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Aniridia is a rare genetic disorder that affects the development of the eye and is caused in most cases by mutations in the PAX6 gene. Patients with a heterozygous mutation in their PAX6 gene are born without irises. Aniridia patients are also prone to other eye diseases over their lifetimes such as cataracts and glaucoma. Aniridia’s progressive nature suggests that therapeutic intervention aimed at restoring PAX6 expression may be effective at ameliorating the progression of this disease. PAX6 is necessary for the development and maintenance not only of the eye, but also the pancreas. Patients with aniridia have an increased likelihood of developing glucose intolerance and diabetes. Indeed, genetic studies in rodents have confirmed that haploinsufficient animals for Pax6 develop glucose intolerance due to an ongoing requirement for Pax6 expression in the pancreas and gut. This thesis is a proof-of-concept study designed to determine the effects of repressing miRNA regulation of murine Pax6. Pax6 is regulated by miRNA-7 and miRNA-375. I hypothesized that repression of miRNA-7 and miRNA-375 would restore Pax6 expression and that this strategy might be useful in treating some of the progressive symptoms that emerge in aniridia patients in adulthood. As a first step towards evaluating miRNA inhibition as a therapeutic strategy for the treatment of aniridia, my first objective was to confirm whether miRNA-7 and miRNA-375 regulate Pax6 expression in pancreatic cells and tissue. My second objective was to determine whether these miRNAs could be efficiently inhibited. My third objective was to determine whether repression of miRNA-7 or miRNA-375 alters endogenous PAX6 protein levels in pancreatic cell lines. My final objective was to determine whether target protectors, delivered to explants of pancreatic islets through an adeno-associated virus (AAV) vector, could be used to restore Pax6 expression in murine haploinsufficient islets. From this study, I have confirmed that miRNA-7 and miRNA-375 regulate Pax6 in pancreatic cells that these miRNAs can be specifically inhibited, and that inhibition leads to an increase in Pax6 on both the reporter and protein levels. I have shown that target protectors against the miRNA-7 and miRNA-375 binding sites within the Pax6 3’UTR are effective at increasing the levels of PAX6 protein in pancreatic cell lines. Finally, I have also shown that a target protector against the miRNA-7 binding site can increase PAX6 protein levels in islets from murine haploinsufficient islets to near wild-type levels. My thesis lays the groundwork for the development of anti-miRNA-based therapies aimed at restoring PAX6 expression in the eye and pancreas. / Graduate

L’usage des écrans tactiles est devenu omniprésent ces dernières années. Ces appareils prennent une place de plus en plus importante dans notre de vie de tous les jours, on les retrouve dans nos maisons, bureaux et même dans nos poches. L’abondance de ces nouvelles interfaces a soulevé l’intérêt pour les technologies permettant une stimulation tactile, et mis en perspective le manque de ce type d’interfaces dans les nouvelles générations d’appareils mobiles avec écrans tactiles. Aujourd’hui, les appareils possédant ce type d’écran se contentent de fournir un retour sensoriel sous la forme de simples vibrations, qui n’ont pas pour objectif de recréer les sensations du toucher de surfaces. La recherche de nouvelles interfaces tactiles, plus performantes, et assurant une restitution plus proche d’une sensation naturelle est donc en cours. Plusieurs solutions technologiques ont prouvé leur efficacité mais posent des problèmes d’industrialisation. Dans cette thèse, une de ces méthodes adaptées aux écrans tactiles, la lubrification ultrasonique, est présentée. Cette méthode de stimulation s’appuie sur la création d’une onde stationnaire ultrasonore au sein d’un résonateur en verre et est capable de faire varier en temps réel la sensation de frottement entre le doigt de l’utilisateur et la surface du résonateur. Grace à des mesures tribologiques, les paramètres influents tels l’amplitude vibratoire, la vitesse d’exploration du doigt, la fréquence de résonance du stimulateur sont mis en évidence. Cette analyse permet la proposition d’une première série de spécifications techniques pour la conception de dispositifs à retour tactile. L’étude est ensuite complétée par l’analyse des problèmes d’alimentation et de contrôle du système, avec en considération les questions d’intégration au sein de dispositifs portables et les problèmes d’industrialisation du processus de fabrication. Enfin, des études psychophysiques sont menées pour affiner du point de vue perceptuel le cahier des charges de conception d’un tel dispositif. / The last few years have seen the emergence of ubiquitous mobile devices and tactile interfaces. These devices take an ever-important place in our daily life, they are present in our home, office, cars and even pockets. The abundance of these novel interfaces raised the interest in touch based human-machine interactions and highlighted the problem of the lack of natural touch feedback in the existing generation of tactile displays. Today’s smartphones all possess basic haptic feedback thanks to low frequency vibrations. However, these vibrations are far from the natural touch sensation of a surface. The search for a better tactile feedback, closer to the natural human perception, is ongoing. Multiple solutions are being explored to deliver improved haptic feedback on existing mobile platforms such as smartphones or tablets. In this thesis, we investigate the tactile feedback thanks to ultrasonic lubrication, well adapted to touch screen. This technology uses the creation of a resonating standing wave in a substrate to modulate in real time the friction perceived by user moving his finger on the resonator surface; the principle is effective even on a flat glass surface. By a series of experimental friction measures, the influence of the relevant parameters such as the vibration amplitude, the exploratory speed, the resonance frequency is highlighted. This analysis is used to build advanced tactile interfaces, based on ultrasonic friction modulation. The control and the supply of the tactile interfaces are also investigated, considering the issues of integration and industrialization of the process. Finally, these new interfaces are used to explore advanced control methods, improving further the quality and reliability of the generated sensation. Psychophysical analysis is performed to fulfil the specifications of these devices on a perceptual point of view.

