Teratogenes are external environmental factors that can cause a developmental or a congenital defect in exposed individuals. The methods used for detecting the embryotoxic effect of substances are the classic when laboratory mammals are used and the alternative which use in vitro and in ovo systems. The main difference between these two is that the alternative methods lack metabolism of maternal organism. The metabolism of maternal organism brings a high variability of results to systems of the classic methods. We used two alternative methods in this thesis, both using chicken embryo. The first of them was in ovo method called CHEST (Jelínek, 1977). CHEST method can be used for administration of tested substances from ED2 to ED6. The disadvantage of this method is due to the dilution of the tested substance after subgerminal application at ED2. Therefore we developed in vitro method called SANDWICH. No dilution occurs while using the SANDWICH method. The aim of this study was to develop in vitro method SANDWICH while using proven teratogene (all-trans retinoic acid) and its solvent (dimethyl sulfoxide), to estimate beginning of the embryotoxicity dose range for both substances using CHEST and SANDWICH, and finally to compare obtained results. We confirmed the embryotoxic effect of all-trans...

Células pluripotentes são aquelas que possuem a capacidade de dar origem às células dos três folhetos embrionários (ectoderma, mesoderma e endoderma), bem como também às células germinativas. As células-tronco embrionárias (CTE) são as células pluripotentes mais conhecidas, as quais apresentam uma elevada capacidade de diferenciação celular e autorenovação. Estas propriedades tornam as CTE potenciais ferramentas para a medicina regenerativa, porém seu uso na prática clínica enfrenta várias barreiras. Neste sentido, o acúmulo de conhecimento a respeito dos mecanismos envolvidos na manutenção da pluripotência, levou ao desenvolvimento de técnicas capazes de induzir a pluripotência em células somáticas adultas. Na maioria das abordagens, isto se dá pela expressão ectópica de fatores de transcrição envolvidos na pluripotência (como Oct4 e Nanog). Com isto em vista, torna-se evidente que estudos que levem a um melhor entendimento destas propriedades biológicas, podem levar ao desenvolvimento desta importante área. Apesar destas inovações, os mecanismos responsáveis pela manutenção ou indução da pluripotência e da autorenovação, continuam largamente inexplorados. Neste sentido, o conjunto de técnicas referidas como High Content Screening (HCS) apresenta características fundamentais que permitiriam a interrogação sistemática e em larga-escala de fatores que possam estar influenciando nestes processos. A técnica de HCS se baseia no uso de microscopia de fluorescência em placas de 96 ou mais poços, permitindo a aquisição e a análise automatizada das imagens, de forma a quantificar alterações fenotípicas nas células. O presente trabalho teve como objetivo estabelecer um modelo experimental para a avaliação funcional e em larga escala de fatores que possam influenciar a diferenciação celular. Tendo em vista a facilidade de cultivo e manuseio, a linhagem humana de células pluripotentes de carcinoma embrionário (CCE) NTera-2, foi utilizada. Para a padronização do modelo, o processo de diferenciação foi avaliado ao longo do tempo (em 2, 4 e 8 dias) na presença ou ausência de ácido transretinóico (atRA), utilizado como indutor de diferenciação celular. Para isso, os níveis transcricionais de Oct4, Nanog (marcadores da pluripotência) e de N-Caderina foram avaliados por PCR em tempo real. Finalmente, a expressão e a distribuição celular de Oct4, Nanog e da alfa-actina foi avaliada por meio de microscopia de fluorescência automatizada, com o uso de anticorpos ou faloidina marcada, utilizando um sistema de HCS (Operetta, Perkin Elmer) para a análise dos resultados. A proliferação celular das células submetidas à diferenciação foi avaliada pelo ensaio do XTT. O atRA inibiu a proliferação e induziu a diferenciação; como demonstrado, respectivamente, pelos resultados do ensaio do XTT, decaimento dos níveis de