Aspergillus fumigatus ist die häufigste Ursache für Lungeninfektionen in immunsuppri-mierten Patienten. Virulenzfaktoren sind häufig an Kontrollmechanismen für Entwick-lung gekoppelt, welche im verwandten Modellorganismus Aspergillus nidulans entdeckt wurden. Diese Arbeit präsentiert die Charakterisierung des F-box Proteins Fbx15 in A. fumigatus, welches einen starken Einfluss auf die Entwicklung in A. nidulans hat. Die Deletion von fbx15 resultierte in starken Wachstumsdefekten unter vielen Stress induzie-renden Bedingungen, welche klassische Virulenz Faktoren beinhalten, wie erhöhte Tem-peratur, oxidativer Stress und Aminosäuremangel, während das Wachstum unter Stan-dardbedingungen nicht beeinflusst war. Oxidativer Stress induziert eine transiente Erhöhung der fbx15 Expression, welche nach 40 Minuten zu einer dreifach erhöhten Pro-teinmenge führte. Fbx15 ist ein stabiles F-box Protein mit einer Halbwertszeit von 90 Minuten. Generell funktionieren F-box Proteine als Substratadapter für SCF-E3-Ubiquitin-Ligasen. Fbx15 liegt unter normalen Bedingungen phosphoryliert vor und in-teragiert mit der Skp1/A Untereinheit des SCF-Komplexes, vorzugsweise in kleineren Subpopulationen im Zytoplasma. Phosphoryliertes Fbx15 wird bevorzugt in SCF-Komplexe eingebaut. Oxidativer Stress führt zu einer schnellen Dephosphorylierung von Fbx15. Fbx15 Varianten, welche nicht phosphoryliert werden können, interagieren mit Skp1/A primär im Kern. Fbx15 rekrutiert drei Untereinheiten des COP9-Signalosoms und Proteine welche in Transkription, Translation, Signalübertragung, Morphologie oder Stoffwechsel involviert sind. Fbx15 bindet die Ssn6/F Untereinheit des konservierten Ssn6/SsnF-Tup1/RcoA Co-Repressors und wird für dessen Kernlokalisation benötigt. Dephosphoryliertes Fbx15 interagiert mit Ssn6/F im Kern und eine Fbx15-Ssn6/F be-dingte Genrepression wird für die Reduzierung der Gliotoxin-Biosynthese benötigt. fbx15 Deletionsstämme sind nicht in der Lage immunsupprimierte Mäuse in einem Model für invasive Aspergillose zu infizieren, was eine essentielle Funktion von Fbx15 für die Viru-lenz bestätigt. Diese Arbeit zeigt, dass Fbx15 nicht nur Teil von SCF-E3-Ubiquitin-Ligasen sein kann, sondern eine zweite neue molekulare Funktion aufweist, welche die physische Interaktion mit der Co-Repressor Untereinheit Ssn6/F und dessen Lokalisa-tionskontrolle beinhaltet. Diese duale Funktion resultiert in einer essentiellen Funktion von Fbx15 für die Kontrolle der oxidativen Stressantwort, des Sekundärmetabolismus und der Virulenz im opportunistischen Humanpathogen A. fumigatus.

