Dégradation des membres de la famille du LDLR par la convertase PCSK9 : troisième locus de l'hypercholestérolémie familiale

Les maladies cardiovasculaires (MCV) sont les principales causes de mortalité et de morbidité à travers le monde. En Amérique du Nord, on estime à 90 millions le nombre d’individus ayant une ou plusieurs MCV, à près de 1 million le nombre de décès reliés par année et à 525 milliards de dollars les coûts directs et indirects en 2010. En collaboration avec l’équipe du Dre. Boileau, notre laboratoire a récemment identifié, le troisième locus impliqué dans l’hypercholestérolémie familiale. Une étude publiée dans le New Engl J Med a révélé que l’absence de la convertase PCSK9 réduit de 88% le risque de MCV, corrélé à une forte réduction du taux de cholestérol plasmatique (LDL-C). Il fut démontré que PCSK9 lie directement le récepteur aux lipoprotéines de faible densité (LDLR) et, par un mécanisme méconnu, favorise sa dégradation dans les endosomes/lysosomes provoquant ainsi une accumulation des particules LDL-C dans le plasma. Dans cet ouvrage, nous nous sommes intéressés à trois aspects bien distincts : [1] Quels sont les cibles de PCSK9 ? [2] Quelle voie du trafic cellulaire est impliquée dans la dégradation du LDLR par PCSK9 ? [3] Comment peut-on inhiber la fonction de PCSK9 ? [1] Nous avons démontré que PCSK9 induit la dégradation du LDLR de même que les récepteurs ApoER2 et VLDLR. Ces deux membres de la famille du LDLR (fortes homologies) sont impliqués notamment dans le métabolisme des lipides et de la mise en place de structures neuronales. De plus, nous avons remarqué que la présence de ces récepteurs favorise l’attachement cellulaire de PCSK9 et ce, indépendamment de la présence du LDLR. Cette étude a ouvert pour la première fois le spectre d’action de PCSK9 sur d’autres protéines membranaires. [2] PCSK9 étant une protéine de la voie sécrétoire, nous avons ensuite évalué l’apport des différentes voies du trafic cellulaire, soit extra- ou intracellulaire, impliquées dans la dégradation du LDLR. À l’aide de milieux conditionnées dérivés d’hépatocytes primaires, nous avons d’abord démontré que le niveau extracellulaire de PCSK9 endogène n’a pas une grande influence sur la dégradation intracellulaire du LDLR, lorsqu’incubés sur des hépatocytes provenant de souris déficientes en PCSK9 (Pcsk9-/-). Par analyses de tri cellulaire (FACS), nous avons ensuite remarqué que la surexpression de PCSK9 diminue localement les niveaux de LDLR avec peu d’effet sur les cellules voisines. Lorsque nous avons bloqué l’endocytose du LDLR dans les cellules HepG2 (lignée de cellules hépatiques pour l’étude endogène de PCSK9), nous n’avons dénoté aucun changement des niveaux protéiques du récepteur. Par contre, nous avons pu démontrer que PCSK9 favorise la dégradation du LDLR par l’intermédiaire d’une voie intracellulaire. En effet l’interruption du trafic vésiculaire entre le réseau trans-Golgien (RTG) et les endosomes (interférence à l’ARN contre les chaînes légères de clathrine ; siCLCs) prévient la dégradation du LDLR de manière PCSK9-dépendante. [3] Par immunobuvardage d’affinité, nous avons identifié que la protéine Annexine A2 (AnxA2) interagit spécifiquement avec le domaine C-terminal de PCSK9, important pour son action sur le LDLR. Plus spécifiquement, nous avons cartographié le domaine R1 (acides aminés 34 à 108) comme étant responsable de l’interaction PCSK9AnxA2 qui, jusqu’à présent, n’avait aucune fonction propre. Finalement, nous avons démontré que l’ajout d’AnxA2 prévient la dégradation du LDLR induite par PCSK9. En somme, nos travaux ont pu identifier que d’autres membres de la famille du LDLR, soit ApoER2 et VLDLR, sont sensibles à la présence de PCSK9. De plus, nous avons mis en évidence que l’intégrité du trafic intracellulaire est critique à l’action de PCSK9 sur le LDLR et ce, de manière endogène. Finalement, nous avons identifié l’Annexine A2 comme unique inhibiteur naturel pouvant interférer avec la dégradation du LDLR par PCSK9. Il est indéniable que PCSK9 soit une cible de premier choix pour contrer l’hypercholestérolémie afin de prévenir le développement de MCV. Cet ouvrage apporte donc des apports considérables dans notre compréhension des voies cellulaires impliquées, des cibles affectées et ouvre directement la porte à une approche thérapeutique à fort potentiel.

