Les protéines solubles sont généralement faiblement immunogènes, ce qui constitue unelimite pour le développement de vaccins sous unitaires à base de protéines. Mes travaux de thèseont eu pour objectif de décrypter certains mécanismes moléculaires et cellulaires qui contribuent àl'immunogénicité et d'en tirer partie pour développer des approches originales permettantd'améliorer la capacité des protéines à déclencher la réponse immunitaire. Pour cela, j'aiprincipalement utilisé le transactivateur transcriptionnel (Tat) du VIH-1. J'ai montré quel'oligomérisation de Tat permet à un mécanisme de collaboration B-TH-2 d'induire la réponseimmunitaire en absence d'adjuvant. J'ai identifié le déterminant minimal responsable de l'effet etmontré qu'il confère la propriété adjuvante à d'autres antigènes. J'ai ensuite montré que laprésentation aux cellules T restreinte aux CMH I et CMH II est accrue lorsque les protéines sontdotées de la capacité à lier des sucres sulfatés d'expression ubiquitaire: les héparanes sulfate. Cestravaux ont permis de définir de nouvelles approches pour améliorer l'immunogénicité de protéinessusceptibles d'être intégrées dans des préparations vaccinales.

The mechanisms underlying cannabinoid impairment of fear memory is not clear. This study investigated the effects of the synthetic cannabinoid HU210 and the endocannabinoid hydrolysis inhibitor JZL 195 on fear memory following contextual fear conditioning (CFC; an animal model of fear). The long-term depression (LTD)-blocking peptide Tat-GluR2 was utilized to investigate whether the expression of cannabinoid-induced LTD (CB-LTD) is required for the cannabinoid impairment of acquisition and consolidation of contextual fear memory. HU210 reduced freezing throughout the test phase of the acquisition protocol, which was not affected by pre-administration of Tat-GluR2. High and moderate doses of HU210 reduced freezing during the first and last half, respectively, of the test phase of the consolidation protocol, which was prevented by pre-treatment with Tat-GluR2. HU210 did not affect freezing during the test phase of the retrieval protocol. Thus, these results suggest that HU210 impairs acquisition and consolidation, but not retrieval of contextual fear memory, and that in vivo CB-LTD expression is required for HU210 impairment of the consolidation, but not acquisition, of contextual fear memory. We also observed that HU210 and JZL 195 do not facilitate the acquisition of contextual fear memory extinction.

HIV-Associated Neurocognitive Disorders (HANDs) are becoming one of the largest problems in patients infected with HIV-1. The ability of infected cells such as monocytes and microglial cells to act as viral reservoirs causes extreme inflammation in the CNS and has led to several different types of neurocognitive problems. Specifically these HIV-1 infected monocytes are able to secrete inflammatory factors such as the regulatory protein Tat which acts as a chemoattractant for monocytes while also promoting the adhesion of leukocytes to the extracellular matrix (ECM). We have shown that one of the major features of the Tat protein is that it promotes cytoskeletal rearrangement resulting in increased adhesion. Specifically integrin and actin visualization was performed using confocal immunofluorescence while cytoskeletal morphology was shown with light and SEM. This microscopy work showed the Tat protein resulted in altered β1-integrin expression and distribution as well as changes in polymerized actin. These cytoskeletal changes resulted in increased adhesion to the ECM. Similarly we have also shown that these cytoskeletal changes of β1-integrin distribution and polymerized actin can be modulated through select cannabinoids THC and CP55940 that bind through the CB2 receptor which inhibits this adhesion as well as the morphological changes. The modulation of this reorganization is characteristic of a signal transduction pathway where a novel convergent point between extracellular Tat and the select cannabinoids THC and CP55940 exists. The aim of this project was to show the cytoskeletal reorganization using different microscopy techniques including light and scanning electron microscopy. This was followed by identifying and characterizing the convergent point along the signal transduction pathway linked to these changes. Different techniques were utilized in order to identify the putative convergent point in the signal transduction cascade including antibody arrays, RT-PCR, and western immunoblotting. The cytoskeletal rearrangements of β1-integrin and actin polymerization were shown successfully via light and scanning electron microscopy in the context of treatment with Tat in the presence and absence of select cannabinoids THC and CP55940. Several different pathways were identified as possibly linked to cannabinoid-mediated inhibition of signal transductional activation consequent of attachment to extracellular matrix proteins. However, the exact molecules implicated in specific signal transductional pathways as targets of cannabinoid-mediated action remain to be defined.

