Manfouo Foutsop Germain (2010). Etude de lexpression des gènes cellulaires dérégulés dans des cellules T CD4+ exprimant Tat de HIV-1 : Focalisation sur la voie de signalisation TCR/CD3 (thèse de doctorat). Université de Liège-Gembloux Agro-Bio Tech, 157 pages, 11 tables, 29 figures. Résumé Nos travaux précédents ont suggéré que les gènes nef (negative factor) et tat (transactivateur de la transcription) du virus de limmunodéficience humaine de type 1 (HIV-1) pourraient induire la perte du récepteur des cellules T (TCR)/CD3 à la surface des lymphocytes T CD4+ infectés par le HIV-1, mais la contribution de tat dans cette perte navait pas été prouvée. Dans la première partie de ce travail nous avons utilisé un vecteur lentiviral exprimant les formes natives [1exon (72aa) ou 2exons (101aa)] ou des formes mutées du gène tat de HIV-1 pour transduire les cellules T CD4+. Ceci nous a permis de montrer que la production de la protéine Tat72aa ou Tat101aa entraîne une perte progressive du complexe TCR/CD3 en surface des cellules T CD4+. Nous avons constaté que cette perte de TCR/CD3 serait due à une diminution du taux des transcrits de CD3γ. Par ailleurs, nous avons montré que certains mutants du gène tat ont perdu leur activité transactivatrice et sont incapables dinduire la perte de TCR/CD3. Dans la deuxième partie des travaux, nous avons identifié par microarray, les gènes cellulaires dont les expressions sont communément altérées dans les cellules T CD4+ infectées par le HIV-1 ou transduites avec le gène tat-1exon ou tat-2exons. Plusieurs de ces gènes sont impliqués dans la voie de signalisation TCR/CD3. Nos travaux nous ont donc permis de montrer limplication du gène tat dans la dérégulation de la voie de signalisation TCR/CD3. Par conséquent, Tat pourrait induire une altération de la réponse immunitaire favorisant ainsi la progression de linfection par le HIV-1/SIDA. Manfouo Foutsop Germain (2010). A study of altered cellular gene expression in CD4+ T cells expressing HIV-1 Tat: Focus on the T cell receptor/CD3-directed pathway (PhD thesis). Université de Liège-Gembloux Agro-Bio Tech, 157 pages, 11 tables, 29 figures. Summary Our previous work has suggested that nef (negative factor) and tat (transactivator of transcription) are responsible for T cell receptor (TCR)/CD3 downmodulation on CD4+ T cells after infection with human immunodeficiency virus type 1 (HIV-1). However, the contribution of tat to this phenomenon has not been investigated. In the first part of this study we used lentiviral vector expressing the native tat gene [1exon (72aa) or two exon (101aa)] and various mutants to transduce CD4+ T cells. We found that Tat expression alone could provoke the progressive downmodulation of surface TCR/CD3 complexes due to the decrease of CD3γ transcripts. We also demonstrated that the majority of mutants lost their transactivation capability and their ability to downregulate the TCR/CD3 complex. In the second part of this work, we used gene expression arrays to identify cellular genes whose expression was altered in CD4+ T cells infected with HIV-1 or transduced with tat at different stages of receptor downregulation. We found progressive alteration of cellular gene expression in parallel with TCR/CD3 downregulation in the tat-transduced cells, which represented a subset of the changes detected in the HIV-1 infected cells, with significant number genes known to play a role in the TCR/CD3 signaling pathway. Our data demonstrate that tat deregulates the TCR/CD3 expression and signaling leading to altered immune function that likely favor HIV-1/AIDS disease progression. Copyright: Aux termes de la loi belge du 30 juin 1994, sur le droit dauteur et les droits voisins, seul lauteur a le droit de reproduire partiellement ou complètement cet ouvrage de quelque façon que ce soit ou dautoriser la reproduction partielle ou complète de quelque manière et sous quelque forme que ce soit. Toute photocopie ou reproduction sous autre forme est donc faite en violation de ladite loi et des modifications ultérieures.

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.