Action de la matrice extra-cellulaire sur le métabolisme de l'hépatocyte infecté par le virus de l'hépatice C

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Étude de la variation de phase des fimbriae F1651, Pap et CS31A et de l'impact des régulateurs homologues de PapI

Les Escherichia coli pathogènes extra-intestinaux (ExPEC) sont responsables d’une grande variété de maladies. Plus particulièrement, certaines souches ExPEC, du sous-groupe d’E. coli uropathogènes, sont porteuses de fimbriae de type P. Cette famille d’adhésines est soumise à une régulation transcriptionnelle appelée variation de phase; un mécanisme du tout ou rien. Il s’agit d’une compétition entre deux protéines régulatrices : la Dam méthylase et la nucléoprotéine Lrp. Ce mécanisme est aussi soumis à l’influence des régulateurs locaux PapB et PapI, deux régulateurs essentiels. Afin d’étudier PapI et ses homologues ainsi que leur impact sur la variation de phase des fimbriae F1651, Pap et CS31A. Grâce à une fusion chromosomique entre la région régulatrice de clp et les gènes lacZYA, nous avons étudié l’effet, en trans, de PapI et FooI qui ont pu restaurer la variation de phase avec une forte tendance pour la phase OFF. Pour étudier l’action de ces protéines sur foo et pap, nous avons utilisé un système utilisant gfp comme gène rapporteur de l’activité des promoteurs des opérons pap et foo. Cela a permis d’observer la variation de phase au niveau cellulaire par cytométrie en flux et en temps réel par microscopie à fluorescence. Ces expériences ont confirmé que la population de cellules F1651 positives a un phénotype d’expression de F1651 partielle alors que les cellules Pap sont en majorité en phase OFF. PapI et FooI n’ont pas la même influence sur la variation de phase, puisque FooI favorise une plus grande fréquence de variation de phase.

Tournefortia L. (Boraginaceae): espécies do Brasil extra-amazônico

Tournefortia possui cerca de 150 espécies distribuídas nas regiões quentes do mundo, principalmente nos Neotrópicos. Está sendo fornecido o tratamento necessário para identificação das espécies de Tournefortia para o Brasil, exceto região Amazônica, incluindo chaves taxonômicas e descrições. Foram encontradas as duas secções: Tournefortia e Cyphocyema, abrangendo 15 espécies e uma variedade. São apresentados comentários sobre a morfologia dos caracteres utilizados na identificação e distribuição geográfica.

Leishmania (V.) braziliensis and L. (L.) amazonensis promote differential expression of dendritic cells and cellular immune response in murine model

The expression of Langerhans cell (LC) and dermal dendritic cell (dDC) as well as T CD4+ and CD8+ immune responses was evaluated in the skin of BALB/c mice experimentally infected by L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb). At 4th and 8th weeks post infection (PI), skin biopsies were collected to determine the parasite load and CD207+, CD11c+, CD4+, CD8+, iNOS+ cellular densities. Cytokine (IFN-?, IL-4 and IL-10) profiles were also analysed in draining lymph node. At 4th week, the densities of CD207+ and CD11c+ were higher in the La infection, while in the Lb infection, these markers revealed a significant increase at 8th week. At 4th week, CD4+ and CD8+ were higher in the La infection, but at 8th week, there was a substantial increase in both markers in the Lb infection. iNOS+ was higher in the Lb infection at 4th and 8th weeks. In contrast, the parasite load was higher in the La infection at 4th and 8th weeks. The concentration of IFN-? was higher in the Lb infection, but IL-4 and IL-10 were higher in the La infection at 4th and 8th weeks. These results confirm the role of the Leishmania species in the BALB/c mice disease characterized by differences in the expression of dendritic cells and cellular immune response.

