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The Distal-less homeobox (Dlx) genes encode a group of transcription factors that are involved in various developmental processes including forebrain development. Dlx genes are arranged in convergently transcribed bigene clusters with enhancer sequences located in the intergenic region of each cluster. The expression patterns of Dlx1/Dlx2 and of Dlx5/Dlx6 are attributed in part to the activity of I12a/I12b and I56i/I56ii intergenic enhancers, respectively. In an effort to determine how Dlx intergenic enhancers interact with the promoter regions of each cluster, I employed the Chromosome Conformation Capture (3C) technique on developing forebrain at E13.5 and E15.5. My 3C analysis provided potential enhancer-promoter interaction, in cis, that are consistent with previously known regulatory mechanisms. Furthermore, trans interactions may exist between Dlx1/Dlx2 and Dlx5/Dlx6 clusters in the developing forebrain at E13.5, thus providing a possible novel cross-regulatory mechanism between these two loci. I have also investigated the phenotypic consequences of Dlx enhancer deletion(s) on forebrain development by characterizing mice with I56ii and I56ii/I12b enhancer deletions. Enhancer deletions significantly impair Dlx expression as well as that of Evf2, Gad2 and of the striatal markers Islet1 and Meis2. Enhancer deletion(s) also reduce the expression of ISLET1 and CTIP2 proteins and Semaphorin 3A, Slit1 and Ephrin A5 that are thought to provide guidance cues in the corridor cells. Overall, these changes may disrupt the guidance of the thalamocortical axons. The data presented here further our understanding of the interactions between Dlx intergenic enhancers and promoter regions. Enhancer deletion(s) furthers our understanding of Dlx regulatory networks necessary that ensure proper Dlx expression, which, in turn may be involved in a genetic pathway underlying the synthesis of GABA, which may be further essential in maintaining the GABAergic phenotype.

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Onychomycosis also known as dermatophytic onychomycosis is the fungal infection of the toenails and fingernails, characterized by discoloration and thickening of the nail and involves the nail plate, nail bed and nail folds. The disease is more than a cosmetic problem, as it severely impacts the patient's quality of life. Onychomycosis is an opportunistic infection in special subpopulations of patients suffering from diabetes, psoriasis, HIV/AIDS etc. The current treatment strategies involve systemic delivery of oral antifungal agents including azoles (e.g. itraconazole) and allylamines (e.g. terbinafine hydrochloride) which are delivered to the nail plate from the nail bed. More recently, topical delivery of drugs including amorolfine and bifonazole/urea (available outside the United States) and Penlac® nail lacquer (ciclopirox) topical solution, 8%, available in the US are an alternative treatment option to the oral antifungal agents. Topical delivery of antifungal agents through the human nail offer several advantages over oral therapy including lower incidence of adverse events and lower potential for drug-drug interaction with drugs used to treat diabetes, HIV/AIDS and psoriasis. The objectives of this project were to: 1) To determine the critical factors affecting the delivery of ciclopirox olamine across the human nail, 2) To screen and select penetration enhancer(s) specific for ciclopirox olamine delivery into the target tissue(s) and 3) To develop a novel transungual formulation containing ciclopirox olamine (CPO) and penetration enhancer(s) for transungual delivery. Ciclopirox olamine, the salt form of the free acid of ciclopirox was used in the study to develop a novel transungual patch formulation and skin and nail permeation from the patch formulation was compared to Penlac® nail lacquer. Various factors such as drug partitioning into the healthy and infected toenail, drug-keratin binding, lateral diffusion, drug-epidermal binding and the formulation components, all play a role in achieving optimum drug penetration and permeation through the nail. Understanding the interplay of these factors helped in the development of an effective topical formulation which was observed to be superior to Penlac® nail lacquer in the in vitro studies. Most cases of onychomycosis show infection and inflammation of the nail folds (skin surrounding the nails). Therefore for an efficient treatment of OM, the antifungal drugs must be delivered to two target tissues - human nail