The oral route of administration remains the preferred route of administrating drugs due to patient acceptance and compliance. Therapeutic proteins are currently mainly administered by means of the parenteral route because of its low intestinal epithelial permeation capability. The major challenges for oral delivery of proteins and peptides are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. The latter can be overcome by including safe and effective absorption enhancers in dosage forms. Aloe vera, Aloe ferox and Aloe marlothii gel materials as well as N-trimethyl chitosan chloride (TMC) were shown to be capable of increasing peptide drug transport across in vitro models such as Caco-2 cell monolayers. The purpose of this study is to investigate binary combinations of chemical drug absorption enhancers and to determine if synergistic drug absorption enhancement effects exist. A. vera, A. ferox and A. marlothii leaf gel materials as well as with N-trimethyl chitosan chloride (TMC) were combined in different ratios and their effects on the transepithelial electrical resistance (TEER) as well as the transport of FITC-dextran across Caco-2 cell monolayers were measured. The isobole method was applied to determine the type of interaction that exists between the absorption enhancers combinations. The TEER results showed synergism existed for the combinations between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions also occurred and can probably be explained by chemical reactions between the chemical permeation enhancers such as complex formation. In terms of FITC-dextran transport, synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism was observed for A. vera and A. ferox. The combinations where synergism was obtained have the potential to be used as effective drug absorption enhancers at lower concentrations compared to single components. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

Transcriptomics is the large-scale study of RNA molecules produced by the genome, in single cells or population of cells using high-throughput methods. With the advances in transcriptomic analysis, the monitoring of genome-wide gene expression provides a powerful approach for determining the action of drugs. In this study, we analyzed the transcriptional responses of cells treated with drugs either alone or in combinations to explore their effects in two different applications: breast cancer therapy and cell conversion. In the first part of this thesis, we aim at modeling the relationship between single and multidrug breast therapy at the transcriptome level. We monitored the effects of three drugs, and their combinations in human breast cancer MCF-7 cells using the cap analysis of gene expression method. We are the first to explore the impact of single and combinatorial treatment on promoter and enhancer expression on a genome-wide scale. After applying and customizing a broad spectrum of regression algorithms, we showed that the transcriptional response to combinatorial drug treatment at both promoters and enhancers is accurately described by a linear combination of the responses to the individual drugs. Our analysis is promising for eliciting the transcriptional reaction to multidrug therapies in an unbiased genome-wide way, which may minimize the need for exhaustive combinatorial screens. Following the drug combination analysis, we explored the possibility to systematically identify drugs that either alone or in combinations facilitate cell conversion. To date, no computational approach prioritizes or suggests chemical compounds promoting cell reprogramming. Using transcriptomic data of human primary cells and drug response expression profiles, we developed a computational framework which accurately predicts single drugs or drug cocktails driving any source cell type towards the desired lineage. Experimental and in-silico validation on human pluripotent stem cells confirms the ability of the top predicted drugs to enhance reprogramming. The introduced method has countless applications in regenerative medicine and can significantly speed up the research in this field.