Effects of seawater pCO2 and temperature on shell growth, shell stability, condition and cellular stress of Western Baltic Sea Mytilus edulis (L.) and Arctica islandica (L.)

Acidification of the World's oceans may directly impact reproduction, performance and shell formation of marine calcifying organisms. In addition, since shell production is costly and stress in general draws on an organism's energy budget, shell growth and stability of bivalves should indirectly be affected by environmental stress. The aim of this study was to investigate whether a combination of warming and acidification leads to increased physiological stress (lipofuscin accumulation and mortality) and affects the performance [shell growth, shell breaking force, condition index (Ci)] of young Mytilus edulis and Arctica islandica from the Baltic Sea. We cultured the bivalves in a fully-crossed 2-factorial experimental setup (seawater (sw) pCO2 levels "low", "medium" and "high" for both species, temperature levels 7.5, 10, 16, 20 and 25 °C for M. edulis and 7.5, 10 and 16 °C for A. islandica) for 13 weeks in summer. Mytilus edulis and A. islandica appeared to tolerate wide ranges of sw temperature and pCO2. Lipofuscin accumulation of M. edulis increased with temperature while the Ci decreased, but shell growth of the mussels only sharply decreased while its mortality increased between 20 and 25 °C. In A. islandica, lipofuscin accumulation increased with temperature, whereas the Ci, shell growth and shell breaking force decreased. The pCO2 treatment had only marginal effects on the measured parameters of both bivalve species. Shell growth of both bivalve species was not impaired by under-saturation of the sea water with respect to aragonite and calcite. Furthermore, independently of water temperatures shell breaking force of both species and shell growth of A. islandica remained unaffected by the applied elevated sw pCO2 for several months. Only at the highest temperature (25 °C), growth arrest of M. edulis was recorded at the high sw pCO2 treatment and the Ci of M. edulis was slightly higher at the medium sw pCO2 treatment than at the low and high sw pCO2 treatments. The only effect of elevated sw pCO2 on A. islandica was an increase in lipofuscin accumulation at the high sw pCO2 treatment compared to the medium sw pCO2 treatment. Our results show that, despite this robustness, growth of both M. edulis and A. islandica can be reduced if sw temperatures remain high for several weeks in summer. As large body size constitutes an escape from crab and sea star predation, this can make bivalves presumably more vulnerable to predation with possible negative consequences on population growth. In M. edulis, but not in A. islandica, this effect is amplified by elevated sw pCO2. We follow that combined effects of elevated sw pCO2 and ocean warming might cause shifts in future Western Baltic Sea community structures and ecosystem services; however, only if predators or other interacting species do not suffer as strong from these stressors.

Catalogue of the magnificent private library of the late Amor L. Hollingsworth of Milton, Mass. ... Magnificent collection of fine and rare books, many in sumptuous bindings by the finest binders in the world. Rare English and American first editions. Extra illustrated books ...

Plates partly printed on both sides.

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm – with or without anchoring them in the plasma membrane –, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised neither by adding a C-terminal stabilisation signal nor by anchoring the protein at the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

Delivery of nucleic acids for cancer gene therapy: overcoming extra- and intra-cellular barriers

The therapeutic potential of cancer gene therapy has been limited by the difficulty of delivering genetic material to target sites. Various biological and molecular barriers exist which need to be overcome before effective nonviral delivery systems can be applied successfully in oncology. Herein, various barriers are described and strategies to circumvent such obstacles are discussed, considering both the extracellular and intracellular setting. Development of multifunctional delivery systems holds much promise for the progression of gene delivery, and a growing body of evidence supports this approach involving rational design of vectors, with a unique molecular architecture. In addition, the potential application of composite gene delivery platforms is highlighted which may provide an alternative delivery strategy to traditional systemic administration.

