. However, these more hydrophobic peptides had only a minor effect on the ATPase activity. The most dramatic changes on the ATPase activity involved peptides with aspartate substitutions. Interestingly, these peptides resulted in a 59% reduction of the ATPase activity in the E. coli chaperone system whereas they stimulated the ATPase activity in the S. aureus system up to 220%. Of particular note is the improvement of the antimicrobial activity against S. aureus from originally >128 μg/mL to as low as 16 μg/mL. Only a single analog exhibited improved activity over the original value of 8 μg/mL against E. coli. Overall, the various moderate-throughput screenings established here allowed identifying (un)favored substitutions on 1) DnaK binding, 2) the ATPase activity of DnaK, 3) the refolding activity of DnaK alone or together with co-chaperones, and 4) the antimicrobial activity against both E. coli and S. aureus.