and the nail folds. The major challenges in developing a topical formulation for treatment on OM are: a) Achieving antifungal drug minimum inhibitory concentration (MIC) in the epidermis of the nail folds. b) Enhancing penetration and permeation of the antifungal drug across the human nail to reach the nail bed and achieve the necessary MIC (tissue underneath the nail). Twelve chemical penetration enhancers (PEs) were screened for their ability to enhance ciclopirox olamine accumulation into the nail folds and permeation through the nail. Propylene glycol (PG) enhanced the levels of the drug in the epidermis of the skin while limiting its permeation across the skin. Thiourea (TU) was selected as the best enhancer to increase ciclopirox olamine penetration into the nail. The diffusion of the antifungal drug across the human nail was studied in vitro using human cadaver toenails mounted in Franz diffusion cells. Pressure sensitive adhesives (PSA) belonging to the polyisobutylene, polysiloxane and polyacrylate classes of adhesives were screened to develop a monolithic drug-in-adhesive-type nail patch. The in vitro release of CPO from the PSA patches were limited and did not improve in presence of hydrophilic plasticizer (propylene glycol) and hydrophobic plasticizers (triacetin and triethyl citrate). Increasing the concentration of TU from 1 % to 10 %, lead to its crystallization in the dry patches. Therefore a change in the patch design was recommended. Other hydrophilic polymers including Polyoxyethylene (POLYOX®) and hydroxyl propyl methyl cellulose (HPMC) were also screened to develop a modified drug-in-hydrophilic matrix patch design. The patch was designed to incorporate CPO, PG and TU in the polymer matrix overlaid on a non-occlusive backing membrane cast with polyacrylate PSA. The HPMC films showed the best drug release profile with 80 % release in 2 to 4 hours using a USP apparatus 5. These patches were characterized for drug penetration into the skin and nail permeation. Penlac® nail lacquer was used as the comparator control product. The prototype HPMC K15M patch containing 10 %w/w each of the drug and TU and 150 % w/w of PG showed 2.8 fold increase in CPO accumulation in epidermis compared to Penlac® nail lacquer in 24 hours. The skin permeation was found to be similar to that of Penlac®. The HPMC K15M patch formulation showed 2.7 fold increase in CPO concentration within the nail and 4.2 fold increase in transungual flux compared to Penlac®. The patch delivered higher levels of ciclopirox olamine into the target tissues with a lower permeation lag-time. The novel nail patch delivery system had the following properties: a) Ease of application, b) Contact with the nail surface, c) Increased concentration of drug in dissolved form within the patch, d) Presence of enhancers. The novel nail patch formulation has shown increased efficiency in topical and transungual drug delivery for treatment of OM, when compared to the commercial formulation, Penlac® nail lacquer in the in vitro studies. The physical characterization of the patch using Scanning Electron Microscopy, Polarized Light Microscopy, Optical Light Microscopy, Differential Scanning Calorimetry, X-Ray Diffractometer and Fourier Transform Infrared Spectroscopy show that ciclopirox olamine exists at a sub-saturation level in a non-crystalline form in the patch without any significant drug-polymer interaction. In conclusion, all the objectives of the study were met by successfully selecting penetration enhancers for CPO delivery into the nail folds and across the nail plate, evaluating the interaction between CPO and target tissues, developing a transungual patch system and characterizing the novel transungual patch. / Pharmaceutical Sciences

Les membres de la famille Homeobox jouent un rôle critique dans le développement hématopoïétique normal. L'expression ectopique des gènes Homeobox provoque des désordres dans l'hématopoïèse et le développement de leucémies. L'oncogène TLX3 s'exprime de manière ectopique exclusivement dans les Leucémies Aiguës Lymphoblastiques T (LAL-T), avec un blocage des thymocytes à un stade de différentiation précoce cortical CD4+CD8+ DP. De nombreuses études ont investigué les mécanismes d'action des oncogènes TLX1/3, mais plusieurs questions restent en suspens. Durant ma thèse, j'ai étudié l'impact de l'expression ectopique de l'oncogène TLX3 sur le développement lymphocytaire T et les mécanismes de transformation leucémique associés. L'expression de TLX3 a provoqué le blocage des thymocytes à un stade DN2 avec une immortalisation