Studio di fattibilità ed implementazione della tecnologia MBR per l'impianto di depurazione acque presso Caviro-Extra (Faenza-RA)

Caviro Extra è un’azienda con sede a Faenza, impegnata nella lavorazione dei sottoprodotti derivanti dalla vinificazione e nella produzione di energia sostenibile ottenuta sia dai propri scarti di lavorazione che da rifiuti agroalimentari di aziende terze. Lo stabilimento svolge autonomamente il trattamento dei reflui abbattendo le sostanze inquinanti e permettendo così lo scarico in pubblica fognatura. Lo scopo della tesi è quello di studiare la fattibilità, tramite lo studio bibliografico, dell’implementazione di un bioreattore aerobico a membrana (MBR) come soluzione impiantistica atta a fronteggiare l’alto carico idraulico ed ammoniacale gestito durante il periodo della campagna di vinificazione. In un’ottica di sostenibilità ambientale si è presentato un secondo scopo , cioè quello di andare a recuperare le acque reflue tramite osmosi inversa. Dallo studio di letteratura scientifica riguardo la capacità di nitrificazione del bioreattore a membrana si hanno risultati favorevoli. Indicando tale sistema come una soluzione efficiente e stabile che può essere applicata nella nitrificazione di acque reflue contenenti un’alta concentrazione di azoto ammoniacale. L’integrazione di un sistema MBR con un’osmosi inversa ha, altresì, dimostrato miglioramenti ulteriori della qualità dell'acqua trattata; dimostrando di fornire un’alta reiezione di nitrato come di cloruri e di solfati ad alta concentrazione iniziale come nel caso in esame e di poter essere la soluzione impiantistica per alleggerire il carico ammoniacale al settore biologico e allo stesso tempo recuperare acqua riducendo la necessità di emungimento da pozzi artesiani. L’azienda verificherà mediante l’attività di testing la reale fattibilità in termini di potenziale miglioramento di depurazione, costi operativi e semplicità di gestione attraverso l’implementazione di impianti pilota sia sulla tecnologia analizzata (MBR+RO) sia su un processo MBBR di tipo ibrido fango attivo/biomassa adesa.

Rilevazione dell'adulterazione di olio extra vergine di oliva con olio di girasole mediante l'impiego di sensori spettrali

Nel corso degli ultimi anni si è assistito ad un aumento di episodi fraudolenti nel settore alimentare. Ciò è vero anche per il settore oleario: per il suo elevato valore economico, nutrizionale e sensoriale, l’olio extra vergine di oliva è un bersaglio perfetto dei frodatori, che realizzano molteplici tipologie di adulterazione. Questo lavoro di tesi si è focalizzato, in particolare, sull’adulterazione dell’olio extra vergine di oliva con olio di semi di girasole. Su campioni preparati in laboratorio per simulare la frode, a partire da due oli extra vergini di oliva addizionati con percentuali diverse di olio di girasole, sono state effettuate determinazioni analitiche utilizzando un sensore spettrale, basato sulla spettroscopia nel vicino infrarosso, seguite da un’elaborazione chemiometrica dei risultati. Lo strumento portatile è stato sviluppato da una start-up olandese presso cui ho svolto il tirocinio curriculare. Il modello di stima risultante dalle risposte fornite dai 16 pixel del sensore ha mostrato una capacità di rilevazione soddisfacente nei confronti di questa adulterazione, conducendo ad un errore medio nella stima della percentuale di olio di girasole aggiunto intorno al 3%. Tale risultato, se verrà confermato da ulteriori analisi grazie alle quali il modello predittivo potrà essere reso più robusto, potrebbe incoraggiare l’utilizzo di questa metodologia analitica come tecnica rapida di screening, facilmente applicabile anche in un contesto industriale, a supporto delle più laboriose e complesse tecniche analitiche tradizionali, nella rilevazione di adulterazioni di oli extra vergini di oliva con oli di semi di girasole.

Partorire in ambiente extra ospedaliero. Un'alternativa poco conosciuta, caratterizzata da grandi vantaggi. L'importanza per le donne di ricevere informazioni adeguate da parte dei servizi territoriali