Oct4 e Nanog e, concomitante aumento de N-Caderina, ao longo do tempo. Também foi observada a diferenciação espontânea da linhagem, na ausência de atRA, porém, de forma reduzida. Finalmente, as avaliações de HCS evidenciaram que, durante o processo de diferenciação, a perda da expressão nuclear de Oct4 e Nanog está associada à alteração do fenótipo celular, com a redistribuição da actina cortical e a formação das stress fibers, caracterizando o processo de transição epitélio-mesenquima (EMT), um importante mecanismo envolvido na diferenciação celular. Os resultados obtidos neste trabalho demonstram a viabilidade do uso da linhagem NTera-2 como modelo para estudos futuros de HCS visando a identificação de moléculas que atuem na modulação de propriedades fundamentais das células tronco pluripotentes. / Pluripotent stem cells are those that possess the ability to generate cells from the three germ layers (ectoderm, mesoderm and endoderm), as well as the germ cells. The embryonic stem cells (ESC) are the best known pluripotent cells that present a high capacity of cell differentiation and self renewal. These properties of the ESC make them potential tools for the regenerative medicine, but their use in clinical practice faces several barriers. In this sense, the accumulation of knowledge about the mechanisms involved in the maintenance of pluripotency led to the development of techniques capable of inducing pluripotency in adult somatic cells. In most approaches, this is achieved by the ectopic expression of transcription factors involved in pluripotency (such as Oct4 and Nanog). With this in mind, it becomes clear that studies that provide a better understanding of these biological properties can lead to the development of this important area. Despite these innovations, the mechanisms responsible for the maintenance or induction of pluripotency and self-renewal remain largely unexplored. In this sense, the set of techniques such as High Content Screening (HCS) has fundamental characteristics that allow systematic and large-scale interrogation of factors that may be influencing these processes. The HCS technique is based on the use of fluorescence microscopy in 96-well or larger plates, allowing the automated acquisition and analysis of images, so as to measure phenotypic changes in the cells. This study aimed to establish an experimental model for functional and large-scale assessment of factors that may influence cellular differentiation. Due its simple cultivation and handling characteristics, a human lineage of pluripotent embryonal carcinoma cell (ECC) NTERA-2 was used. To standardize the model, the process of differentiation was evaluated over time (at 2, 4 and 8 days) in the presence or absence of all-trans retinoic acid (atRA), used as an inducer of cellular differentiation. The transcriptional levels of Oct4, Nanog (pluripotency markers) and Ncadherin were assessed by real time PCR. Finally, the expression and cellular distribution of Oct4, Nanog and alpha-actin was assessed by fluorescence microscopy, using antibodies or labelled phalloidin, using a HCS platform (Operetta, Perkin Elmer) for the analysis of the results. The proliferation of cells undergoing differentiation was assessed by XTT assay. atRA inhibited proliferation and induced differentiation, as shown by the XTT assay results, and the decay of Oct4 and Nanog, and concomitant increase of N-cadherin levels over time, respectively. It was also observed spontaneous differentiation in the absence of atRA although in less extent. Finally, the HCS results showed that during the differentiation process, the loss of nuclear expression of Oct4 and Nanog is associated with alteration of cell phenotype, with redistribution of cortical actin and formation of stress fibers, characterizing the epithelialmesenchymal transition (EMT), an important mechanism involved in cell differentiation. The results of this study therefore demonstrate the feasibility of using the NTERA-2 cell line as a model for future HCS studies aiming identification of molecules that act in the modulation of fundamental properties of pluripotent stem cells.