During development, oligodendrocytes (OLGs), the myelinating cells of the central nervous system (CNS), undergo a stepwise progression during which OLG progenitors, specified from neural stem/progenitor cells, differentiate into fully mature myelinating OLGs. This progression along the OLG lineage is characterized by well-synchronized changes in morphology and gene expression patterns. The studies presented in this dissertation identified the extracellular factor Autotaxin (ATX) as a novel upstream signal modulating HDAC1/2 activity and gene expression in cells of the OLG lineage. Using the zebrafish as an in vivo model system, as well as rodent primary OLG cultures, this functional property of ATX was found to be mediated by its lysoPLD activity, which has been well-characterized to generate the lipid signaling molecule lysophosphatidic acid (LPA). LPA binds to Gprotein-coupled LPA receptors (LPARs) on the surface of OLGs to initiate downstream signaling events. ATX’s lysoPLD activity was found to modulate HDAC1/2 regulated gene expression during a time window coinciding with the transition from OLG progenitor to early differentiating OLG. When looking further downstream of the ATX-LPA axis, down-regulation of LPA receptor 6 (LPA6) was found to reduce the expression of OLG differentiation genes as well as the overall process network area of OLGs. Thus, LPA6 plays a role in both the gene expression and morphology changes seen in OLG differentiation. These findings prove useful for future therapeutic targets needed for demyelinating diseases of the CNS such as Multiple Sclerosis (MS), in which OLGs fail to differentiate into mature OLGs, needed for remyelination. Additionally, white matter injury has been frequently reported in HIV+ patients. Previous studies showed that HIV-1 Tat (transactivator of transcription), a viral protein that is produced and secreted by HIV-infected cells, is a toxic factor to OLGs. We show here that Tat treatment reduces the expression of OLG differentiation genes and the overall process network area of OLGs. Additionally, Tat-treated OLGs have reduced ATX lysoPLD activity and there is a physical interaction between Tat and ATX. Together, these data strongly suggest functional implications of Tat blocking ATX’s lysoPLD activities and thus the ATX-LPA signaling axis proves to play a significant role in the development of OLGs.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

SLAVÍČKOVÁ, T. Tématicko apercepční test u uživatelů drog. Pedagogická fakulta Univerzity Karlovy v Praze, 2012. 140 s. Diplomová práce. Diplomová práce měla za cíl odhalit specifika použití TAT u problémových a závislých uživatelů drog a na základě srovnání s výsledky, kterých v testu dosáhla kontrolní skupina, přinést evidenci pro zhodnocení diagnostického potenciálu TAT u uživatelů drog. Výzkumné šetření bylo provedeno v Kolíně na klientech nízkoprahového kontaktního centra, které poskytuje služby harm reduction skryté populaci uživatelů nealkoholových drog. Těžiště výzkumu bezprostředně souvisí s metodou analýzy příběhů, jejichž vyprávění bylo u probandů vyvoláno předložením tabulí TAT. K analýze příběhů byl použit psychometricky zakotvený skórovací systém SCORS (Social Cognition - Object Relation Scale) současného amerického autora D. Westena. Použití TAT u uživatelů drog tak bylo zaměřeno na zhodnocení úrovně jejich fungování v sociálních vztazích. Metodologický princip diplomové práce tkví v induktivním usuzování a statistickém zpracování dat. Způsob zpracování dat, který je až na několik oblastí výhradně v režii kvantitativních metod, spolu s dostatečně velkým souborem zkoumaných osob umožňuje zobecnění důležitých rozdílů ve výsledcích TAT u výzkumné a kontrolní skupiny. V rámci analýzy...

ANGLICKÝ ABSTRAKT Thesis Title: The SCORS Scale - An Assessment of Processing of Social Information and Object Relations of Men with Pathological Sexual Aggression Author: Mgr. Bc. Lucie Táborská Department: Department of Psychology Thesis Advisor: PhDr. Tereza Soukupová, Ph.D. Email of Thesis Advisor: tereza.soukupova@pedf.cuni.cz This empirical Thesis Essay titled The SCORS Scale - An Assessment of Processing of Social Information and Object Relations of Men with Pathological Sexual Aggression focuses on study of social skills of men with the diagnosis mentioned. The theoretical portion of the Thesis centers around the subject of sexual deviation as such, namely around pathological sexual aggression and projective methods; in particular, the TAT method and its specific SCORS scoring system. The fundamental research question was whether or not the answers of sample respondents would fall into the sphere of the pathological. Furthermore, the Thesis explores whether the differences between individual segments of the scale are significant or, on the contrary, corresponding. Subsequent examination of the phenomena follows. There were 20 participants in the survey, the individuals belonging to three different psychiatric asylums. D. Westen's SCORS was the method chosen for acquisition of data. The research has...