HIV-1 infections can be treated but not cured by the current antiretroviral therapy regimens. One of the major barriers to HIV-1 eradication is the persistence of the virus in treated HIV+ individuals under the form of reservoirs. A continuum of molecular mechanisms, at the epigenetic, transcriptional and post-transcriptional levels maintains HIV-1 gene expression silent in its reservoirs. A better understanding of HIV-1 molecular mechanisms of persistence thus allows to devise novel therapeutic approaches to eradicate the virus. In this context, our thesis aimed at characterizing the molecular mechanisms of HIV-1 persistence in its T-lymphoid and myeloid reservoirs. More specifically, we have studied the epigenetic and transcriptional mechanisms of HIV-1 persistence at three different levels. First, we have investigated the DNA methylation-mediated mechanisms underlying the heterogeneity of the DNA methylation inhibitor 5-aza-2’-deoxycytidine potency in the reactivation of HIV-1 gene expression from latently-infected CD4+ T cells. Second, we have studied the contribution of the intragenic binding sites for the cellular PU.1 transcription factor in the specific regulation of HIV-1 gene expression in myeloid lineages. Finally, in a third study, we have designed a new tool to study the transcriptional landscape of HIV-1 in LTRs-suppressed proviruses. Collectively, our work has offered individual insights into the molecular mechanisms underlying the heterogeneity of HIV-1 T-lymphoid and myeloid reservoirs, with the ultimate goal of developing new HIV-1 curative strategies and improving the quality of life of HIV+ individuals. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Cell signaling pathways are frequently used in multiple tissue and stage-specific contexts during multicellular development. The integration of these signaling pathways by transcriptional enhancers controls the tissue specific gene expression necessary for proper development. Enhancers are segments of DNA that interpret developmental signals to produce patterns of gene expression. A set of operational rules defines how different enhancers targeted by the same signals interpret and act on these signals. Using the Drosophila model system, my thesis work focuses on determining the operational rules used by developmental enhancers that integrate the Notch signaling pathway with other pathways. During development, the Notch signaling pathway in used to pattern cell territories involved in cell fate determination, and plays a role in differentiation. I first used a computational approach to identify a set of candidate Notch-target enhancers. From this set I carefully studied one specific enhancer from the nab gene that integrates the Notch and Bone Morphogenetic Protein (BMP) signaling pathways in the developing wing. This nab enhancer is a part of a cluster of enhancers that work together to drive the global nab expression pattern during development. Each of these enhancers drives the expected expression patterns as well as atypical expression patterns, which are silenced by adjacent enhancers. These results suggest that Notch targeted enhancers are involved in both tissue specific gene activation and gene silencing.