This thesis presents a detailed investigation of the performance limiting factors of long wavelength infrared (LWIR) and very long wavelength infrared (VLWIR) p on n HgCdTe detectors through numerical simulations at 77 K incorporating all considerable generation-recombination mechanisms including trap assisted tunneling (TAT), Shockley-Read-Hall (SRH), Auger and radiative processes. Numerical simulations under dark and illuminated conditions were performed with different absorber layer thicknesses, material compositions (cut-off wavelengths), trap density, and trap energy level. The results identify the relative strength of the dark current generation mechanisms by numerically extracting the contribution of each G-R mechanism on the detector characteristics with various cut off wavelengths and practically achievable material parameters. While the provided information can be used as a guide for optimizing the device processing conditions and detector structure, it also enlights the importance of various intrinsic mechanisms on the detector sensitivity. The results show that the dominant sensitivity degrading trap level depends on the detector cut-off wavelength being about 0.7Eg for LWIR HgCdTe sensors (cut-off wavelength=10 &micro / m) instead of 0.5Eg which is generally believed to be the most efficient R-G level. TAT related 1/f noise dominates the sensor noise even under small reverse bias voltages at a trap density as low as 1E14 cm-3 for sensors with cut-off wavelength &gt / 11 &micro / m. Considering the fact that trap densities below this level are rarely reported for HgCdTe material, exceptionally trap-free material is required to achieve desirable imaging performance with these sensors. Simulation results show that Auger mechanism has twofold effect on the sensitivity of the sensor by increasing the dark current and decreasing the photo current of the detector. As to our knowledge, this work is one of the most comprehensive simulation based investigations of the HgCdTe detector performance providing important results that can be used as a guide for optimization of the detector performance in order to meet the demanding requirements of the third generation thermal imagers.

Major advances in highly active antiretroviral therapies (ARVs) have extended the lives of people living with HIV, but there still remains an increased risk of death by cardiovascular diseases (CVD). HIV proteins and ARVs have been shown to contribute to cardiovascular dysfunction with effects on the different cell types that comprise the arterial wall. In particular, HIV-1 transactivating factor, Tat, is a cationic polypeptide that binds to endothelial cells, inducing a range of responses that have been shown to contribute to vascular dysfunction. It is well established that hemodynamics also play an important role in endothelial cell mediated atherosclerotic development where upon exposure to low or oscillatory shear stress, such as that found at branches and bifurcations, endothelial cells contribute to proteolytic vascular remodeling, by upregulating cathepsins, potent elastases and collagenases. The results of this work demonstrate that upregulation of cathepsins in vivo and in vitro is caused by a synergism between pro-atherogenic shear stress and HIV-1 proteins, elucidates pathways that are activated by HIV-1 Tat and pro-atherogenic shear stress - leading to cathepsin-mediated ECM degradation, and identifies cathepsins as novel biomarkers to monitor the adherence of patients on efavirenz- and tenofovir-containing antiretroviral regimens.

Prevalence of HIV-associated cognitive impairment is rising, the worst form of which is HIV-associated dementia (HAD). The disease is fuiled by a chronic innate type pro-inflammatory response in the brain which is highly dependent upon the activation of microglia. We first created an in vitro model of HAD composed of cultured microglial cells synergistically activated by the addition of IFN-gamma and the HIV-1 coat glycoprotein, gp120. This activation, as measured by TNF-alpha and NO release, is synergistically attenuated through the alpha7nAChR and p44/42 MAPK system by pretreatment with nicotine, and the cholinesterase inhibitor, galantamine. As these medications have been FDA approved, and over time, have shown only minor improvement in neurodegenerative disease for a limited period, we next sought to explore natural compounds that may attenuate HAD mediated inflammation and related pathology. This inflammation is a key moderator of A-#914; plaque deposition in the brain. Indeed it is likely a contributing factor as epidemiological data suggests significant numbers of HIV survivors are at elevated risk of developing Alzheimer's disease (AD). HIV-1 Tat-induced A-beta deposition, tau phosphorylation, and subsequent neuronal death could be risk factors for subsequent AD and/or HAD. Recent reports suggest green tea-derived (-)-epigallocatechin-3-gallate (EGCG) can attenuate neuronal damage mediated by conditions such as brain ischemia. In order to investigate the therapeutic potential of EGCG to mitigate the neuronal damage characteristic of HAD, IFN- gamma was evaluated for its ability to enhance well-known neurotoxic properties of HIV-1 proteins gp120 and Tat in primary neurons and mice. Indeed, IFN-gamma enhanced the neurotoxicity of gp120 and Tat via increased JAK/STAT signaling. Additionally, primary neurons pretreated with a JAK1 inhibitor, or those from STAT1-deficient mice, were largely resistant to the IFN- gamma-enhanced neurotoxicity of gp120 and Tat. Moreover, EGCG treatment of primary neurons from normal mice reduced IFN-gamma-enhanced neurotoxicity of gp120 and Tat by inhibiting JAK/STAT1 pathway activation. EGCG was also found to mitigate the neurotoxic properties of HIV-1 proteins in the presence of IFN-#947; in vivo. To explore the mechanism by which HIV may augment AD-like pathology, we found HIV-1 Tat protein inhibits microglial uptake of A-beta-1-42 peptide, a process enhanced by IFN-#947; and rescued by EGCG. To mimic the HAD clinical condition, we generated mice with HIV-1 Tat-induced AD-like pathology by cross-breeding HIV-1 Tat expressing mice (expressed under control of GFAP, Doxycline inducible promoter) with the PSAPP mouse model of AD. To simulate chronic Tat secretion over we used an optimized dose of 54 mg/kg/day on a biweekly basis over three months Tat significantly induced neuron degeneration and tau phosphorylation in Tat transgenic mice, dox dependently (P<0.001). Similar effects at the chronic 54 mg/kg/day dose were observed in PSAPP/Tat mice induced with dox. These mice also showed significantly more A-beta deposition (P < 0.05), neurodegeneration, neuronal apoptotic signaling, and phospho-tau than PSAPP mice (P < 0.05). In conclusion, HIV-1 Tat significantly promotes AD-like pathology in PSAPP/Tat mice. This model may provide a framework in which to identify new mechanisms involved in cognitive impairment in the HIV infected population, and possible treatments. Additional works will be needed to fully characterize the mechanism(s) of HIV- induced amyloid deposition, and to uncover viral mechanisms promoting AD-like pathology in general.