Dalla letteratura si evince come, in una gravidanza fisiologica, il parto extra ospedaliero sia un’alternativa sicura, al pari dell’assistenza ospedaliera. In Italia avviene in una bassa percentuale di casi, nonostante sul territorio siano presenti strutture per poter attuare questa scelta. Un piccolo aumento è avvenuto negli ultimi due anni probabilmente dovuto anche alla pandemia da Covid. In particolare in Emilia Romagna, sono state indagate le informazioni e le modalità che i servizi territoriali e ospedalieri offrono alle gestanti in merito ai luoghi del parto. Un secondo obiettivo è stato capire, tramite le testimonianze delle donne, come avessero reperito le informazioni e a chi si fossero rivolte per la pianificazione del parto. Si è posta l’attenzione sull’opinione di Ostetric* del SSN circa il parto extra ospedaliero. Sono state effettuate 8 interviste semi-strutturate, e raccolti 27 questionari tra Ostetriche operanti nei Consultori e nei punti nascita ospedalieri. È stata raccolta, inoltre, l’opinione di 62 donne che hanno partorito in ambiente extra ospedaliero. I dati sono stati utilizzati in forma anonima. Dallo studio emerge la necessità di ampliare gli spazi e i tempi dedicati alla scelta del luogo del parto al fine di offrire alle coppie ogni alternativa possibile; le Ostetriche e le donne coinvolte suggeriscono diversi metodi per facilitare la conoscenza e il contatto con la realtà extra ospedaliera: da una più ampia diffusione di informazioni da parte delle strutture del SSN a momenti di formazione condivisa con chi opera a domicilio o in casa di Maternità. Le donne sottolineano la difficoltà di reperire informazioni dettagliate e di come la scelta sia spesso influenzata da pregiudizi. I consultori dovrebbero fornire informazioni dettagliate su tutti i luoghi del parto a tutte le donne per permettere una scelta libera da opinioni soggettive. Dovrebbero essere presi accordi specifici tra chi pratica l’assistenza in ospedale e chi in altri ambiti.

Transmission Of Intra-cellular Genetic Information: A System Proposal.

One of the great challenges of the scientific community on theories of genetic information, genetic communication and genetic coding is to determine a mathematical structure related to DNA sequences. In this paper we propose a model of an intra-cellular transmission system of genetic information similar to a model of a power and bandwidth efficient digital communication system in order to identify a mathematical structure in DNA sequences where such sequences are biologically relevant. The model of a transmission system of genetic information is concerned with the identification, reproduction and mathematical classification of the nucleotide sequence of single stranded DNA by the genetic encoder. Hence, a genetic encoder is devised where labelings and cyclic codes are established. The establishment of the algebraic structure of the corresponding codes alphabets, mappings, labelings, primitive polynomials (p(x)) and code generator polynomials (g(x)) are quite important in characterizing error-correcting codes subclasses of G-linear codes. These latter codes are useful for the identification, reproduction and mathematical classification of DNA sequences. The characterization of this model may contribute to the development of a methodology that can be applied in mutational analysis and polymorphisms, production of new drugs and genetic improvement, among other things, resulting in the reduction of time and laboratory costs.

Clozapine Promotes Glycolysis And Myelin Lipid Synthesis In Cultured Oligodendrocytes.

Clozapine displays stronger systemic metabolic side effects than haloperidol and it has been hypothesized that therapeutic antipsychotic and adverse metabolic effects of these drugs are related. Considering that cerebral disconnectivity through oligodendrocyte dysfunction has been implicated in schizophrenia, it is important to determine the effect of these drugs on oligodendrocyte energy metabolism and myelin lipid production. Effects of clozapine and haloperidol on glucose and myelin lipid metabolism were evaluated and compared in cultured OLN-93 oligodendrocytes. First, glycolytic activity was assessed by measurement of extra- and intracellular glucose and lactate levels. Next, the expression of glucose (GLUT) and monocarboxylate (MCT) transporters was determined after 6 and 24 h. And finally mitochondrial respiration, acetyl-CoA carboxylase, free fatty acids, and expression of the myelin lipid galactocerebroside were analyzed. Both drugs altered oligodendrocyte glucose metabolism, but in opposite directions. Clozapine improved the glucose uptake, production and release of lactate, without altering GLUT and MCT. In contrast, haloperidol led to higher extracellular levels of glucose and lower levels of lactate, suggesting reduced glycolysis. Antipsychotics did not alter significantly the number of functionally intact mitochondria, but clozapine enhanced the efficacy of oxidative phosphorylation and expression of galactocerebroside. Our findings support the superior impact of clozapine on white matter integrity in schizophrenia as previously observed, suggesting that this drug improves the energy supply and myelin lipid synthesis in oligodendrocytes. Characterizing the underlying signal transduction pathways may pave the way for novel oligodendrocyte-directed schizophrenia therapies.