NCube/papyrus huggingface dataset:
Étude du trafic cellulaire de la convertase de proprotéine PCSK9 responsable de la dégradation du récepteur des lipoprotéines de faible densité (LDLR)

Coronary heart diseases (CHD) are a leading cause of death in Western societies. Hypercholesterolemia is a major risk factor for CHD. It is characterized by high levels of circulating low-density lipoprotein cholesterol (LDL, also called "bad cholesterol"). The prolonged presence of elevated levels of LDL in the circulation increases the risk of formation of atherosclerotic plaques, which can lead to obstruction of arteries and myocardial infarction. LDL is normally cleared from the blood through the binding of its sole protein constituent apolipoprotein B100 to hepatic LDL receptor (LDLR), which mediates its endocytosis in the liver. Human genetic studies have identified PCSK9 as the third gene responsible of autosomal dominant hypercholesterolemia after LDLR and its ligand apolipoprotein B100. PCSK9 interacts with the LDLR and induces its degradation thereby causing plasma LDL levels to rise. PCSK9 gain-of-function (GOF) mutations are associated with elevated plasma LDL levels and premature CHD while PCSK9 loss-offunction (LOF) mutations reduce the risk of CHD up to ~88% owing to reduction of circulating LDL. Accordingly, PCSK9 is recognized as a major pharmacological target to lower the risk of CHD. PCSK9 binds the LDLR at the cell surface and/or in the Golgi apparatus of hepatocytes and causes its degradation in lysosomes by a mechanism not yet clearly understood. The goal of this study was to determine why some human PCSK9 mutations fail to induce LDLR degradation while others increase it in lysosomes. Several PCSK9 LOF and GOF mutations were fused to the fluorescent protein mCherry to study their molecular mobility in living human liver cells. Our quantitative analysis of fluorescence recovery after photobleaching (FRAP) showed that PCSK9 GOF mutations S127R and D129G have a higher protein mobility (>35% compared to WT) at the trans- Golgi network (TGN). Our quantitative analysis of inverse fluorescence recovery after photobleaching (iFRAP) showed that PCSK9 LOF mutation R46L presented a much slower protein mobility (<22% compared to WT) and a much slower mobile fraction (<40% compared to WT). In addition, our confocal and electron microscopy analyses demonstrate for the first time that PCSK9 is localized and concentrated at the TGN of human hepatocytes. Furthermore, our results demonstrate that PCSK9 localization in the TGN is mediated through its C-terminal cysteine and histidine-rich domain (CHRD), which is essential for LDLR degradation. Also, our live-cell analyses demonstrate for the first time that the CHRD is not required for internalization of PCSK9. These results provide important new information on the mechanism of action of PCSK9 and may ultimately help in the development of inhibitors of the PCSK9-induced LDLR degradation.

Keyphrases: PCSK9, CHRD, FRAP, iFRAP, LDLR degradation, Endocytosis, Cellular trafficking, Trans Golgi Network (TGN), Hypercholesterolemia, Live-cell confocal microscopy
Étude des mécanismes moléculaires impliquant l'homéoprotéine MEIS1 dans le développement de leucémies myéloïdes aigües

Acute myeloid leukemias are the result of a perturbed hematopoietic process and regroup heterogeneous diseases with distinct clinical and genetic profiles. Identifying and understanding the faulty cellular processes would allow the development of targeted and efficient therapeutic tools. Over the last 15 years, a growing number of disease-linked genetic anomalies have been correlated with abnormal expression levels of HOX genes and their cofactors MEIS and PBX. Mouse model experimentations revealed a direct role for these proteins in leukemogenesis. Indeed, the protein MEIS1 collaborates with HOXA9 in the acceleration of leukemia development. This specific function requires the presence of three different domains, two of which are highly conserved in PREP1, another member of the MEIS class of homeoproteins. Using a gain-of-function approach, I confirmed the importance of the C-terminal domain of MEIS1 in the acceleration of HOXA9-induced leukemias. I also correlated the activity of this domain with a transcriptional signature related to cell proliferation. Furthermore, I performed a high-throughput screen to identify antagonists of the MEIS-PBX interaction, also required for acceleration of HOX-induced leukemogenesis. In this regard I developed an assay that exploits bioluminescence resonance energy transfer (BRET) to monitor the MEIS-PBX dimerization in living cells. More than 115 000 compounds were tested and upon confirmation of their activity using an orthogonal assay, 20 small molecules were identified as potential inhibitors. These compounds will be rapidly tested on proliferation of primary leukemic cells in a preclinical setting. Finally two complementary proteomic approaches allowed the identification of new potential partners of MEIS1 and PREP1. The functional clustering of these candidates suggests a new role for homeoproteins in mRNA splicing and methylated DNA recognition.