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows selective and efficient killing of neuroblastoma cell lines in vitro, and delays human neuroblastoma xenograft tumor growth on nude mice. To further enhance the viral oncolytic efficacy, we also switched the fiber 5 to fiber 35 to generate Ad5PTDf35. This vector shows dramatically increased transduction capacity of primary human cell cultures including hematopoietic cells and their derivatives, pancreatic islets and exocrine cells, mesenchymal stem cells and primary tumor cells including primary cancer initiating cells. Ad5PTDf35-based adenovirus could be a useful platform for gene delivery and oncolytic virus development. Viral oncolysis alone cannot completely eradicate tumors. Therefore, we further armed the Ad5PTDf35-D24 virus with a secreted form of Helicobacter pylori Neutrophil Activating Protein (HP-NAP). Expression of HP-NAP recruits neutrophils to the site of infection, activates an innate immune response against tumor cells and provokes a Th1-type adaptive immune response. Established tumor on nude mice could be completely eradicated in some cases after treatment with this virus and the survival of mice was significantly prolonged.

Le système Tat (pour Twin Arginine Translocation) exporte des protéines repliées depuis le cytoplasme vers le périplasme des bactéries. L'adressage des protéines à exporter au système Tat repose sur une séquence signal spécifique amino terminale clivée après exportation. Chez le phytopathogène Dickeya dadantii, l'homologue de pectine lyase PnlH possède une séquence signal Tat qui assure son adressage au système Tat mais qui n'est pas clivée après exportation et ancre la protéine dans la membrane externe. Chez les protéobactéries, la majorité des protéines de membrane externe sont soit des lipoprotéines soit des protéines intégrales de membrane en tonneau β. L'adressage de ces protéines à la membrane externe repose sur des voies spécifiques du type de protéine : la voie Lol pour les lipoprotéines et la combinaison des chaperons périplasmiques SurA, Skp et DegP et du complexe de membrane externe Bam (β barrel assembly machinery) pour les protéines en tonneau β. Au cours de ce travail, l'étude de l'adressage de PnlH à la membrane externe a montré que SurA se liait à la séquence signal hydrophobe de PnlH pour la protéger de l'environnement hydrophile au cours de son transit dans le périplasme. La séquence signal de PnlH (41 acides aminés) porte l'intégralité de l'information nécessaire à son adressage à la membrane externe. La nature de l'information adressant les protéines au système Tat est bien connue et dans ce travail nous nous sommes efforcés d'identifier les informations requises pour les deux dernières étapes de l'adressage de PnlH à la membrane externe : la traversée du périplasme et l'insertion dans la membrane externe. La délétion d'une région conservée comprise entre les résidus 28 et 41 de la séquence signal de PnlH affecte l'adressage de cette dernière à la membrane externe. Des substitutions des acides aminés conservés de cette région ne semblent pas affecter l'adressage de PnlH, indiquant que l'information nécessaire à l'adressage de PnlH à la membrane externe après exportation ne réside pas dans la séquence en acides aminés de la séquence signal de PnlH. En revanche, nos données suggèrent que la présence d'une hélice α hydrophobe dans la séquence signal de PnlH est importante pour son adressage à la membrane externe. Cette observation est particulièrement intéressante puisqu'une telle structure est généralement considérée comme une caractéristique des protéines de membrane interne.