The Arabidopsis SON1 F-box protein was implicated in regulating a pathogen defense pathway, but its exact function in wild-type plants is unknown. As an F-box protein it was predicted that SON1 would assembles into a SON1-SCF ubiquitin ligase complex that recruits specific plant defense-related proteins for proteolysis. The yeast 2-hybrid assay was used to screen for potential substrates for a putative SON1-SCF ligase, leading to the identification of Arabidopsis LNK2 as a SON1-binding factor. Comprehensive protein-protein interaction analysis has shown that the binding of SON1 to LNK2 protein is specific, because closely related, full-length Arabidopsis F-box proteins do not interact with LNK2. However, when tested in isolation, some fragments derived from the paralog proteins did bind SON1, suggesting that higher order structure or inter-domain interference affects the ability of SON1 to recruit substrate. When analyzed for interaction domains, three regions of SON1 were identified that bind to LNK2 and a LNK2 binding region spans a conserved amino acid region. Phylogenetic analysis revealed that there is a paralogous gene called LNK1 in Arabidopsis, and both LNK1 and LNK2 are restricted to the plant kingdom. LNK2 and LNK1 are seen to be widely distributed in embryophyte seed and spore plants. Genetic analysis and complementation tests showed that LNK1 and LNK2 regulate flowering and photo-morphogenesis redundantly. Though lnk1 and lnk2 mutants look similar to wild-type plants, lnk1 lnk2 double mutant plants possess a long hypocotyls and flower late compared to wild-type plants. Because SON1 is implicated in plant defense, lnk mutants were assessed for susceptibility to a virulent oomycete pathogen, Hyaloperonospora arabidopsidis (Hpa). Interestingly, lnk mutants supported less disease development, suggesting a role of the wild-type LNK proteins in the enabling of pathogen colonization or in repressing host defenses. To confirm that each of the phenotypes described were a consequence of lnk1 and lnk2 mutations, wild type LNK1 and LNK2 were introduced into lnk1 lnk2 plants and examined. For most phenotypes, genetic complementation and thus restoration of a WT phenotype was observed. However, differences were uncovered in the ability of LNK genes to rescue the phenotypes, indicating specialization of function. The interaction of LNK2 with SON1 suggests that SON1-dependent ubiquitination and proteasomal degradation may control LNK2 abundance. I show by a cell free protein degradation assays that proteasome-based degradation of LNK2 as well as LNK1 is possible. Data showed that SON1 binds to ASK1 in-planta suggest the existence an SCFSON1 complex that targets LNK2 for polyubiquitination and its subsequent degradation by the proteasome. The data presented in this dissertation indicates a role for LNKs in flowering and plant defense and also suggest that proteasome-base regulation of LNK turnover may be utilized to regulate LNK protein abundance and proper maintenance of the circadian clock.

En 1996, les autorités publiques ont décidé de réformer la comptabilité algérienne pour l’adapter aux changements induits par l'environnement économique. Cela a eu lieu dans le cadre de réformes économiques engagées par l’État algérien dans le but de passer d’une économie planifiée à une économie de marché. A cet effet, une réflexion a été conduite sur l’élaboration d’un nouveau référentiel comptable compatible avec ce nouveau contexte de libération de l’économie ; il désormais nommé Système Comptable Financier (SCF), entrée en vigueur depuis le 1er janvier 2010. Le projet de recherche porte sur le processus de mise en œuvre du SCF 2010 au sein des entreprises algériennes. Il tente de décrire et d’expliquer la pratique organisationnelle des entreprises lors du changement comptable. A ces fins, nous nous appuyons sur un cadre théorique cohérent afin de garantir une meilleure compréhension du phénomène : courant d’innovation et courant du changement organisationnel. Par ailleurs, pour enrichir notre recherche, nous avons fait également des emprunts à la Théorie Néo-Institutionnelle sociologique. Ainsi, notre recherche est qualitative et consiste à mettre en lumière les qualités et les caractéristiques de l’information examinée. Pour cela, nous avons retenu la méthodologie de l’étude de cas longitudinale au sein de deux entreprises du secteur publique : SONATRACH et ERO. L’étude de ces deux études de cas démontre que le processus de mise en œuvre peut être décomposé en trois phases successives à savoir : la phase de pré-adoption & adoption, la phase d’implantation et la phase d’intégration. L’observation et la description de ce processus à suggérer des propositions destinées à en garantir le succès de son intégration au sein des entreprises y sont concernées. / In 1996, algerian public authorities have initiated a number of reforms in view of adjusting Algeria to the new economic changes and promoting the country’s integration into world economy. Reforming accounting practices reports to this will to change. To this end, new accounting guidelines, compatible with the new context of economic liberalization, have been elaborated. It was labelled the Financial Accounting System (FAS) and made effective since January 1st 2010. This paper then examines Algerian firms’ implementation of the FAC 2010. It tries to describe and explain firms’ organisational practices in these accounting changes. Moreover, to enrich our study, we invoked also the sociological component of the Neo-Institutionalist Theory. Our study is qualitative and consists of highlighting the qualities and characteristics of the examined issue. To this end, we chose as methodology a longitudinal case study of two public firms: SONATRACH and ERO. Examining these two case studies, we found that the implementation process follows three major phases: a pre-adoption and adoption phase, an implementation phase and an embedded ness phase. The study and description of the process led us to propose recommendations on the successful embedded and implementation of the FAS 2010 by targeted firms.

