Today, cancer is understood as an epigenetic as well as a genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. The latter changes may be an optimal target for novel anticancer agents. The main goal of using histone deacetylase inhibitors (HDACIs) would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promoter-associated histone deacetylation. However, HDACIs have pleiotropic effects that we are only just starting to understand. These may also be responsible for the induction of differentiation, cell-cycle arrest and pro-apoptotic effects. There are now so many HDACIs available, with such different chemical structures and biological and biochemical properties, that it is hopeful that at least some of them will succeed, probably in combination with other agents or therapies. In our studies we focussed ourselves on studies some new HDACIs, that can be useful for treating cancers, including leukemia and epithelial cancer. To do that, we used novel HDACIs, like BML-210, and their combination with the differentiation inducer all-trans retinoic acid (ATRA). Cell differentiation and proliferation in general, and specific gene expression require de novo protein synthesis and/or post-translational protein modifications. So, we tried to identify proteins in general and specifically the proteins that could be important for the cell differentiation process, and when and where in the cell the proteins appear. We delineated that HDACIs inhibited leukemia (NB4 and HL-60) cell growth in a time- and dose-dependent way. Moreover, BML-210 blocked HeLa cell growth and promoted apoptosis in a time-dependent way. Combining of BML-210 with ATRA induced a differentiation process in leukemia cell lines that lead to apoptosis. This correlated with cell cycle arrest in G0/G1 stage and changes in expression of cell cycle proteins (p21, p53), transcription factors (NF-κB, Sp1) and their binding activity to consensus or specific promoter sequences. We also assessed histone modifications, i.e. H3 phosphorylation and H4 hyperacetylation due to HDACI, leading to chromatin remodeling and changes in gene transcriptions. We have also studied changes in protein maps caused by HDACIs and differentiation agents, identifying differences for a few proteins due to growth inhibition and induction of differentiation in NB4 cells using BML-210 alone or in combination with ATRA. These proteins are involved in cell proliferation and signal transduction, like Rab, actin and calpain. One of them was alpha-dystrobrevin (α-DB). To further study possible roles of the latter, we determined changes of α-DB protein isoform expression that correlated with induction of differentiation. We thus identified a novel ensemble of α-DB interacting proteins in promyelocytic leukemia cells, including tropomyosin 3, actin, tubulin, RIBA, STAT and others, being important in cytoskeleton reorganization and signal transduction. Using confocal microscopy, we determined that α-DB co-localizes with HSP90 and F-actin in NB4 and HeLa cells. We also revealed that it changes sub-cellular compartment after treatment with ATRA and/or BML-210. α-DB silencing affected F-actin expression in HeLa cells, further supporting the idea that α-DB is involved in cytoskeleton reorganization in cells. Altogether, our results suggest that α−DB may work as a structural protein during proliferation and differentiation processes of human cancer cells. Based on our findings, we suggest that HDACIs, like BML-210, can be promising anticancer agents, especially in leukemia treatment, by inducing apoptosis and regulating proliferation and differentiation through the modulation of histone acetylations and gene expression.

Proper embryonic development requires precise genetic regulation of cell growth and differentiation. Organogenesis, the origin and formation of internal organs, must be exquisitely choreographed to ensure correct temporal and spatial patterning of functional organs within the developing organism. The liver is a vital organ responsible for hundreds of essential metabolic functions, but the intricate pathways controlling organ specification, differentiation, and positioning have not been fully elucidated. Uncovering the molecular mechanisms involved in hepatogenesis will enhance our understanding of normal liver development as well as inform the design of therapeutics to combat liver disease. Nuclear receptors are evolutionarily recent signal transducers that occupy a special niche in gene regulation, acting as direct connections between a ligand and its downstream transcriptional target. Nuclear receptor signaling governs many physiological processes, however its impact on liver development is not well understood.

