Les corps nucléaires PML (CNs) sont des structures sphériques organisées par la protéine PML où, de nombreuses protéines impliquées dans divers processus biologiques tels que la sénescence, la mort cellulaire ou la défense virale, y sont recrutées. Ces protéines ont été identifiées individuellement dans des études spécifiques mais aucune étude n’avait permis la purification des CNs et l’établissement de liste des partenaires de PML de façon systématique. La protéine PML est sensible à l’oxydation et le stress oxydant promeut son assemblage en CNs permettant le recrutement de l’enzyme E2 clé de la conjugaison, UBC9. Les interférons (IFNs), cytokines aux propriétés antivirales induisent l’expression de PML. L’arsenic et les IFNs augmentent l’assemblage des CNs.Dans cette étude nous avons exploré, in vivo, le rôle de PML dans la sumoylation en réponse au stress en utilisant deux modèles de souris et un modèle cellulaire, les cellules souches embryonnaires de souris (mESC), exprimant des versions taguées de SUMO1 ou SUMO2. Nous avons montré que PML augmente rapidement la sumoylation dans les foies de souris KI His6-HA-SUMO1 en réponse aux traitements arsenic/IFN. De façon similaire, dans la leucémie aiguë promyélocytaire (LAP), où les CNs sont désorganisés, l’arsenic promeut la reformation des CNs et la conjugaison par SUMO. L’analyse par spectrométrie de masse quantitative des conjugués His10-SUMO2 a permis de mettre en évidence une liste de protéines sumoylées en réponse au traitement thérapeutique à l’arsenic. Parmi ces protéines, TRIM28/KAP1 et d’autres protéines appartenant au même complexe régulant la chromatine ont été identifiées. TRIM28, qui joue un rôle clé dans la répression des rétrovirus endogènes (ERV) dans les mESC, est sumoylée de façon dépendante de PML dans ces cellules. Les CNs régulent également l’ubiquitinylation et la dégradation dépendante du protéasome, de certaines de ces protéines. Enfin, nous avons montré que les CNs favorisent la formation de chaînes SUMO2/3, expliquant le rôle des CNs dans la dégradation des protéines. Cette étude nous a permis de mettre en évidence que les CNs sont des compartiments nucléaires qui contrôlent la conjugaison de diverses protéines dans des conditions physiologiques de stress cellulaire, montrant pour la première fois leur activité de contrôle de MPT in vivo. / PML drives assembly of PMLNuclear Bodies (NBs) where it recruits hundreds of serendipitously-identified proteins, among which the key UBC9 E2-sumoylation enzyme. Interferons (through transcriptional PML induction) and arsenic or oxidative stress (through PML aggregation) dramatically enhance NB assembly. Here we directly investigated any role for PML in stress-responsive sumoylation in vivo. We demonstrate that PML very rapidly promotes arsenic/interferon-responsive sumoylation in mouse liver or mouse embryonic stem cells. Similarly, in Acute Promyelocytic Leukemia (APL), where PML NBs are disorganized in the basal state, arsenic therapy promoted NB-reformation and broad SUMO-conjugation. Label free quantitative proteomic analysis of His10-SUMO2-conjugates revealed a comprehensive list of therapy-responsive sumoylated proteins, among which TRIM28 and other proteins belonging to the same epigenetic complex. PML NBs-regulated sumoylation also drives ubiquitination and proteasome-dependent degradation of some targets. Finally, by expressing conjugation-resistant SUMO2, we demonstrate that PML NBs promotes processive SUMO2/3 chain elongation, thus explaining PML role in partner degradation. Collectively, our findings highlight the key activity of NBs in stress-regulated sumoylation/degradation in vivo.

Arsenic trioxide is a clinically effective treatment for the disease acute promyelocytic leukaemia (APL) which is caused by the chromosomal translocation t(15;17) which fuses the promyelocytic leukaemia (PML) protein to the retinoic receptor alpha (RARa). The PML-RARa oncoprotein disrupts normal retinoic acid signalling and the function of PML nuclear bodies (PML-NBs), subnuclear protein complexes with roles in control of apoptosis and cellular senescence. Treatment with arsenic induces rapid post translational modification of PML and with the small ubiquitin like modifier (SUMO). SUMO modification of PML recruits the SUMO targeted ubiquitin E3 ligase RNF4 via four SUMO interaction motifs within the N-terminal region of RNF4. PML is then ubiquitylated and targeted for proteasomal degradation. In APL, these events trigger degradation of PML-RARa, curing the disease. To further investigate the process of arsenic induced degradation of PML, a high content siRNA screen was designed to monitor the fate of a YFP linked version of PML after siRNA mediated knockdown of components of the ubiquitin system and arsenic treatment. RNF4 depletion prior to arsenic treatment prevented PML degradation and resulted in accumulation of PML in large, bright PML-NBs. This was used as a positive control. A library of siRNAs targeting 1067 gene products were screened to identify those which perturbed the process of arsenic mediated degradation of PML, and those which affected the stability of PML in untreated cells. A number of putative hits were identified. Depletion of the cullin RING ligase scaffold CUL3, and the NEDD8 E3 ligase DCUN1D1 resulted in striking accumulation of PML, suggesting PML may be a substrate of a CUL3 RING ligase complex. Further experiments using the inhibitor of neddylation, MLN4924 support this hypothesis. PML is expressed as a various isoforms which encode a unique C-terminal region, due to alternative splicing. The second part of this study investigated the role of this variable C-terminal region in the response of the six major PML isoforms to arsenic treatment. Using a system in which only a single eYFP-linked PML isoform is expressed, differences in the localisation of PML isoforms following arsenic treatment were identified, with PML I, II and VI found to accumulate in the cytoplasm following arsenic treatment, whereas PML III, IV and V did not. A high content imaging assay identified PML V as the isoform most readily degraded following arsenic treatment, and PML IV as relatively resistant to degradation. Using siRNA it was demonstrated that arsenic induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 resulted in marked accumulation of PML V, suggesting this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment.