Der humane Stammzellfaktor (SCF) ist ein zentraler Wachstumsfaktor für Mastzellen in der Dermis und für Melanozyten in den Basalzellschichten der Epidermis. Er wird u. a. von Keratinozyten produziert. In dieser Arbeit wurde die mögliche Regulation der Expression von SCF aus Keratinozyten durch All-Trans-Retinsäure (RA) und Dexamethason in vitro an Hand der HaCaT-Zelllinie untersucht. Die HaCaT-Zellen wurden mit den beiden o. g. Substanzen (10 hoch -5 M bis 10 hoch -9 M) über 24 Stunden und 11 Tage inkubiert. Die Auswertung der HaCaT-Zellzahl, des Gesamt-Proteins SCF, dessen Splicevarianten (mSCF, sSCF) und der Rezeptoren von RA (RAR-alpha, -beta, -gamma) und von Dexamethason (GR-alpha, -beta) erfolgte mittels ELISA und RT-PCR. Dabei ergaben sich folgende Resultate: RA bewirkt einen Anstieg von SCF, Dexamethason bewirkt bei Kurzinkubation eine deutliche Zunahme von SCF, bei Dauerinkubation einen starken Abfall. Die RA-Rezeptoren RA-alpha und -gamma waren nach Inkubation mit RA verstärkt nachzuweisen; die Glukokortikoid-Rezeptoren GR-alpha und -beta zeigten nach Inkubation mit Dexamethason ebenfalls eine vermehrte Expression. Die Expression des Mastzellwachstumsfaktors SCF könnte deshalb unter physiologischen, pathologischen und therapeutischen Bedingungen durch Retinoide und Glukokortikoide reguliert sein. / The human stem cell factor (SCF) is a crucial growth factor for mast cells in the dermis and for the melanocytes in the basal layers of the epidermis. SCF is produced, among others, by keratinocytes. This study examines the possible regulation of the expression of SCF from keratinocytes by all-trans retinoic acid (RA) and dexamethasone in vitro by the keratinocyte cell line HaCaT. The HaCaT-cells were incubated for 24 hours or 11 days, respectively, with one of the above mentioned substances (10 to the power of -5 M to 10 to the power of -9 M). The analysis of the number of HaCaT-cells, of the total SCF protein, its splice variants (mSCF, sSCF), the receptors of RA (RAR-alpha, -beta, -gamma), and of the dexamethasone (GR-alpha, -beta) was done by ELISA and RT-PCR. The following results were found: RA induces an increase of SCF, dexamethasone at a short incubation period a considerable increase of SCF, and at long-term incubation a strong decrease. The RA-receptors RA-alpha und -gamma expression is increased after incubation with RA, and the glucocorticoid-receptors GR-alpha and -beta after the incubation with dexamethasone. Therefore, it is probable that the increase of the mast cell growth factor SCF under physiological, pathological and therapeutic conditions could be regulated by retinoic acid and glucocorticoids.

Made available in DSpace on 2014-12-17T14:03:36Z (GMT). No. of bitstreams: 1 BrunaLLM_TESE.pdf: 3672151 bytes, checksum: 3a91fdec3101fa0f0ebdb652c2960bda (MD5) Previous issue date: 2013-12-20 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Nutritional status is an important determinant to the response against Leishmania infection, although few studies have characterized the molecular basis for the association found between malnutrition and the disease. Vitamin A supplementation has long been used in developing countries to prevent mortality by diarrheal and respiratory diseases, but there are no studies on the role of vitamin A in Leishmania infection, although we and others have found vitamin A deficiency in visceral Leishmaniasis (VL). Regulatory T cells are induced in vitro by vitamin A metabolites and are considered important cells implicated T CD4+ cell suppression in human VL. This work aimed to examine the correlation of nutritional status and the effect of vitamin A in the response against Leishmania infantum infection. A total of 179 children were studied: 31 had active VL, 33 VL history, 44 were DTH+ and 71 were DTH- and had negative antibody to Leishmania (DTH-/Ac-). Peripheral blood monuclear cells were isolated in a subgroup of 10 active VL and 16 DTH-/Ac- children and cultivated for 20h under 5 different conditions: 1) Medium, 2) Soluble promastigote L. infantum antigens (SLA), 3) All-trans retinoic acid (ATRA), 4) SLA + ATRA and 5) Concanavalin A. T CD4+CD25highFoxp3+, T CD4+CD25-Foxp3- and CD14+ monocytes were stained and studied by flow cytometry for IL-10, TGF-β and IL-17 production. Nutritional status was compromised in VL children, which presented lower BMI/Age and retinol concentrations when compared to healthy controls. We found a negative correlation between nutritional status (measured by BMI/Age and serum retinol) and anti-Leishmania antibodies and acute phase proteins. There was no correlation between nutritional status and parasite load. ATRA presented a