Proper embryonic patterning requires precise spatio-temporal regulation of retinoic acid (RA) activity. Morphogenesis can be regulated at the level of RA distribution, mainly via its synthesis and catabolism by the RALDH and CYP26 enzymes respectively, and at the level of RA-mediated transcription through activation of its cognate nuclear receptor, the retinoic acid receptors (RARs) α, β, and γ. Loss of Cyp26b1 leads to increased local levels of RA in tissues such as the limb and craniofacial structures, and results in neonatal lethality. Visible gross phenotypic defects in neonates include phocomelia (shortening of the limbs), adactyly (missing digits), micrognathia (shortened lower jaw), and open eyes at birth. In addition, these embryos exhibit cleft palate and have a paucity of vibrissal (whisker) and pelage (hair) follicles. We have previously shown that ablating the gene encoding RARγ in a Cyp26a1-null background was able to rescue the caudal abnormalities associated with improper RA exposure in these embryos by limiting aberrant RA signalling, and thus rescuing expression domains of target genes involved in caudal development. I show here that ablating Rarg in a Cyp26b1-null background is able to partially rescue the defects associated with loss of CYP26B1. These include a reduction in the severity of limb defects, rescued vibrissae, fused eyelids, and recovered aspects of axial skeletal development. This compound-null murine model illustrates that RARγ plays a specific role in transducing the RA signal within tissues that are affected by the loss of CYP26B1. Further molecular analysis of the pathways responsible for directing limb bud outgrowth and eyelid fusion provided insight into pathways regulated by RARγ in these rescued tissues. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-04-01 15:38:52.05

Skeletal muscle fibers develop from mono-nucleated myoblasts that fuse to form multinucleated myotubes. In embryonic growth, this process occurs concurrently with the formation of the early cartilaginous skeleton and innervation by migrating nerve cells. The goal of my research was to explore co-culture conditions that encourage proliferation, differentiation and maturation of myoblasts to myotubes. A variety of co-culture experiments tested the influence of three basic tissues types (murine neural, cartilage and kidney primary cells) on the formation of myotubes in the C2C12 myoblast cell line. Three plating strategies were used: 1) C2C12 myoblasts were plated first, grown for two days before the addition of a second cell type; 2) both cell types were mixed and plated simultaneously; and 3) C2C12 myoblasts were added to a pre-established, 10 day old neural, cartilage or kidney cell culture. In addition, a parallel set of experiments were treated with all-trans retinoic acid, a potent myogenic activator and embryonic patterning signaling molecule. Myotube formation was consistently highest in C2C12 and cartilage co-cultures across all three plating strategies with a 277% increase in myotube area compared to controls. These effects were further enhanced when grown in 1 µg/mL all-trans retinoic acid. Co-cultures with neural or kidney cells consistently exhibited fewer myotubes when compared to C2C12 controls. It is postulated that the enhanced muscle growth in cartilage co-cultures was due to a chondrocyte-secreted extracellular matrix that facilitated myotube attachment to the substratum. / Graduate

Sensory neurons are key mediators of the transduction of external stimuli from the ear to the brain, essential for the sense of balance and hearing. Understanding when, where and how the sensory nervous system is assembled during development can provide insights on deafness and balance disorders. Here, I show in zebrafish that Her9 transcription factor is a key element in the regulation of the otic neurogenesis. Loss of Her9 function leads to the ectopic expression of neurogenic genes neurod and neurod4. Moreover, I show that Her9 acts downstream of Tbx1, and both genes are activated by retinoic acid signaling emanating from the paraxial mesoderm and negatively regulated by Hedgehog signaling. Altogether, the data demonstrates a role of retinoic acid in axial patterning and the establishment of a neurogenic domain through Tbx1 and Her9. At later stages, retinoic acid has an additional role by regulating neuronal differentiation in the statoacoustic ganglion. / Les neurones sensorials de l’oïda interna són mediadores claus en la transducció dels estímuls externs des de l’oïda interna al cervell. Entendre a on, quan i com el sistema nerviós sensorial s’organitza durant el desenvolupament embrionari pot ajudar en l’estudi de les malalties neurosensorials. En el present treball, mostro en peix zebra que el factor de transcripció Her9 és un element clau en el control de la neurogènesi òtica i que Her9 es troba sota el control directe del factor Tbx1. A més, ambdos factors estan regulats de manera positva per la via de senyalització de l’àcid retinoic i negativament per la vía de hedgehog. En resum, la tesis demostra un paper de l’àcid retinoic en la regionalització axial del primordi òtic en l’eix anteroposterior i l’establiment d’un domini neurogènic a través de Tbx1 i Her9. En estadis tardans, l’àcid retinoic regula la diferenciació neuronal en el gangli estato-acústic.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Cornell University, January, 2009. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references (Leaves 188-223).