Keyphrases: BRET, Transcriptome, Acute myeloid leukemia, Homeoprotein, Functional study, BRET, Transcriptome, High-throughput screening
Étude pharmacogénomique de la warfarine et de l'activité physique

Warfarin is an oral anticoagulant agent with a narrow therapeutic index. Dosing of warfarin is highly variable among patients and it may also vary in time for the same patient. All factors influencing warfarin response are not known and this study aims to elucidate if regular physical activity is one important factor. We evaluated whether warfarin dosage and the time in therapeutic range (TTR) were associated with RPA. RPA was measured via 2 different questionnaires.  The study was conducted by using 1,064 patients in the Quebec Warfarin Cohort (QWC) and 618 patients from the Genetic-Hospital Cohort. Both cohorts are hosted at the Montreal Heart Institute.   In both cohorts, we found that active patients required higher weekly doses of warfarin than inactive patients. The association was maintained when the model was adjusted for variables known to influence warfarin response, i.e. CYP2C9 and VKORC1 genotypes, age, height, weight and targeted INR. The targeted INR is fixed by the physicist and is usually between 2.0 and 3.0 or between 2.5 or 3.5. Active patients from the QWC were more susceptible to have a TTR inferior to 60%, i.e. to be unstable.  RPA influences warfarin response and intensive RPA is associated with a greater instability in treatment response.

Keyphrases: Warfarin, Physical activity, Personalized medicine, Drug interaction, Pharmacogenomics
Facilitation de la procédure d’ablation de fibrillation auriculaire persistante après restauration du rythme sinusal pré-procédure : une étude multicentrique comparative

Abstract Catheter ablation of persistent atrial fibrillation (AF) is associated with longer procedure-times and lower long-term success rates than paroxysmal AF. We hypothesized that restoration/maintenance of sinus rhythm (SR) pre-ablation would facilitate AF-termination and improve outcomes in patients with persistent AF.  Methods: We conducted a two-group cohort study of consecutive patients with persistent AF and SR restored for at least one month prior to ablation (SR Group; N=40) and controls matched by age, sex, and AF duration (Control Group; N=40). Radiofrequency (RF) step-wise catheter ablation was performed in AF for both groups (induced and spontaneous, respectively). Success was defined as freedom from atrial tachyarrhythmia without antiarrhythmic drugs beyond one year of follow-up.  Results: During the index ablation-procedure, AF cycle length was longer in the SR versus Control Group (183±32 vs 166±20 ms, P=0.06) suggestive of reverse remodeling. In the SR Group, AF more frequently terminated during ablation (95.0% vs 77.5%, P<0.05) and required less extensive ablation of complex fractionated electrograms (40.0% vs 87.5%, p<0.001) and linear lesions (42.5% vs 82.5%, p<0.001). Mean procedural (199.8±69.8 vs 283.5±72.3 minutes, P<0.001), fluoroscopy (51.0±24.9 vs 96.3±32.1 minutes, P<0.001), and RF-energy delivery (47.5±18.9 vs 97.0±30.6 minutes, P<0.001) times were shorter in the SR Group. Clinical success rates were similar between groups for first (55.0% vs 45.0%, P=0.28) and last (80.0% vs 70.0%, P=0.28) procedures, during similar follow-up periods (21.1±9.7 months).  Conclusions: Restoration of SR prior to catheter ablation for persistent AF whenever possible decreases the extent of ablation with the same high clinical efficacy.