dual effect in Treg cells and monocytes: In healthy children (DTH-/Ac-), it induced a regulatory response, increasing IL-10 and TGF-β production; in VL children it modulated the immune response, preventing increased IL-10 production after SLA stimulation. Furthermore, we found a positive correlation between BMI/Age and IL-17 production and negative correlation between serum retinol and IL-10 and TGF-β production in T CD4+CD25highFoxp3+ cells after SLA stimulus. Our results show a potential dual role of vitamin A in the immune system: improvement of regulatory profile during homeostasis and down modulation of IL-10 in Treg cells and monocytes during symptomatic VL. Therefore, the use of vitamin A concomitant to VL therapy might improve recovery from disease status in Leishmania infantum infection / O estado nutricional ? importante determinante da resposta ? infec??o por Leishmania. No entanto, s?o poucos os trabalhos que caracterizem as bases moleculares das associa??es encontradas entre a desnutri??o e a doen?a. A suplementa??o de vitamina A ? utilizada em pa?ses em desenvolvimento para reduzir a mortalidade por diarreia e doen?as respirat?rias. Apesar disso, n?o existem estudos sobre o papel da vitamina A na infec??o por Leishmania apesar de nosso grupo e outros terem demonstrando a defici?ncia de vitamina A durante a leishmaniose visceral (LV). As c?lulas T regulat?rias s?o consideradas c?lulas supressoras durante a LV e s?o induzidas por metab?litos de vitamina A. Este trabalho teve como objetivo verificar a correla??o do estado nutricional e o efeito da vitamina A na resposta frente ? infec??o por Leishmania infantum. Foram estudadas 179 crian?as, sendo 31 casos de LV ativa, 33 com hist?ria pregressa de LV, 44 DTH+ e 71 DTH- e Anticorpo anti-Leishmania negativo (DTH-/Ac-). C?lulas mononucleadas de sangue perif?rico foram isoladas em um subgrupo de 10 crian?as com LV e 16 DTH-/Ac-, sendo cultivadas por 20h sob as seguintes condi??es: 1) Meio, 2) Ant?genos sol?veis de promastigotas de L. infantum (SLA), 3) ?cido all-trans retin?ico (ATRA), 4) SLA + ATRA e 5) Concanavalina A. As c?lulas T CD4+CD25highFoxp3+, T CD4+CD25-Foxp3- e mon?citos CD14+ foram marcadas e estudadas por citometria de fluxo quanto ? produ??o de IL-10, TGF-β e IL-17. O estado nutricional apresentou-se comprometido nas crian?as com LV, que apresentaram menor IMC/idade e baixas concentra??es de retinol s?rico quando comparadas aos controles sadios. Observou-se correla??o negativa entre o estado nutricional (medido por ?ndice de Massa Corporal/Idade e retinol s?rico) e anticorpos anti-Leishmania e prote?nas de fase aguda. N?o foi encontrada correla??o entre o estado nutricional e a carga parasit?ria. O ATRA apresentou efeito distinto nas c?lulas Treg e mon?citos: Em crian?as saud?veis (DTH-/Ac-), induziu resposta regulat?ria, com aumento na produ??o de IL-10 e TGF-β; e, em crian?as com LV, modulou a resposta imune, diminuindo a produ??o de IL-10 ap?s o est?mulo com SLA. Foi encontrada correla??o positiva entre o IMC/Idade e a produ??o de IL-17 e correla??o negativa entre o retinol s?rico e a produ??o de IL-10 e TGF-β nas c?luas T CD4+CD25highFoxp3+ ap?s est?mulo com SLA. Os dados deste estudo permitem concluir que o estado nutricional comprometido durante a LV ? correlacionado com a resposta imune e inflamat?ria frente ? Leishmania. Al?m disso, possivelmente, a vitamina A apresenta duplo efeito na resposta imune: em crian?as sadias, promove resposta regulat?ria; durante a LV, reduz a produ??o de IL-10 em c?lulas Treg e mon?citos. Dessa forma, o uso de vitamina A durante a LV pode promover a recupera??o de pacientes em tratamento para a doen?a

Células pluripotentes são aquelas que possuem a capacidade de dar origem às células dos três folhetos embrionários (ectoderma, mesoderma e endoderma), bem como também às células germinativas. As células-tronco embrionárias (CTE) são as células pluripotentes mais conhecidas, as quais apresentam uma elevada capacidade de diferenciação celular e autorenovação. Estas propriedades tornam as CTE potenciais ferramentas para a medicina regenerativa, porém seu uso na prática clínica enfrenta várias barreiras. Neste sentido, o acúmulo de conhecimento a respeito dos mecanismos envolvidos na manutenção da pluripotência, levou ao desenvolvimento de técnicas capazes de induzir a pluripotência em células somáticas adultas. Na maioria das abordagens, isto se dá pela expressão ectópica de fatores de transcrição envolvidos na pluripotência (como Oct4 e Nanog). Com isto em vista, torna-se evidente que estudos que levem a um melhor entendimento destas propriedades biológicas, podem levar ao desenvolvimento desta importante