des clones preleucémiques. Les souris transplantées avec les cellules TLX3 ont développé des tumeurs similaires aux LAL-T. Les analyses de ChIP-Seq et d'expression génique ont identifié un recrutement de TLX3 sur les enhancers spécifiques aux cellules T par le motif de fixation Ets/Runx1. Nos résultats suggèrent que la fixation de TLX3 sur les éléments cis-régulateurs peut contribuer à la transformation maligne des thymocytes en perturbant les réseaux transcriptionnels responsables de l'oncogenèse LAL-T. / It is now well established that members of the homeobox gene family play a critical role in normal hematopoietic cell development and that their unbalanced or ectopic expression can lead to characteristic perturbations in haemopoiesis and the onset of leukaemia. TLX3 expression in human haematologic malignancies is exclusive to T-ALL, where it is almost universally associated with transformation of early cortical CD4+CD8+ DP thymocytes. Multiple studies intensively investigated the mechanisms by which TLX1/3 oncogenes could promote complex tumor development, but many questions remain still unclear. During my thesis I investigated the impact of ectopic TLX3 expression on T cell development, and the initiating mechanisms of T-cell transformation leading to leukemia onset. Forced expression of TLX3 disrupted the thymic develoment at DN2-like stage giving rise to immortalized preleukemic clones. Following the transfer into immunodeficient mice TLX3 preleukemic cells initiated malignant cell transformation resulting into leukemia-like disease. Applying a combination of ChIP sequencing and gene expression profiling, we identified TLX3 recruitment onto T-cell specific enhancers via interaction with Ets1/Runx1 composite motif sites as preferential molecular events in the initial steps of TLX3-induced transformation. Thus our findings suggest that the genome-wide binding properties of TLX3 on cis-regulatory elements may contribute to its ability to promote thymocyte preleukaemic state via perturbation of transcriptional regulatory networks responsible for T-ALL oncogenesis.

AN ABSTRACT OF THE DISSERTATION OF TITLE: (OPTIMIZATION PROTECTION OF CARBOB-CARBON COMPOSITES DISC-BRAKES MATERIAL BY COATING WITH ANTI-OXIDANTS) Developing glass enhancer mixture solutions (Ki’s), which promote the formation of a stable glass layer, homogenous clear liquid solution, and low viscosity liquid form, are easy to apply, and penetrating. They are compatible with ceramic liquid glass based anti-oxidants for treating surfaces of carbon/carbon composites material, and significantly increase the rate of protection against oxidation. Ki’s’ are comprised of mixing chemical compositions at standard temperature and pressure conditions from group one and two such as Na, K, Ca, Mg, etc. of 5 to 25 wt. %, deionized water from 95 to 75 % by weight, and adding up to 1 % by weight of surfactants such as DF-16, DF-20, and CF-10 with specific proportions, and followed by thorough stirring to produce a homogeneous blend of mixture solution. The glass enhancers, which are aqueous mixture solutions, are applied to the surfaces of carbon/carbon (C/C) composites by dipping, brushing, spraying, or other painting application techniques, followed by annealing, or a heat-treating range of 80 to 110 ℃ for a minimum of 8 hours, and allowing cooling time of the coated C/C composites of a minimum of 12 hours to room temperature. Preferential compatibility of the glass enhancer mixture solutions (Ki's) is with liquid glass former's, anti-oxidants comprised mostly of borate and phosphate glasses. The glass enhancer solution mixtures (Ki’s) are supplemental additions to ceramics’ liquid anti-oxidants coatings used for carbon-carbon composites protection against oxidation, and it will increase the rate of protection against oxidation for low, and moderate temperature’s range from 400 to 900 ℃. The glass enhancer Ki’s mixture solutions should be used with liquid glass former's’ anti-oxidants, such as SiO₂, GeO₂, B₂O₃, and P₂O₅. A series of glass enhancer’s Ki’s, heat treatment cycle (char-cycle) ranged between 700 to 900 ℃, and application methods, were developed and tested experimentally. Two arbitrary isothermal temperatures of 650 ℃, and 871 ℃ were selected for thermal oxidation testing, and a temperature of 650 ℃ was selected, and tested against catalytic thermal oxidation. Additions of glass enhancer Ki’s improved protection of C/C composites disc-brakes against oxidation by double, and triple amount of time in hours versus the use of anti-oxidant coatings alone.