Keyphrases: Atrial fibrillation, Catheter ablation
Criblage génétique et caractérisation fonctionnelle des mutations dans les différentes sous-unités du récepteur GABAA associées à l'épilepsie génétique généralisée

Genetic generalized epilepsy (GGE) syndrome is a group of epilepsy disorders that occur early in childhood and adolescence. Genetics generalized epilepsies (GGE) account for approximately 30 % of all epilepsy syndromes. There is currently no cure for GGE. Although patients with GGE typically have no anatomical brain abnormalities, the root cause of these conditions is considered to be genetic in origin. An increasing number of genes predisposing to epilepsy have been identified over the past ten years. It has emerged in many cases that the causative genes for inherited epilepsies code for ion-channels such as the GABAA receptor (GABAAR). Among these genes, mutations in four subunits of the GABAA receptor appear to be an important cause of familial epilepsy. The main aim of the present thesis is to better characterize the genetic component of our GGE cohort explain by GABRA genes and evaluated the critical role of these variants in the pathogenesis of this phenotype.  The first part of our project was to investigate the impact of rare variants of GABAAR in GGE, we screened the coding regions of 19 genes encoding for all the known subunits of the GABAAR in unrelated GGE patients, including familial cases. Overall, approximately 8% of our GGE individuals have novel GABRA mutations, including 19 missenses and 3 nonsenses including 1 frameshift mutation. By sequencing those genes in a large cohort of cases and controls, we were able to establish the profile of rare variants for these genes. Our data suggest that a significant proportion of GGE patients share rare variants in GABRA genes. The second part of the work builds on the genes bearing mutations identified in the sequencing analysis. Among this group, five novel mutations have been so far associated to this syndrome (GABRA1 and GABRG2). We characterized the gating properties of GABA-evoked currents and the subcellular localization of the mutated subunits by expressing recombinant GABAA receptors in vitro. The third part of the work aimed to characterize the impact of mutated GABAA receptors on synapse formation and development of neuronal networks. By knocking down these genes in cortical organotypic slices, we provided a better understanding of the specific and distinct neural circuit alterations caused by different GABRA1 mutations and help define the pathophysiology of genetic generalized epilepsy syndromes.  We believe that these findings will allow a better understanding of the genetic mechanisms underlying the disease and involve mutations in GABAA receptors in critical mechanisms leading to epilepsy. Eventually, our results could lead to a better diagnosis and counteract the devastating effects of some GGEs early on before this complex condition has had the opportunity to be established.

Keyphrases: Genetic generalized epilepsy, GABAA receptor, Genetics
Caractérisation du rôle de MCAM dans la sclérose en plaques

Objective: In multiple sclerosis (MS), pro-inflammatory lymphocytes use adhesion molecules to cross the blood-brain barrier (BBB) and accumulate in central nervous system (CNS) lesions. CD4 T lymphocytes polarized into auto-aggressive encephalitogenic TH17 (IL-17 secreting) are known to partake in MS lesion formation. Much less is known about the role of CD8 TC17. Identification of specific surface markers and adhesion molecules expressed by TH17 and TC17 lymphocytes would allow further characterization of these pathogenic subsets and would provide new therapeutic targets in MS. Methodology: We identified MCAM in a proteomic screen of human BBB endothelial cells (ECs) and on a subset of T lymphocytes. We characterized the phenotype and function of MCAM-expressing cells ex vivo, in vitro and in situ using human and mouse material obtained from controls, MS subjects and Experimental Autoimmune Encephalomyelitis (EAE) animals. Results: MCAM is expressed by human BBB-ECs and by human effector memory CD161+ and CCR6+ T lymphocytes. Both CD4 and CD8 MCAM+ lymphocytes express more IL-17, IL-22, GM-CSF and Gz B than MCAMneg lymphocytes. Moreover, MCAM is strikingly up-regulated in human on CD4+ and CD8+ T lymphocytes during MS relapses, while treatment decreases MCAM expression. In situ, MCAM+ CD8 and CD4 T lymphocytes are present in perivascular infiltrates of MS and EAE CNS specimens, while MCAM expression is up-regulated on BBB-ECs within lesions. In vitro, MCAM+ CD8 T lymphocytes display higher killing capacity of oligodendrocytes, and MCAM blockade reduces CD8 TC17 and CD4 TH17 transmigration across human BBB-ECs. In vivo, depletion of MCAM+ cells from reactivated CD4 T lymphocytes and from CD8 T lymphocytes decreases clinical symptoms in adoptive transfer EAE. Furthermore, expression of MCAM is up-regulated on CD4 and CD8 T lymphocytes in the TCR1640 transgenic mice, a model of spontaneous EAE. Finally, blocking MCAM in both MOG35-55-induced and spontaneous primary progressive EAE attenuates chronic neurological deficits. Conclusions: Our data demonstrate that encephalitogenic IL-17-producing lymphocytes with high effector and migratory capacity express MCAM, and that MCAM could serve as a biomarker for MS and a valuable target for the treatment neuroinflammatory conditions.

