No description available.

<p>The following work describes the methodology and materials used to study three human protein complexes involved in the etiology and progression of cancer. The first, ubiquitin-specific protease 7 (USP7) is an isopeptidase that employs a unique auto-regulatory mechanism. The second is another ubiquitin-specific protease, USP28, which forms higher order states in solution. Lastly, the third case was a protein complex that utilizes an oxidation-sensitive dimeric protein, Keap1, and two components of an E3 ligase – Cul3-Rbx1. Each of these studies involved overcoming unique challenges for cryo-EM sample optimization. Not all yielded the quality of data that would result in high-resolution (< 6 Å) densities. Despite this, new information was discovered about each system.</p> <p>USP7 has a unique mechanism of intramolecular regulation that stems from a hypothesized tethered-rheostat, whereby the c-terminal distal domains activate the catalytic domain via a hypothetical wide degree of conformational movement. My cryo-EM work, done in collaboration with the Wen Jiang lab, is the first comprehensive structural data that provides structural evidence for the movement of the tethered-rheostat. The particle set showed a great degree of conformational heterogeneity, even after a strategy was employed with a chemically-modified ubiquitin substrate to ameliorate these issues. The data showed that during the ubiquitin-bound state, after the release of a hypothetical substrate, but prior to the release of mono-ubiquitin, the HUBL4-5 domains do not remain engaged with the catalytic domain. This information suggests a change to existing models of catalysis. </p> <p>Additionally, the structural model built from the cryo-EM density has revealed an interfacial region between domains that were previously not thought to interact. This interfacial region between the TRAF domain and HUBL1-3 represents a candidate location of binding for a mixed, non-competitive inhibitor of USP7 previously identified in the lab. Enzyme kinetics, DSF, and Glide molecular docking experiments all yielded data that corroborate this idea.</p> <p>Structural studies on USP28 have been difficult as the multi-domain enzyme adopts oligomers in solution and is generally not amenable to crystallographic analysis. Prior to the work described herein, the only structural data were a solution NMR structure describing a few alpha-helical motifs in the N-terminus. During my graduate studies, two articles were published of the USP28 catalytic domain crystallographic structure. Both corroborated the existence of a dimer. The USP28 catalytic domain migrates during analytical gel filtration assays with the apparent molecular weight of a tetramer. Furthermore, glutaraldehyde crosslinking experiments show the catalytic domain appears to adopt a tetrameric state, like the USP25 tetramer. The USP25 tetramer was published alongside the USP28 catalytic domain dimer, concluding that a USP28 tetrameric state was not observed. Upon cryo-EM data collection and single particle analysis, it was observed that the compositional heterogeneity of the dataset was too great for any meaningful reconstruction. Although, the dataset appeared to how the presence of the <i>E. coli</i> GroEL chaperone complex. Co-expression experiments confirmed that the GroEL chaperone complex migrates with USP28 throughout the purification and may be useful for purifying USPs for structural studies.</p> <p>Currently, our lab has a single-angle X-ray scattering (SAXS) model of the Keap1-Cul3 E3 ligase complex. But, the field does not fully agree on the molecular stoichiometry or the overall structure-function of this oxidation sensor – E3 ligase complex. It is hypothesized that Keap1 forms a dimer through its BTB domain, and a single Cul3 molecule then binds this dimer. The oxidation state of Keap1 cysteines appears to be critical to the interaction, but the field remains uncertain about which residues are responsible for the interaction with the Cul3-Rbx1 E3 ligase. To better understand this interaction and to obtain structural information to corroborate the SAXS model, recombinant Keap1 and Cul3-Rbx1 were purified and their interaction was tested by ITC, gel filtration assay, and a new technique called <i>mass photometry</i>. </p> <p>It was found that the Keap1 Cys151 residue is not the oxidation sensor critical to the interaction, contrary to what some in the field anticipated. Additionally, it was found that under oxidative conditions, WTKeap1 could not form a complex with Cul3-Rbx1. The complex was successfully purified and was measured by SDS-PAGE, gel filtration assay, and mass photometry, and then used for cryo-EM single particle analysis. Full data collection and analysis has not yet been completed. It is anticipated that like the data from mass photometry, analytical SEC, and cryo-EM single particle analysis will show the complex appears to show a 1:1 Keap1-Cul3 stoichiometry, as opposed to the anticipated 2:1 ratio.</p>

The aim of the thesis is to assess the extent of individual factors’ influence on the thickness growth of retention oak trees in the middle forest. The factors include height in 2008, habitat, release rate, vegetation age, the rate of epicormic branching and treetop health state and quality. To analyze the data, MS Excel and Statistica were used. The results were interpreted with the use of multidimensional regression analysis. The analyses resulted in regression coefficients which represent the relative magnitude of the increase of the thickness gain by the action of the individual factors. Not all the factors were shown to be statistically significant; their significance also differs over time.