Made available in DSpace on 2014-12-17T14:16:36Z (GMT). No. of bitstreams: 1 CandidaMSM_DISSERT.pdf: 2674854 bytes, checksum: 35ec01e6e19e4129b6100e813363df78 (MD5) Previous issue date: 2014-03-25 / Universidade Federal do Rio Grande do Norte / Retinoic acid (RA) and hydroquinone (HQ) assets are widely used in pharmaceutical and cosmetic formulations, for having depigmenting properties and are largely produced in drugstores. To assist in the development of formulations containing the active RA and HQ National Forms of Brazilian Pharmacopoeia (2005 and 2012 ) proposes formulations with different excipients such as cetyl alcohol (AC), cetostearyl alcohol (ACT), methylparaben (MTP), propyl paraben ( PPB), glycerin (GLY), dipropylene glycol (DPG), imidazolidinil urea ( IMD ), cyclomethicone (CCM ), butylated hydroxytoluene (BHT), octyl stearate (ETO), EDTA, decil oleate (ODC) and hydroxipropymethyl celullose (HPMC). One of the difficulties found in most cosmetic formulations is the large number of incompatibilities between the components of the formulations, so the aim this study was to evaluate thermal stability and interactions between these active pharmaceutical ingredients and excipients. The depigmenting agents were analyzed by DSC and TG and excipients were analyzed by TG. The dynamic thermogravimetric curves were obtained on a SHIMADZU thermobalance, model DTG-60, using an alumina crucible, at the heating rate of 10?C min-1, in the temperature range of 25-900 ?C, under an atmosphere of nitrogen at 50 mL min-1. The DSC curves were obtained using Shimadzu calorimeter, model DSC-60, using aluminum crucible, at the heating rate of 10?C min-1, in the temperature range of 25-400?C. The thermogravimetric and calorimetric curves were analyzed using TASYS software SHIMADZU. In this study no were found interactions between AR and the following excipients: MTP, PPB, IMD, ODC, EDTA, CCM, ETO, HPMC. However, were found interactions with the following excipients: AC, ACT, BHT, GLI and DPG. For HQ were found interactions with IMD and DPG. Interactions remained even changing proportions of the mixtures and the ternary. Thus, the studies conducted with excipients of National Formulary from 2005 and 2012 showed that these new excipients do not interact by thermogravimetry with the active pharmaceutical ingredients of this study / A Termogravimetria (TG) e a Calorimetria Explorat?ria Diferencial (DSC) s?o t?cnicas usadas em estudos farmac?uticos para a caracteriza??o de f?rmacos, determina??o da pureza, compatibilidade de formula??es, identifica??o de polimorfismo, avalia??o da estabilidade, decomposi??o t?rmica de f?rmacos e formula??es farmac?uticas. A Hidroquinona (HQ) e produtos contendo HQ t?m sido largamente utilizados como agentes despigmentantes para o clareamento da pele. Os retin?ides, que tamb?m exibem propriedades despigmentantes, s?o compostos que apresentam em sua estrutura o n?cleo b?sico da vitamina A. O ?cido retin?ico (AR) ? um exemplo de despigmentante dessa classe e ? muito utilizado em formula??o cosm?ticas. Para auxiliar no desenvolvimento de formula??es contendo os ativos AR e HQ o Formul?rio Nacional da ANVISA (2005 e 2012) prop?e formula??es com diferentes excipientes como: ?lcool cet?lico, ?lcool cetoestear?lico, parabenos (metil e propil), glicerina, dipropilenoglicol, imidazolidinilur?ia, ciclometicona, BHT, estearato de octila, EDTA, oleato de decila, hidroxipropimetilcelulose. Os agentes despigmentantes e excipientes foram analisados por TG e DSC. As curvas din?micas foram obtidas atrav?s de uma termobalan?a SHIMADZU, modelo DTG-60, usando cadinho de alumina, em uma raz?o de aquecimento de 10 ?C min-1 no intervalo de temperatura 25-900 ?C sob a atmosfera de nitrog?nio com fluxo de 50 mL min-1. As massas das amostras foram aproximadamente 10 ? 