The objective of this Ph.D. study was to develop new and improved miniaturized particle-based optochemical sensors for the analysis of biological fluid and cellular components. This is highly important because current sensing systems can be biologically toxic and incompatible, invasive, and have limited responsiveness. To accomplish this goal we defined three tasks. The first was to develop lipobead-based sensors for chloride. The halide-specific fluorescence dye, lucigenin, was immobilized into the phospholipid membrane of the lipobeads to enable chloride ion detection. The fluorescence intensity of lucigenin decreases with increasing chloride ion concentration due to dynamic quenching. To stabilize the lipobeads we co-immobilized hexadecanesulfonate molecules into the phospholipid membrane. We also immobilized the chloride ionophore [9] mercuracarborand-3 (MC-3) into the lipobeads membrane. The study resulted in a unique submicrometric chloride ion sensor, which is suitable for chloride ion measurements in biological fluids. The second task was to develop for the first time lipobeadbased biosensors. Urea was chosen as a model substance since the urea/urease biosensing system is well known. Fluorescence sensing lipobeads were characterized by coating carboxylfunctionalized silica microspheres with phospholipids for the measurement of urea in aqueous samples. The enzyme urease and the pH indicator Fluorescein-5-thiosemicarbazide were attached covalently to the phospholipid membrane of the lipobeads. We prepared improved fluorescence sensing lipobeads by utilizing covalent chemistry to bind the phospholipid membrane to the silica particles and the fluorophores to the membrane. It led to improvement in the stability of the newly developed urea sensing lipobeads compared to previously developed micrometric fluorescence sensors. The final task of this study was to coat particle-based sensors with cell penetrating peptides to enable their permeation into cells. This step is essential for the use of particles as intracellular sensors. Streptavidin coated microspheres were modified by the strongest noncovalent interaction between avidin and biotin. Tat peptide and nonfluorescence indicator flubida were attached to the surface of the microspheres. These nanoparticles were delivered into MCF7 and Hela cancer cells for pH measurement. Before penetrating into the cells, flubida did fluoresce in cell medium; however it did not convert to fluorecein in Phosphate Buffered Saline (PBS) buffer.

The Tat protein export system transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. In Escherichia coli, the Tat system is composed of the TatA, TatB and TatC proteins. TatB and TatC assemble into a multimeric receptor complex that recognises and binds the substrate, before the TatA protomers cluster at the TatBC complex to facilitate substrate transport. A genetic screen was devised to explore the oligomeric state of TatC, reasoning that the isolation of dominant negative TatC variants that inactivate the Tat system in the presence of a functional copy of wild type TatC would provide strong evidence TatC is an obligate oligomer. Single dominant negative TatC substitutions were isolated that were located in the first and second periplasmic loops of TatC. These substitutions did not prevent TatC from interacting with TatB, TatA, itself or with a Tat substrate. Blue Native PAGE analysis showed that the TatC variants were unable to form the 440 kDa TatBC complex. Surprisingly, the substitutions did not prevent TatC:TatC self-interactions in the periplasmic regions, detected by disulphide cross-linking, but they did abolish a substrate-induced interaction at the fifth transmembrane helix of TatC. Fluorescence microscopy experiments revealed that the dominant negative TatC variants prevented the polymerisation of TatA-YFP in vivo. These results show that TatC possesses at least two interaction interfaces and imply that the periplasmic loops are critical for the transition between substrate binding and TatA polymerisation. Accessibility of single cysteine substitutions in TatC was probed by PEG-Mal labelling in intact cells. TatB was shown to be important for the proper insertion of TatC into the membrane. The absence of TatA led to accessibility changes in the vicinity of the fifth transmembrane domain of TatC, where both TatA and TatB are known to dock. This suggests that TatA and TatB may share an overlapping binding site.

Human immunodeficiency (HIV) infection can result in neurocognitive deficits in about one-half of infected individuals. Despite excellent systemic effectiveness, restricted antiretroviral penetration across the blood-brain barrier (BBB) is a major limitation in fighting HIV infection within the central nervous system (CNS). Drug abuse exacerbates cognitive impairment and pathologic CNS changes in HIV-infected individuals. This work investigates the effects of the HIV-1 protein, Tat, and drugs of abuse on factors affecting drug penetration into the brain. Firstly, an in vitro model of the blood-brain barrier was built to study effects of HIV-1 Tat and methamphetamine (Meth) on integrity and function of the BBB, in turn how HIV-1 Tat and meth will affect antiretroviral penetration into the brain. We found that co-exposure HIV-1 Tat and Meth results in inhibition or impairment of P-glycoprotein activity at the BBB. Also, simultaneous inhibition of P-glycoprotein (P-gp) and Multidrug Resistant Protein -1 (MRP-1), by verapamil and MK-571 causes an increase in accumulation of atazanavir inside the primary human brain endothelial cells. Secondly, we developed and validated the method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell extracts of CNS cells. This method was used to study how HIV-1 Tat and/or morphine affects antiretroviral penetration in CNS cells like human brain microvascular endothelial cells, human astrocytes, human microglia, and human pericytes. We found that in untreated cells, accumulation of antiretroviral drugs was higher in hCMEC/D3 cells compared to other CNS cell types. Also, HIV-1 Tat and/or morphine had no significant effect on antiretroviral penetration amongst these cell types. Overall, the rank order of intracellular accumulation observed in treated and untreated cells was dolutegravir > emtricitabine > tenofovir.