Subfossil trunks are unfossilized woods which are discovered rarely in the Czech Republic. The aim of this work was to find, sampled and dated subfossil trunks from the Polabí region. Available Czech and foreign standard chronologies were used for dendrochronological dating of tree-ring series. Simultaneously, earlier sampled subfossil trunks from the study area were assembled. The chosen tree-ring series were used to extension and improvement of the Czech oak standard chronology. Selected samples, which were not possible to dendochronologically date, were sent for radiocarbon dating. For a purpose of this work, 122 new subfossil findings were dendrochronologically processed from the Polabí region and three samples were prepared and sent for radiocarbon dating. The major part of subfossil trunks was identified as an oak and only 5 samples as an elm. In total, 96 samples were successfully dated mostly into two periods: 3500 - 3200 BC and 133 BC - 155 AD. In Kostomlátky location, the oldest oak subfossil trunk in the Czech republic was found the tree-ring series covers the period 8181 7971 BC. Thanks to these subfossil findings, the Czech oak standard chronology could be extended.

The aim of the thesis was to make a constructional-historical research and dendrochronological dating of the roof frame (truss) of St. James´s church in Stará Ves near Bílovce (the district of Novy Jicin). It is the case of the roof frame structure of hammerbeam type which was made from fir timber. The result of the dendrochronological analysis has shown that the trees used for the construction of the roof of the nave were fell down between 1712 and 1713. The trees used for the construction of the roof over the chancel were also fell down between 1712 and 1713. The oak bell stool could not be reliably dendrochronologically dated. The results of the dendrochronological analysis correspond to the historical development of the church found out from the literary and historical sources

No description available.

No description available.

Les lymphomes non hodgkiniens (LNH) regroupent un panel hétérogène de pathologies originaires de cellules lymphatiques. Parmi les LNH à cellules B matures, la leucémie lymphoïde chronique (LLC) constitue la forme de leucémie de l’adulte la plus fréquente en Occident. La physiopathologie des LNH à cellules B matures est marquée par l’inhibition des mécanismes de la mort cellulaire, notamment via la surexpression de la protéine MCL-1. Une première partie de ce travail de thèse a été de déterminer quelles pouvaient être les enzymes de déubiquitination (DUBs) impliquées dans la survie des LNH à cellules B matures et la stabilisation de MCL-1. Notre étude a permis d’identifier la DUB USP14, qui est liée au système ubiquitine-protéasome, comme capable de réguler MCL-1 et la survie cellulaire. Nos travaux montrent également pour la première fois que l’activité DUB des cellules, ainsi que l’activité d’USP14, sont directement régulées par la signalisation du BCR via l'activité de la tyrosine kinase SYK. Le FTY720, un analogue de la sphingosine utilisé comme immunosuppresseur dans la sclérose en plaques, a montré un effet cytotoxique dans des hémopathies malignes sans toutefois que son mécanisme d’action soit clairement expliqué. Une deuxième partie de ce travail de thèse a été de caractériser la mort induite par le FTY720. Notre étude montre que la caractérisation de la morphologie cellulaire et des marqueurs induits par la mort due au FTY720 dans les LLC correspond en fait à une nécrose cellulaire programmée indépendante de RIPK1, mais dépendante d'une enzyme régulatrice de la fission mitochondriale, DRP1. / Non-Hodgkin lymphoma (NHL) include a diverse range of pathologies originate from the lymphatic cells. Among the mature B-cell NHL, chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The pathophysiology of mature B-cell NHL is marked by the inhibition of cell death mechanisms, particularly through the overexpression of MCL-1 protein. The first part of this thesis was to determine which deubiquitinating enzymes (DUBs) are involved in the survival of mature B-cell NHL and in the stabilization of MCL-1. Our study identified the DUB USP14, which is linked to the ubiquitin-proteasome system, as able to regulate MCL-1 and cell survival. Our work also shows for the first time that the DUB activity of the cells and the activity of USP14 are directly regulated by BCR signaling through the activity of the SYK tyrosine kinase. FTY720, a sphingosine analog used as an immunosuppressive drug in multiple sclerosis, showed a cytotoxic effect in hematological malignancies but its mechanism of action is not well understood. A second part of this thesis was to characterize the death induced by FTY720. Our study shows that the characterization of the cellular morphology and markers induced by death due to FTY720 in the LLC corresponds in fact to a programmed RIPK1-independent necrosis cell death, but dependent on DRP1, a regulatory enzyme of the mitochondrial fission.