0,05 mg. As curvas DSC foram obtidas usando o calor?metro SHIMADZU, modelo DSC-60, em cadinho de alum?nio sob raz?o de aquecimento de 10 ?C min-1, em uma temperatura de 25-400 ?C. As curvas termogravim?tricas e calorim?tricas foram analisadas usando o software TASYS da SHIMADZU. Neste estudo n?o foram encontradas intera??es entre AR e os seguintes excipientes: MTP, PPB, IMD, ODC, EDTA, CCM, ETO, HPMC. No entanto, foram encontradas intera??es com os seguintes excipientes: AC, ACT, BHT, GLI e DPG. Para a HQ foram encontradas intera??es com a IMD e DPG. As intera??es permanecem mesmo alterando as propor??es das misturas, bem como nas tern?rias. Desta forma, os estudos realizados com os excipientes do formul?rio nacional de 2012 da ANVISA mostram que esses novos excipientes n?o interagem por termogravimetria com os ativos deste estudo

L'acide rétinoïque (AR) joue un rôle important dans plusieurs processus cellulaires à travers la régulation de la différentiation cellulaire, de la prolifération et de l'apoptose. Ces propriétés sont à la base de l'utilisation de l'AR dans le traitement de plusieurs cancers dont la leucémie aiguë promyélocytaire. Décrypter comment l'AR contrôle l'expression de gènes spécifiques est un défi permanent pour l'étude des cancers. Les effets de l'AR sont médiés in vivo principalement par les récepteurs à l'acide rétinoïque (RARs). Il a été récemment démontré que la phosphorylation des RARs par différentes kinases est une étape nécessaire dans la régulation de leurs fonctions. Dans ce contexte, ma thèse a porté sur l’étude des mécanismes moléculaires de la régulation par phosphorylation des RARs. Nous nous sommes intéressés en particulier à deux aspects : l’effet de la phosphorylation sur le domaine de liaison au ligand (LBD) et sur le domaine N-terminal (NTD). Dans le cas du LBD, la phosphorylation induit la fixation de la Cycline H qui est une sous-unité du facteur de transcription TFIIH, alors que la phosphorylation du NTD induit une diminution d’affinité de liaison à la Vinexine beta qui est un co-répresseur. Nous avons étudié les effets de la phosphorylation par des simulations de dynamique moléculaire. Cette technique permet de caractériser la dynamique structurale et de quantifier les interactions qui stabilisent les états phosphorylés et non phosphorylés. Ce projet a permis de définir les bases moléculaires de la communication entre le RA et les cascades de phosphorylation et d’obtenir des informations originales sur des mécanismes régulateurs d’une grande importance. / Retinoic Acid (RA) plays a critical role in many cellular processus through regulatory effects on cellular differentiation, proliferation and apoptosis. This proprety is at the basis of RA therapy in the treatment of several diseases and cancers such as Acute Promyelocytic Leukemia. Deciphering how RA controls the expression of specific subsets of genes is therefore a permanent challenge in oncology. The effects of RA are mediated in vivo by the retinoic acid receptor (RAR), which consistsof three subtypes. A new concept has recently emerged according to which phosphorylation of RARs by different kinases is a necessary step in the regulation of their function. In this context, the specific aim of this thesis was the elucidation of the molecular mechanisms of the regulation of RAR mediated by phosphorylation. In particular, we focused on two aspects, the effects of phosphorylation of the ligand binding domain (LBD) and the effects on the N-terminal domain (NTD). In the case of the LBD, phosphorylation enhanced binding to cyclin H, a component of the TFIIH transcription factor, while phosphorylation of the NTD decreased binding to vinexinB, a corepressor protein. We used molecular dynamics simulations to characterize the structural dynamics of these proteins in both phosphorylated and unphosphorylated states and to quantify theirinteractions. From this project, we were able to define the molecular basis of the communication between RA-induced phosphorylation cascades and regulatory mechanisms of high importance.