Maintenance of genomic integrity is dependent on the duplication of chromosomes, only once per cell cycle. Highly conserved mechanisms for the regulation of chromosome replication exists to ensure that the genome is copied only once. The Cdc45-MCM-GINS (CMG) DNA helicase which is the core of the eukaryotic replication complex, has been shown to be extensively regulated by post translational modifications, during its assembly. Therefore, it is not inconceivable that the process to unload the replication complex would also be a conserved and regulated process. In 2014, our lab discovered that the CMG complex undergoes post-translational modification in the form of ubiquitylation on one of the subunits of CMG, leading to its disassembly from the chromatin. Though the main players in the disassembly of CMG were known, viz the E3 ligase SCFDia2 and segregase Cdc48, very little was known about the mechanism of CMG disassembly. In the process of learning more about the disassembly of the replicative helicase from chromatin, I reconstituted the ubiquitylation of CMG and thereafter the disassembly of CMG helicase in vitro. My work resulting in the reconstitution of CMG disassembly in vitro is the first example of the disassembly of a multi-subunit physiological substrate of Cdc48. Though CMG is ubiquitylated in yeast extracts in vitro, it does not lead to its disassembly and therefore led me to find conditions necessary for the efficient ubiquitylation of CMG. I have further shown that purifying the E3 ligase associated CMG can be efficiently ubiquitylated in a semi-reconstituted system consisting of purified factors, necessary for the ubiquitylation of substrate. I investigated whether this efficiently ubiquitylated CMG can be disassembled by purified Cdc48 and associated co-factor Ufd1/Npl4 in vitro and found that disassembly is dependent on K48 linked poly-ubiquitylation of CMG. I have found that the reconstituted poly-ubiquitylation of CMG is restricted to the Mcm7 subunit of CMG, recapitulating the ubiquitylation of CMG in vivo, and my data points out that there are multiple sites of ubiquitylation on Mcm7. Through this work, I have also found that ubiquitylated Mcm7 no longer associates with the rest of the CMG components after disassembly of CMG. My assays and findings, open the door towards dissecting the molecular mechanism of the disassembly of CMG in greater detail.

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers, including melanoma, ovarian cancer, and colon cancer. Madoz-Gurpide et al. recently observed Bmp4 in the nuclei of a minor percentage of cells in colon cancer tissues. In addition, our lab has recently discovered a nuclear variant of Bmp2 (nBmp2), the TGF-β family member most closely related to Bmp4. These observations led us to hypothesize that a nuclear variant of Bmp4 (nBmp4) also exists. The results of chapter one report the existence of a nuclear variant of Bmp4. nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. Chapter 2 and 3 further report that nBmp4 interacts with several subunits in the SCF E3 ubiquitin ligase, namely two Regulator of Cullins (ROC) proteins, five Cullin proteins, and two F-box proteins. Due to the known role of the SCF E3 ubiquitin ligase in regulating the cell cycle, the effect of nBmp4 on cell cycle progression was analyzed and the results show that nBmp4 affects the cell cycle by causing cells to accumulate in G0/G1. The association of nBmp4 and the SCF E3 ubiquitin ligase components and the affect that nBmp4 has on the cell cycle suggest that nBmp4 functions in the nucleus by inhibiting the SCF E3 ubiquitin ligase from ubiquitinating target proteins that are involved in regulating cell cycle progression. Finally, the initial stages in the generation of an nBmp4 over-expression mouse are described. The results of this research clearly change the traditional paradigm that Bmp4 performs all of its functions via extracellular signaling and introduce the existence of a nuclear variant that is involved in cell cycle regulation.