Keyphrases: Th17, Tc17, IL-23, transmigration leucocytaire, MCAM (CD146), Multiple sclerosis, Blood-brain barrier, experimental autoimmune encephalomyelitis (EAE), leukocyte transmigration, adhesion
The regulatory roles of APE1 and Prdx1 interaction

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein, which play important roles in base excision repair (BER) pathway and serve as transcriptional co-activator. APE1 is also involved in RNA metabolism and redox regulation. APE1 can cleave abasic sites and process 3’-blocking termini into 3’-OH for DNA repair replication as it posseses several DNA repair activities including AP endonuclease, 3’-phosphodiesterase and 3’ to 5’-exonuclease. Mammalian cells knockdown for APE1 are very sensitive to various DNA damaging agents. APE1 has a unique cysteine C65, which is required to maintain the reduced state of several transcriptional activators such as p53, NF-кB, AP-1, c-Jun, and c-Fos and therefore is involved in the regulation of gene expression. APE1 also undergoes at least four types of post-translational modifications that include acetylation, deacetylation, phosphorylation and ubiquitylation. How APE1 is being recruited to execute the various biological functions remains a challenge, although this could be directly related to its ability to interact with multiple different partners. Under normal growth conditions, APE1 has been shown to interact with a number of proteins that are involved in various functions. We propose that the oxidative state of APE1 governs its interacting partners thereby allowing the protein to perform specific functions. In this study we find that APE1 interactome alters in response to hydrogen peroxide. One novel APE1 interacting partner Prdx1, a member of the peroxiredoxin family that can scavenge hydrogen peroxide is characterized. We demonstrate that knockdown of Prdx1 did not impair APE1 DNA repair activity, but alters APE1 detection, and subcellular distribution in HepG2 cells leading to the induction of interleukin 8 (IL-8). IL-8 is a pro-inflammatory chemokine involved in cellular stress, under physiological and iv oxidative stress conditions. It has been shown that the induction of IL-8 is dependent on APE1 indicating Prdx1 may regulate APE1 transcriptional activity. Prdx1 has been discovered to be involved in the redox regulation of cell signaling initiated by hydrogen peroxide, which has important roles as a signaling molecule in the regulation of a variety of biological processes. Prdx1 exists as a dimer in the cells and we show that Prdx1 is required to reduce APE1 in the cytoplasm in response to H2O2. During this process, the dimeric form of Prdx1 is converted to the oxidized monomeric form. Interestingly, the H2O2-induced conversion of Prdx1 to the monomeric form is dependent upon the presence of APE1. These observations imply that there is a tight regulatory network existing between APE1 and Prdx1.

Keyphrases: APE1, Prdx1, IL-8, Hydrogen peroxide, Cell signaling
Caractérisation de stratégies stimulant l’immunité cellulaire par l’étude de la présentation antigénique d’une nanoparticule vaccinale et du blocage d’un mécanisme d’immunosuppression