The promyelocytic leukaemia protein (PML) is a tumour suppressor initially identified in acute promyelocytic leukaemia (APL). In APL, PML and the retinoic acid receptor alpha (RARalpha) genes are fused as a consequence of the translocation t(15;17). The product of the chimeric gene is the oncogenic PML-RARalpha protein. The PML gene encodes multiple nuclear and cytoplasmic isoforms. PML nuclear isoforms (nPML) are the main components of the PML nuclear bodies (PML-NBs), sub-nuclear structures involved in the modulation of essential cellular players including the tumour suppressor p53. Nuclear PML has been intensively studied, while, the role of cytoplasmic PML remains poorly understood. Increasing evidence indicates that PML could bear cytoplasmic functions in both physiological and pathological settings. This study aims to gain more insights into the function of PML and PML-RARalpha cytoplasmic pool of proteins. Recently, two missense mutations resulting in truncated PML cytoplasmic protein (Mut PML) have been identified in aggressive APL cases. We found that Mut PML alters the structure and the function of the PML-NB mainly through the cytoplasmic relocation of nPML. Remarkably, Mut PML inhibits p53 transcriptional, growth suppressive and apoptotic functions. In the cytoplasm, Mut PML interacts and stabilizes PML-RARalpha, thus potentiating its block of RA-induced transcription and differentiation. A mutant of PML-RARalpha (Delta2) accumulating in the cytoplasm is able to inhibit RA-dependent transcription and differentiation, suggesting that cytoplasmic localization of PML-RARalpha may contribute to transformation. Finally, we found that Delta2 expression blocks G-CSF-dependent myeloid differentiation and causes partial transformation of primary haematopoietic progenitor cells.

We consider computation of resonances in open systems and acoustic scattering problems. These problems are posed on an unbounded domain and domain truncation is required for the numerical computation. In this paper, a perfectly matched layer (PML) technique is proposed for computation of solutions to the unbounded domain problems. For resonance problems, resonance functions are characterized as improper eigenfunction (non-zero solutions of the eigenvalue problem which are not square integrable) of the Helmholtz equation on an unbounded domain. We shall see that the application of the spherical PML converts the resonance problem to a standard eigenvalue problem on the infinite domain. Then, the goal will be to approximate the eigenvalues first by replacing the infinite domain by a finite computational domain with a convenient boundary condition and second by applying finite elements to the truncated problem. As approximation of eigenvalues of problems on a bounded domain is classical [12], we will focus on the convergence of eigenvalues of the (continuous) PML truncated problem to those of the infinite PML problem. Also, it will be shown that the domain truncation does not produce spurious eigenvalues provided that the size of computational domain is sufficiently large. The spherical PML technique has been successfully applied for approximation of scattered waves [13]. We develop an analysis for the case of a Cartesian PML application to the acoustic scattering problem, i.e., solvability of infinite and truncated Cartesian PML scattering problems and convergence of the truncated Cartesian PML problem to the solution of the original solution in the physical region as the size of computational domain increases.

Loss of the promyelocytic leukemia (PML) protein is associated with genomic instability/cancer. There are several isoforms of the PML protein that localize in PML nuclear bodies (PML NBs). How each individual isoform contributes to the functions of PML NBs is unknown. The objective of this study was to identify and characterize PML isoform-I (PML-I) specific protein-protein interactions. Using yeast two-hybrid screens, several interacting partners of PML-I were identified that play roles in translational regulation, including eukaryotic initiation factor 3 subunit K (eIF3K). Our studies demonstrated that eIF3K interacts with PML-I in vitro and in vivo. Through its interaction with eIF3K, overexpression of PML-I resulted in the concomitant increase in eIF3K protein levels in mammalian cells. This suggests that PML-I may be involved in regulating eIF3K protein translation or stability, which in turn could affect translation of specific mRNAs or global translation in cancer cells with reduced expression of PML-I.

No description available.