Alzheimers disease (AD) represents one of the greatest health problems in industrialized countries considering the ageing population. Only four drugs are currently approved for the treatment of this disease. As these drugs are characterized with a limited time efficacy, it has become urgent to develop additional innovative AD treatments. Amongst the approaches that are actively investigated, the one consisting in potentiating a subclass of glutamate receptors appears attractive. This well advanced pharmacological approach includes three major classes of compounds amongst which appear the benzothiadiazine 1,1-dioxides. The present thesis is a pursuit of the preliminary efforts that were published in 1998 and 2001 by our team. Based on promising in vitro results obtained with the lead compound 59, pharmacomodulations around 59s structure have been achieved in order to enhance its in vivo activity and to optimize its pharmacokinetic parameters. First efforts were devoted to exploratory synthesis where attention was paid to the impact of the substituent introduced at the 7-position. Moreover, some pyridothiadiazine dioxides as well as thienothiadiazine dioxides were prepared. The most important part of our pharmacomodulations was focused on the thiadiazine ring system. Considering that the poor in vivo results obtained with 59 could be due to a metabolic weakness of the latter, the introduction of fluorine atoms was tempted as a lead optimization strategy. This approach was successful, since it led to the synthesis of 95b which was selected for further pharmacological evaluations. This new lead compound was shown to exert significant cognitive-enhancing effects in vivo after oral administration to Wistar rats. Moreover, the study of the metabolic degradation of 95b allowed the assessment of the starting hypothesis that had dictated the pharmacomodulations philosophy. Finally, additional exploratory pharmacomodulations were achieved notably leading to the preparation of a quinazolinone series and 1,4-benzothiazine compounds. This research allowed to significantly improve the pharmacokinetic profile of our series and led to the identification of 95b as a new lead compound. However, many pharmacomodulations remain to be explored. The data collected during this thesis are appealing further studies. Efforts in the near future should lead to the design of novel drug candidates among which a future innovative AD treatment could emerge. Au vu du vieillissement de la population dans les pays industrialisés, la maladie dAlzheimer constitue un problème majeur en termes de santé publique. A lheure actuelle, seuls quatre médicaments sont utilisés pour traiter cette maladie. Sachant que ces substances nont quune efficacité limitée, la conception de nouveaux médicaments actifs contre cette pathologie apparaît comme une priorité. Dans cette optique, la potentialisation des récepteurs glutamatergiques de sous-type AMPA semble une approche thérapeutique intéressante. Parmi les composés étudiés dans cette voie, apparaissent les benzothiadiazine dioxydes. Cette thèse sinscrit dans la continuité des recherches publiées en 1998 et 2001 par notre équipe et poursuit les pharmacomodulations entamées autour du composé leader 59. Ce travail sest principalement focalisé sur lamélioration de lactivité in vivo de ce dérivé et sur loptimisation de ses paramètres pharmacocinétiques afin dobtenir un candidat médicament potentiel. Notre attention sest tout dabord portée sur la subsitution aromatique en position 7. Par ailleurs, plusieurs séries pyrido- et thiénothiadiazines ont été également préparées. Ensuite, en postulant que le manque dactivité in vivo du composé 59 pouvait être dû à une faiblesse métabolique de ce composé, lintroduction judicieuse datomes de fluor a été choisie comme stratégie doptimisation. Cette approche nous a notamment amené à lidentification du composé 95b. Ce dérivé sest montré particulièrement actif dans un test de reconnaissance dobjets chez le rat Wistar. Ce résultat encourageant a amené à réaliser toute une série dévaluations pharmacologiques sur ce produit, afin de caractériser son mécanisme daction. Les données récoltées à lissue de ces investigations suggèrent que le composé 95b présente un intérêt potentiel en tant quagent procognitif. Dautre part, létude du profil de dégradation métabolique de ce dérivé a permis de confirmer lhypothèse qui avait dicté lintroduction datomes de fluor. Enfin, des pharmacomodulations supplémentaires ont été réalisées de façon exploratoire et ont entre autres débouché sur une série de quinazolinones et une série de 1,4-benzothiazines. Les travaux réalisés au cours de cette thèse ont permis daméliorer grandement le profil pharmacocinétique des séries explorées. Néanmoins, de nombreuses pharmacomodulations restent à explorer. Des études complémentaires savèrent nécessaires et devraient déboucher sur de nouveaux candidats médicaments innovants, parmi lesquels pourraient figurer un futur traitement de la maladie dAlzheimer.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 114-136).

Troponin I (TnI), like many striated muscle contractile proteins, consists of multiple isoforms encoded by a multigene family whose members are differentially expressed in the different striated muscle cell types. Two TnI genes, TnIfast and TnIslow, are expressed in skeletal muscle the former in fast muscle fibers, the latter in slow fibers. The tissue- and fiber-type-specificities of the TnI fast and slow genes are driven by transcriptional enhancer elements: a Slow Upstream Regulatory Element (SURE) upstream of the TnIslow gene and a fast Intronic Regulatory Element (IRE) within the first intron of the TnIfast gene. Within the 144 bp IRE, there are 4 known cis elements, and the aim of this work was to initiate the studies to map the element(s) that are chiefly responsible for directing the fast-fiber-specificity of IRE-driven gene expression. This was approached by making IRE end-deletion constructs lacking either the left-most or right-most IRE cis-element. These IRE derivatives were coupled to a reporter gene consisting of a minimal (enhancer-dependent) TnIfast promoter linked to E. coli beta-galactosidase coding sequences. The transcriptional activity of these constructs was first evaluated in cell culture transfection experiments, and then by in vivo gene transfer into adult mouse skeletal muscles. The conclusion of these experiments was that fast-fiber-specificity of IRE-driven gene expression resides in the left-most 30 bp of the IRE, a region including an E-box binding site for myogenic regulatory factors of the MyoD family.

Thesis (M.S.)--University of Toledo, 2007 / "In partial fulfillment of the requirements for the degree of Master of Science in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 37-48, 62-84.

Thesis (M.Sc.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/01/14). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (p. 233-246).