Impaired cell mediated immunity is the clinical hallmark of HIV infection yet the manner in which CD8 T-cells are disabled is not yet fully understood. IL-7 signalling is essential for normal CD8 T-cell development and function. Our lab has previously shown decreased expression of the IL-7 receptor a-chain (CD127) on circulating CD8 T-cells in HIV+ patients is mediated by the HIV Tat protein which results in poor CD8 T-cell function. Soluble Tat protein is secreted by infected CD4 T-cells and taken up by neighbouring uninfected CD8 T-cells through endocytosis. Once in the cytoplasm, Tat translocates to the inner leaflet of the cell membrane where it binds directly to the cytoplasmic tail of CD127 inducing receptor aggregation, internalization, and degradation by the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is able to reduce IL-7 signaling and impair CD8 T-cell proliferation and function. To determine which domain(s) in the cytoplamic tail of CD127 are required for interaction with Tat, a series of plasmids encoding for CD127 deletion mutants were successfully created. These series of mutant CD127 coding sequences were transfected into a eukaryotic expression system, the Jurakt cell line, where CD127 mutants were successfully expressed. Before determine which region on CD127 is required for Tat binding, an optimized Ni-NTA column system was used to successfully isolate histidine-tagged HIV-1 Tat at a high yield and purity from E. coli. This HIV Tat protein was used to treat the lysates of the Jurakt cells transfected with the panel of CD127 mutants. CD127 was then immunoprecipitated, followed by Western analysis of the immune complexes to detect Tat protein. Tat was immunoprecipitated with all CD127 mutants suggests neither tyrosine 449, box 1, the acidic region, serine region nor C-tail are specifically required for Tat binding to CD127.