Neural stem cell self-renewal and differentiation are tightly regulated events during CNS development, leading to cell division into new neural stem cells or the formation of neurons and glial cells. This thesis focuses on the cellular responses induced by known and novel factors in neural stem and progenitor cells (NSPCs). Platelet-derived growth factor (PDGF) signaling has previously been implicated in NSPC regulation as well as in tumor formation. In order to evaluate the differentiation process and find new regulators of NSPCs a micro-array screen was performed, evaluating transcription during normal differentiation and the effect of PDGF-AA in this process. The transcriptional profile of PDGF-AA treated NSPCs was shown to be an intermediate between the profiles of neural stem cells and their progeny. The NSPC transcriptome was also found to have similarities with that of experimental glioma. A previously non-characterized transcript, the nuclear receptor binding protein 2 (NRBP2), was identified and found to be expressed in the developing and adult mouse brain and in medulloblastoma. NRBP2 down-regulation rendered neural progenitors sensitive to induced cell death. Different PDGF ligands interact with different combinations of PDGF receptors. Therefore NSPCs were stimulated with either PDGF-AA or -BB to further evaluate cellular responses with regard to the two specific isoforms. A divergent effect between the two isoforms in long-term proliferation and cell survival was found, with PDGF-BB being the most efficient stimulator. Stem cell factor (SCF) has previously been identified as a regulator in the hematopoietic system and we showed that SCF induces a migratory response in NSPCs. In addition, SCF positively affected cell survival but had no effect on NSPC differentiation. Insights into the regulatory mechanisms involved in neural stem cell signaling are needed to develop diagnostic tools and novel treatments.

F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.

In this work we study properties of branched polymers in a good solvent. We focus on problematic related to the size exclusion chromatography and predicting elution behavior of randomly branched polymers. We developed a software for generating self-avoiding walks (SAW) of any given non-looping architecture on a cubic lattice using Monte Carlo (MC) simulation and vali- date its reliability by presenting the scaling of different architectures: linear, 3-arm star and 6-arm star and asymmetric star. We calculate distribu- tion coefficients and calibration curves for size exclusion chromatography for various architectures to validate that the hydrodynamic radius is more suitable for predicting elution volume than the radius of gyration. Then we propose a new method for, although approximate, a very fast estimation of radius of gyration and hydrodynamic radius for different architecture using a graph method. It is done by comparing MC results with results obtained from graph theory. Then we introduce a correction to graph-theory results to fit the MC. At the end we present depletion layer calculation from MC and self-consistent field (SCF) method for polymers and their comparison. We show how calculation of depletion layer using SCF can be improved to get significantly better agreement with MC results. v

The topic of this thesis is the investigation of the local states of stress resulting from the introduction of av-notch in a coaxial circle on the pressurized surface of a circumferentially clamped plate subject to axisymmetric loading. The understanding of the fracture behavior of a component experiencing such a condition is of particular interest to the aerospace and defense industries where circular plate components are often utilized. In such applications, it is imperative that the designer be able to predict the loading conditions facilitating dynamic fracture. As a step towards solving such problems, the quasi-static analogy is studied. Specifically, the purpose of this research is to examine and model the precise effects a stress raiser will have on the fracture behavior and strength reduction of a circular plate machined from Ultem 1000. Parametric FEM simulations were employed to determine the correlation between notch geometry and the resulting maximum stress and stress distribution in the notch root vicinity. Stress concentration factor (SCF) relationships were developed which characterize the effect individual geometric parameters have on the notch root stresses. Mathematical models were developed to provide the elastic stress concentration factor for any combination of geometric parameters within the range studied. Additionally, the stress distributions along the notch root and ahead of the notch were characterized for a variety of geometric configurations. Test coupons were employed to not only characterize the mechanical behavior of the material, but also characterize the correlation between simple and axisymmetric loading, respectively. The development of a predictive approach for designers of such circular components to be able to accurately determine the fracture behavior of these components was the motivating factor of this study.

No description available.