(ENGLISH) Ubiquitin signaling is a key regulatory mechanism for many important cellular processes such as transcription, differentiation and cell division. Cell division requires duplication of all genetic material during S-phase followed by its precise partitioning between two daughter cells during mitosis. Misregulation of the complex mitotic machinery may lead to aneuploidy and genomic instability, known drivers of tumorigenesis. Indeed, systematic genetic analysis of many cancer tissues over the last decades, indicates the presence of severe chromosome abnormalities in thousands of cancer tissue samples. In this work, I investigated the function of two components of ubiquitin signaling, the deubiquitinating enzyme UCHL3 and the E3 ubiquitin ligase TRIM15. The hypothesized role of E3 ligase TRIM15 in the cell cycle regulation could not be confirmed by our experiments, but I observed an effect on cell adhesion and motility instead. UCHL3 was identified using high-content visual siRNA screen, as a critical factor controlling genome segregation and integrity. Interestingly, it has been previously reported that UCHL3 levels are altered in various cancer types, especially colon cancer. My data demonstrate that UCHL3 drives proper alignment of chromosomes at the metaphase plate by facilitating...

La signalisation Notch est une voie extrêmement conservée depuis Caenorhabditis elegans jusqu'aux mammifères en passant par Drosophila melanogaster. Elle est requise dès le développement embryonnaire et contrôle de nombreux processus comme la différenciation et le choix du destin cellulaire, la prolifération, ou encore le maintien de stocks de cellules souches et l'apoptose. La voie est basée sur l'activité du récepteur Notch, un hétérodimère transmembranaire qui est activé lors de contacts intercellulaires par la liaison de ses ligands qui sont également des protéines transmembranaires. Suite à cette activation, le récepteur subit une série de deux clivages protéolytiques internes, respectivement catalysés par une métalloprotéase ADAM et par le complexe multi-protéique γ-secrétase. Ce dernier clivage libère le domaine intracellulaire de Notch dans le cytoplasme, cette forme étant ensuite transportée dans le noyau où elle active directement la transcription des gènes cibles de la voie Notch en se liant à ses co-facteurs de transcription, CSL et Mastermind. Le récepteur Notch est régulé à diverses étapes par des processus d'ubiquitination : la monoubiquitination du récepteur Notch activé contrôle son clivage γ-secrétase, la polyubiquitination du récepteur Notch non activé et endocytosé contrôle sa dégradation dans les lysosomes. Les processus d'ubiquitination sont réversiblement contrôlés par la déubiquitination. Or, aucune déubiquitination n'a été identifiée dans la voie de signalisation Notch. Mon projet consistait à identifier les déubiquitinases - les enzymes responsables de la déubiquitination - impliquées dans les deux processus d'ubiquitination décrits précédemment. Nous avons pour cela mis au point deux cribles en immunofluorescence permettant de tester une banque de shRNA qui cible l'expression des 91 déubiquitinases connues ou putatives du génome humain. Le premier crible m'a permis d'identifier eIF3f, une sous-unité du facteur d'initiation de la traduction eIF3, comme une nouvelle protéine portant une activité déubiquitinase. eIF3f est recrutée au récepteur Notch via l'E3 ubiquitine ligase Deltex, et régule positivement la voie de signalisation Notch en déubiquitinant le récepteur Notch activé avant le clivage γ-secrétase, favorisant ainsi la production de la forme transcriptionnellement active de Notch. Par ailleurs, le deuxième crible a identifié plusieurs déubiquitinases candidates, dont le rôle dans la régulation du trafic intracellulaire du récepteur Notch non activé est en cours de validation.