Interleukin (IL) -7 is an essential non-redundant cytokine that influences T-cell differentiation, proliferation, homeostasis and T-cell functions. In T-cells, IL-7 signals are transduced via IL-7's heterodimeric receptor composed of a common, γ chain (CD132) and an IL-7 specific, α chain (CD127). In light of the many roles that IL-7 plays in T-cell biology, it is no surprise that CD127 expression is tightly regulated in T-cells. In this study, I explore the effects that disease specific mutations in CD127 have on CD127 expression, regulation and signal transduction using an in vitro T-cell model. Here I specifically examined four disease associated mutations of CD127: P132S associated with severe combined immunodeficiency; L242_L243insNPC associated with T-cell acute lymphoblastic leukemia; I356V & T244I associated with autoimmune diseases like multiple sclerosis, rheumatoid arthritis and type 1 diabetes. In developing my model, I decided to use Jurkat cells because they expressed high endogenous surface levels of CD132, low endogenous surface levels of CD127 and endogenous STAT5. Jurkat cells were transduced with lentiviruses that induced expression of either WT or one of the four mutant CD127. I found that transduced Jurkat cells produced the WT and all four mutant CD127 proteins. I also found that wild type CD127, I356V, L242_L243insNPC and T244I mutant CD127 proteins were all expressed at the same level on the cell surface. However, I could not detect P132S mutated CD127 protein in its native state on the surface or intracellularly. I also found no differences between the mutant CD127 and wild type CD127 with regards to the level of soluble CD127 transcripts. I found that cell lines expressing L242_L243insNPC, I356V and T244I mutant CD127 protein, down-regulated surface CD127 at high IL-7 doses (25ng/mL) to the same extent as in the cell line expressing wild type CD127 protein. Interestingly, at the low IL-7 dose (1ng/mL) these mutant CD127 cell lines down-regulated surface CD127 to a lesser degree the wild type CD127 cell line. Further studies are required to elucidate whether P132S mutated CD127 is expressed on the surface and if T224I and I356V mutations in CD127 enhance signaling. By understanding CD127 dysregulation and dysfunction in disease states, we can potentially develop therapeutics that can return the function of CD127 to normalcy.

Interleukin (IL)-7 is an essential non-redundant cytokine and throughout the life-span of a T cell signaling via the IL-7 receptor influences cell survival, proliferation and function. It is therefore no surprise that expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. In this study I establish IL-7 down regulates CD127 gene transcription and surface protein expression in primary human CD8 T cells through two mechanisms. Upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at the critical tyrosine residue Y449. Concurrent activation of the JAK/STAT5 pathway stimulates expression of CIS, a member of the SOCS family of proteins. CIS protein already expressed at basal levels and induced by IL-7 bind directly to CD127 as demonstrated by Coimmunoprecipitation assays and colocalize with both CD127 and the early endosomal marker EEA1. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase. Through siRNA-mediated knockdowns I confirm CIS plays a predominant role in the IL-7 mediated degradation of CD127. The mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells was also examined. Through qPCR and nuclear run-on assays I illustrate that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor of the CD127 gene. Through PCR arrays, qPCR and Western blot analysis the IL-7 inducible transcription factor c-Myb was identified as a candidate repressor. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains three putative c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, I illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells. A thorough understanding of the mechanisms by which IL-7 regulates CD127 expression is imperative and may reveal novel insights into the contribution of abnormal IL-7 signaling to diseases affecting immune function.