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic infection of oligodendrocytes by JC Virus (JCV). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. JCVCPN was isolated from the brain of a JCVE patient. JCVCPN contains a unique 143 base pair deletion in the agnogene and has an archetype-like regulatory region (RR), of the type typically found in the kidneys. In this dissertation, we studied the JCVCPN virus to better understand the role of the agnogene and the RR in JCV replication. We used kidney, glial and neuronal cell lines to compare the replication of JCVCPN to the prototype virus JCVMad-1. JCVCPN was able to replicate viral DNA in all cell lines tested, but was unable to establish the high level of infection seen with JCVMad-1. Levels of VP1 capsid protein were undetectable in JCVCPN transfected cells, and few infectious virions were produced. JCVCPN did not have a replication advantage in the neuronal cell line tested. To determine if the agnogene deletion or the archetype-like RR was responsible for the observed phenotype of JCVCPN, we generated a series of chimeric viruses between JCVCPN and JCVMad-1. We found that the phenotype of JCVCPN was due predominantly to the deletion in the agnogene, in particular the loss of the DNA and not the lack of a full length agnoprotein. To further study the role of the agnogene DNA in JCV replication, we introduced a series of small agnogene deletions into a virus with a start codon mutation which prevents agnoprotein expression. We characterized the replication of these additional mutants and found that nucleotides 376-396 are crucial for the expression of VP1 capsid protein. Previous studies have provided evidence for the binding of host cell proteins to the agnogene DNA. We used DNA-Immunoprecipitations with the agnogene to identify candidate binding proteins, but were unable to confirm any candidate proteins as binding specifically to the JCV agnogene. Studying this naturally occurring pathogenic variant of JCV provided a valuable tool for understanding the functions of the agnogene and RR form in JCV replication.

This thesis investigates the use of a combined finite element and perfectly matched layer approach in modeling guided elastic wave motion in infinite plates and cylinders and its potential applications to non-destructive evaluation. Underlying principles of the per-fectly-matched, absorbing layer are demonstrated on one-dimensional wave propagation in a semi-infinite elastic rod. Feasibility of using the perfectly matched layer as absorbing boundary condition in the finite-element modeling of guided elastic wave propagation and scattering is studied for the canonical problem of shear horizontal wave motion in isotropic plates. Numerical re-sults in this study are validated against exact analytical solutions. Excellent agreement has motivated the endeavour to take the technique to the next level of pressure, shear-vertical wave motion in isotropic and transversely isotropic plates. Time-domain, finite-element formulation of the perfectly matched layer for pressure, shear-vertical wave motion was validated through comparisons with semi-analytical lit-erature data and reciprocity checks. Numerical implementation of the model was em-ployed in studying the effect of crack presence on the time of arrival in a pitch-catch, non-destructive inspection arrangement. Predictions made confirmed previously-reported experimental findings. Extensions into three-dimensional, Cartesian and cylindrical spaces were validated against reported data. Practical examples of wave scattering in damaged concrete beams, oil and gas pipelines, and composite shells demonstrated the potential use of the proposed model in simulating elastic-wave based non-destructive inspection. Up to 80 % of the computational time needed to run an extended-mesh, finite-element model can be saved by introducing the perfectly-matched, absorbing layer to the finite-element model as the current thesis proposes. This significant saving in computational time by the proposed FE-PML model can accelerate the production of artificial neural network training data or help tackle complicated non-destructive testing applications.

This thesis investigates the use of a combined finite element and perfectly matched layer approach in modeling guided elastic wave motion in infinite plates and cylinders and its potential applications to non-destructive evaluation. Underlying principles of the per-fectly-matched, absorbing layer are demonstrated on one-dimensional wave propagation in a semi-infinite elastic rod. Feasibility of using the perfectly matched layer as absorbing boundary condition in the finite-element modeling of guided elastic wave propagation and scattering is studied for the canonical problem of shear horizontal wave motion in isotropic plates. Numerical re-sults in this study are validated against exact analytical solutions. Excellent agreement has motivated the endeavour to take the technique to the next level of pressure, shear-vertical wave motion in isotropic and transversely isotropic plates. Time-domain, finite-element formulation of the perfectly matched layer for pressure, shear-vertical wave motion was validated through comparisons with semi-analytical lit-erature data and reciprocity checks. Numerical implementation of the model was em-ployed in studying the effect of crack presence on the time of arrival in a pitch-catch, non-destructive inspection arrangement. Predictions made confirmed previously-reported experimental findings. Extensions into three-dimensional, Cartesian and cylindrical spaces were validated against reported data. Practical examples of wave scattering in damaged concrete beams, oil and gas pipelines, and composite shells demonstrated the potential use of the proposed model in simulating elastic-wave based non-destructive inspection. Up to 80 % of the computational time needed to run an extended-mesh, finite-element model can be saved by introducing the perfectly-matched, absorbing layer to the finite-element model as the current thesis proposes. This significant saving in computational time by the proposed FE-PML model can accelerate the production of artificial neural network training data or help tackle complicated non-destructive testing applications.

No description available.