There are several challenges to the development of therapies aimed at stimulating cellular immunity. In viral infection prevention and in cancer immunotherapy, the induction of specific T lymphocytes is, however, of paramount importance. In the first part of this study, we focused our attention on understanding the major histocompatibility complex class I (MHC I) cross-presentation mediated by virus-like particles (VLP) composed of potexvirus coat protein, in which we had inserted an epitope from the M1 protein of the Influenza virus or an epitope from gp100, a tumour antigen of melanoma. This particular VLP is characterized by its ability to stimulate the immune system with no adjuvant and its cross-presentation of the inserted epitope independently of proteasome activity. First, we compared the antigenic cross-presentation properties of Malva mosaic virus (MaMV) VLPs to that of Papaya mosaic virus (PapMV) VLPs. The results confirm that cross-presentation mechanisms are shared among different members of the potexvirus family despite marked differences in their sequences (Hanafi et al. Vaccine 2010). Furthermore, we have conducted experiments to clarify the mechanism leading to the cross-presentation of the inserted epitope in PapMV VLPs. The results confirm a vacuolar pathway dependent on cathepsin S activity and on lysosomal acidification for antigen presentation. Autophagy induction by VLPs is also important to PapMV VLP antigen cross-presentation. We have herein described a new vacuolar MHC I cross-presentation pathway dependent on autophagy (Hanafi et al. in preparation).  Secondly, in cancer immunotherapy, it is crucial to control immune evasion mechanisms that are initiated by the tumour. We have specifically studied the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) (human cancer literature review in Hanafi et al. Clin. Can. Res. 2011), and its inhibition in tumour cells. To this end, we inhibited its expression using fludarabine, a chemotherapeutic agent previously studied for its inhibitory effect on STAT1 (signal transducers and activators of transcription 1) phosphorylation. Surprisingly, our results demonstrate that IDO inhibition in cancer cells by fludarabine was independent of STAT1 phosphorylation. We showed that the mechanism of action was rather dependent on the induction of IDO degradation by the proteasome (Hanafi et al. PlosOne 2014).  The work presented in this thesis provides a better understanding of how a new vaccine platform can mediate cytotoxic CD8+ T lymphocytes activation and the control of the problem of immunosuppression by tumour cells for the evading of the immune system. These two main strategies are to key for the consideration of cancer immunotherapy in combination with other existing therapies, as these should allow a better clinical response to cancer treatment.

Keyphrases: fludarabine, Cellular immunity, MHC I cross-presentation, Vaccine nanoparticles, indoleamine 2,3-dioxygenase
Dissecting the dynamic of Noc2p and its partners in pre-60S particles maturation

Several studies have been performed to characterize the ribosome as far as to understand its structure and its function.  However, major aspects of ribosome biogenesis remain elusive or gave only a static picture of the process.  In fact, ribosome biogenesis involves dynamic processing and assembly pathways that are required for rRNA modification and folding, in addition to rRNA binding with some ribosomal proteins. One set of assembly factors, the Noc proteins, allowed one of the first indications about the spatio-temporal ordering of ribosome maturation. By using yeast as model, our objective is to provide a dynamic picture of the Noc proteins complexes exchange and nuclear localization by determining the nature of Noc2p interactions with Noc1p and Noc3p and by studying the influence of reversibly arrested intranuclear transport on these proteins and on Rix7p, an AAA-ATPase. In order to achieve these aims, inducible promoter, fluorescent microscopy, western blot, qRT-PCR and affinity purification analyses were used.

Keyphrases: Ribosome, Noc1p, Noc2p, Noc3p, Rix7p, Assembly factors, Spatio-temporal ordering, Ribosome biogenesis, Ribosome maturation
Évaluation du besoin et de la pertinence de l'implantation d'un service d'injection supervisée en Montérégie

This research project aimed to conduct a strategic analysis of the implementation of a supervised injecting facility (SIF) in Montérégie. Using a mixed design, we first completed a portrait of the injection drug user (IDU) population. We then explored the perceptions of IDU and stakeholders with regard to the relevance of implementing a SIF in the region. Although some similarities were found with the IDU populations of Montreal and the province of Quebec, this population in Montérégie is characterized by a lower frequency of injections in public, less homeless people and lower rates of HIV and HCV infections. Despite these differences, the IDU population in Montérégie was found to have important physical and psychosocial needs. Although the relevance of a SIF in Montérégie is undeniable, improvements regarding the accessibility, continuity and appreciation of the actual services dedicated to IDU remain a priority.

Keyphrases: Montérégie, Substances psychoactives, Injection, Supervised injection facility (SIF), Injection drug users (IDU), Substance use, Drug, Syringe, Human immunodeficiency virus (HIV), Hepatitis C virus (HCV), Homeless