Le rôle des gangliosides complexes di- ou trisialylés (GD3, GD2, GT3) dans le développement des cancers d’origine neuro-ectodermique (mélanome, neuroblastome) a été clairement démontré. Ces gangliosides sont également surexprimés dans 50% des carcinomes mammaires canalaires infiltrants et l’expression de la GD3 synthétase (GD3S) contrôlant leur biosynthèse, est augmentée dans les tumeurs de sein ER-négatives, avec une diminution de la survie des patientes. Notre laboratoire a montré que l’expression de la GD3S dans les cellules cancéreuses de sein MDA-MB-231 induit une augmentation de la migration et de la prolifération en conditions de sevrage, liée à une activation constitutive du récepteur c-Met et des voies de signalisation intracellulaires Erk/MAPK et PI3K/Akt par le GD2. Nous avons développé un autre modèle cellulaire dérivant des cellules cancéreuses mammaires MCF-7 sur-exprimant la GD3S. Ces cellules accumulent principalement les gangliosides GD1b et GT3. Nous avons pu montrer une augmentation de la migration mais aucun effet sur la prolifération n’a été observé. L’analyse en spectrométrie de masse des glycosphingolipides a montré que les MCF-7 expriment des quantités élevées de globosides et de faibles quantités de gangliosides par rapport aux cellules MDA-MB-231. Dans les cellules MCF7 GD3S+, nous avons démontré que la GD3S était capable de synthétiser du GT3 à partir du GD3, mais également des gangliosides inhabituels tétra- et penta-sialylés. Nos résultats montrent que les cellules cancéreuses mammaires présentent des profils gangliosidiques différents et que la modification du phénotype cellulaire induite par l'expression de la GD3S dépend du type cellulaire. / The role of di- and trisialylated complex gangliosides (GD3, GD2 and GT3) in the development of neuro-ectoderm derived cancers (melanoma, neuroblastoma) has been clearly demonstrated. Complex gangliosides are also over-expressed in 50% of breast invasive ductal carcinomas and the expression of the GD3 synthase (GD3S) that controls their biosynthesis, is increased in ER-negatives breast cancer tumors, associated with a decreased overall survival of the patients. Our lab has previously demonstrated that GD3S expression in MDA-MB-231 breast cancer cells induced an increase of the cell migration and proliferation in serum-free conditions, associated to a constitutive GD2-dependent constitutive activation of c-Met receptor and Erk/MAPK et PI3K/Akt signaling pathways. We have developed another cellular model deriving from MCF-7 breast cancer cells, over-expressing the GD3S. These cells mainly express GD1b and GT3 gangliosides. We have shown the increased migration of MCF7 GD3S+, but no effect on the proliferation was observed. Mass spectrometry analysis of total glycosphingolipids has shown that MCF-7 cells express a high level of globosides and a low level of gangliosides compared to MDA-MB-231 cells. In MCF7 GD3S+ cells, we demonstrated that the GD3S was able to synthesize GT3 from GD3, but also other unusual tetra- and penta-sialylated gangliosides. Our results show that breast cancer cell lines express different gangliosidic profiles and that the modification of the cellular phenotype resulting from the expression of the GD3S is depending on the cell type.

In today’s fast paced world, as the number of stations of television programming offered is increasing rapidly, time accessible to watch them remains same or decreasing. Sports videos are typically lengthy and they appeal to a massive crowd. Though sports video is lengthy, most of the viewer’s desire to watch specific segments of the video which are fascinating, like a home-run in a baseball or goal in soccer i.e., users prefer to watch highlights to save time. When associated to the entire span of the video, these segments form only a minor share. Hence these videos need to be summarized for effective presentation and data management. This thesis explores the ability to extract highlights automatically using MPEG-7 features and hidden Markov model (HMM), so that viewing time can be reduced. Video is first segmented into scene shots, in which the detection of the shot is the fundamental task. After the video is segmented into shots, extraction of key frames allows a suitable representation of the whole shot. Feature extraction is crucial processing step in the classification, video indexing and retrieval system. Frame features such as color, motion, texture, edges are extracted from the key frames. A baseball highlight contains certain types of scene shots and these shots follow a particular transition pattern. The shots are classified as close-up, out-field, base and audience. I first try to identify the type of the shot using low level features extracted from the key frames of each shot. For the identification of the highlight I use the hidden Markov model using the transition pattern of the shots in time domain. Experimental results suggest that with reasonable accuracy highlights can be extracted from the video.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Chemistry and Drug Control Student: Pavla ábelová Supervisor: Assoc. Prof. RNDr. Veronika Opletalová, Ph.D. Supervisor specialist: PharmDr. Jan Korábe ný, Ph.D. Title of diploma thesis: Synthesis, biological evaluation and in silico studies in the series of novel 7-methoxytacrine-donepezile like compounds Alzheimer's disease (AD) is an irreversible neurodegenerative disorder of the brain characterized clinically by loss of memory, deterioration of activities of daily living and cognition. The pathological hallmarks of AD include neuritic plaques composed of extracellularly stored fibrils of amyloid- peptide, intracellular deposits of hyperphosphorylated tau and neurotransmitter deficits. The aim of the study was to design and synthesize 7-methoxytacrine-donepezil-like compounds as potential inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8). New compounds consist of 7-methoxytacrine representing less toxic derivative of tacrine and benzylpiperazine moiety corresponding to donepezil fragment. To determine the potential of new derivatives, AChE and BChE inhibitory activities of the new molecules were assessed in vitro according to the method of Ellman et al....

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