Functionalized polymeric membrane based techniques are becoming increasingly popular in biotechnology, food and pharmaceutical industries due to their versatility and hydrodynamic benefits over traditional materials and methods. This research work has been directed towards the development of functionalized polymeric membranes, extensive experimental and theoretical analyses of some of the fundamental aspects of accessibility, membrane fouling and enzyme catalysis, and applications in affinity based bioseparation and biocatalysis. In this research work, the impact of different types of functionalization techniques, such as functionalization of different membrane materials, covalent and electrostatic immobilization, on interaction of various biomolecules and active sites in membrane has been studied in detail. Avidin was used as model biomolecule, and covalently immobilized within acyl anhydride derivatized nylon based membrane. Quantification of the accessibility of covalently immobilized avidin sites was carried out by model biotinylated probe molecules, such as biotin 4-amidobenzoic acid and biotinylated-BSA. This study has been further extended to separate and purify a target protein, HIV-Tat, from a complex mixture of proteins (97-99 % unwanted protein) using avidin-biotin affinity interaction. It has been demonstrated that covalent immobilization of avidin in membranes reduces the accessibility of active sites for probe molecules. Accessibility decreases further for the biotinylated target protein present in the mixture of other unwanted proteins. Affinity based membrane separation of proteins is also associated with decrease in permeate flux due to fouling in membrane structure. Fouling in the membrane has been discussed by analyzing the characteristics of adsorbed protein layer in membrane. In order to improve the accessibility and fouling behavior of affinity separation of Tat protein, a pre-filtration step has been introduced prior to affinity separation. Significant enhancement in accessibility and reduction in fouling has been observed for pre-filtered cases as it removes unwanted proteins prior to affinity interaction. Contribution of the pre-filtration step in reduction of fouling has been elucidated by simple model equations. Improvement in accessibility and fouling behavior reflects in higher separation efficiency (protein recovery) and lower processing time for the pre-filtered cases. Quality of membrane purified Tat protein was examined by different analytical techniques, such as SDS-PAGE, Western Blot and biotin analysis, and then compared with that purified by traditional packed-bead column chromatography. It has been demonstrated that membrane based technique was able to isolate superior quality of pure monomeric Tat protein compare to column chromatographic technique. The other study carried out as a part of this dissertation, has involved development of high capacity, highly active, stable and reusable functionalized membrane domains for electrostatic immobilization of enzymes. Glucose oxidase (GOX) was used as a model enzyme to study the oxidation of glucose to gluconic acid and hydrogen peroxide under convective flow condition. Two different approaches of functionalization of membranes have been presented. In the first approach, alternative electrostatic attachment of cationic and anionic polyelectrolytes was carried out using Layer-By-Layer (LBL) assembly technique within a functionalized nylon based membrane. In the second one, a hydrophobic PVDF membrane was functionalized by in-situ polymerization of acrylic acid. Kinetics of glucose oxidation, effect of pH and flow rate on the activity of GOX was discussed. A comparative study was presented between the activity of free GOX, electrostatically immobilized GOX and covalently immobilized GOX, along with the advantage of convective mode of operation over soaking mode. A novel study has also been conducted on detachment and reattachment of GOX in the same membrane matrix. Further study has been directed towards implementation of the above mentioned immobilized enzymatic system for oxidative dechlorination of chloro-organics. A first time attempt was made to use a 2-stack functionalized membranes system for simultaneous enzymatic production of hydrogen peroxide in first membrane, and oxidative dechlorination of 2, 4, 6-trichlorophenol (TCP) in the Fe+2 immobilized (by ion exchange) second membrane by Fenton reaction. The technique was efficient in destruction of TCP as evident from the overall dechlorination of 70-80 %. This technique provides additional benefit of reusing the same membrane matrices by reattaching fresh GOX and Fe+2.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 108-121).

The mixed-signal and analog design on a pre-diffused array is a challenging task, given that the digital array is a linear matrix arrangement of minimum-length transistors. To surmount this drawback a specific discipline for designing analog circuits over such array is required. An important novel technique proposed is the use of TAT (Trapezoidal Associations of Transistors) composite transistors on the semi-custom Sea-Of-Transistors (SOT) array. The analysis and advantages of TAT arrangement are extensively analyzed and demonstrated, with simulation and measurement comparisons to equivalent single transistors. Basic analog cells were also designed as well in full-custom and TAT versions in 1.0mm and 0.5mm digital CMOS technologies. Most of the circuits were prototyped in full-custom and TAT-based on pre-diffused SOT arrays. An innovative demonstration of the TAT technique is shown with the design and implementation of a mixed-signal analog system, i. e., a fully differential 2nd order Sigma-Delta Analog-to-Digital (A/D) modulator, fabricated in both full-custom and SOT array methodologies in 0.5mm CMOS technology from MOSIS foundry. Three test-chips were designed and fabricated in 0.5mm. Two of them are IC chips containing the full-custom and SOT array versions of a 2nd-Order Sigma-Delta A/D modulator. The third IC contains a transistors-structure (TAT and single) and analog cells placed side-by-side, block components (Comparator and Folded-cascode OTA) of the Sigma-Delta modulator.