área. Apesar destas inovações, os mecanismos responsáveis pela manutenção ou indução da pluripotência e da autorenovação, continuam largamente inexplorados. Neste sentido, o conjunto de técnicas referidas como High Content Screening (HCS) apresenta características fundamentais que permitiriam a interrogação sistemática e em larga-escala de fatores que possam estar influenciando nestes processos. A técnica de HCS se baseia no uso de microscopia de fluorescência em placas de 96 ou mais poços, permitindo a aquisição e a análise automatizada das imagens, de forma a quantificar alterações fenotípicas nas células. O presente trabalho teve como objetivo estabelecer um modelo experimental para a avaliação funcional e em larga escala de fatores que possam influenciar a diferenciação celular. Tendo em vista a facilidade de cultivo e manuseio, a linhagem humana de células pluripotentes de carcinoma embrionário (CCE) NTera-2, foi utilizada. Para a padronização do modelo, o processo de diferenciação foi avaliado ao longo do tempo (em 2, 4 e 8 dias) na presença ou ausência de ácido transretinóico (atRA), utilizado como indutor de diferenciação celular. Para isso, os níveis transcricionais de Oct4, Nanog (marcadores da pluripotência) e de N-Caderina foram avaliados por PCR em tempo real. Finalmente, a expressão e a distribuição celular de Oct4, Nanog e da alfa-actina foi avaliada por meio de microscopia de fluorescência automatizada, com o uso de anticorpos ou faloidina marcada, utilizando um sistema de HCS (Operetta, Perkin Elmer) para a análise dos resultados. A proliferação celular das células submetidas à diferenciação foi avaliada pelo ensaio do XTT. O atRA inibiu a proliferação e induziu a diferenciação; como demonstrado, respectivamente, pelos resultados do ensaio do XTT, decaimento dos níveis de Oct4 e Nanog e, concomitante aumento de N-Caderina, ao longo do tempo. Também foi observada a diferenciação espontânea da linhagem, na ausência de atRA, porém, de forma reduzida. Finalmente, as avaliações de HCS evidenciaram que, durante o processo de diferenciação, a perda da expressão nuclear de Oct4 e Nanog está associada à alteração do fenótipo celular, com a redistribuição da actina cortical e a formação das stress fibers, caracterizando o processo de transição epitélio-mesenquima (EMT), um importante mecanismo envolvido na diferenciação celular. Os resultados obtidos neste trabalho demonstram a viabilidade do uso da linhagem NTera-2 como modelo para estudos futuros de HCS visando a identificação de moléculas que atuem na modulação de propriedades fundamentais das células tronco pluripotentes. / Pluripotent stem cells are those that possess the ability to generate cells from the three germ layers (ectoderm, mesoderm and endoderm), as well as the germ cells. The embryonic stem cells (ESC) are the best known pluripotent cells that present a high capacity of cell differentiation and self renewal. These properties of the ESC make them potential tools for the regenerative medicine, but their use in clinical practice faces several barriers. In this sense, the accumulation of knowledge about the mechanisms involved in the maintenance of pluripotency led to the development of techniques capable of inducing pluripotency in adult somatic cells. In most approaches, this is achieved by the ectopic expression of transcription factors involved in pluripotency (such as Oct4 and Nanog). With this in mind, it becomes clear that studies that provide a better understanding of these biological properties can lead to the development of this important area. Despite these innovations, the mechanisms responsible for the maintenance or induction of pluripotency and self-renewal remain largely unexplored. In this sense, the set of techniques such as High Content Screening (HCS) has fundamental characteristics that allow systematic and large-scale interrogation of factors that may be influencing these processes. The HCS technique is based on the use of fluorescence microscopy in 96-well or larger plates, allowing the automated acquisition and analysis of images, so as to measure phenotypic changes in the cells. This study aimed to establish an experimental model for functional and large-scale assessment of factors that may influence cellular differentiation. Due its simple cultivation and handling characteristics, a human lineage of pluripotent embryonal carcinoma cell (ECC) NTERA-2 was used. To standardize the model, the process of differentiation was evaluated over time (at 2, 4 and 8 days) in the presence or absence of all-trans retinoic acid (atRA), used as an inducer of cellular differentiation. The transcriptional levels of Oct4, Nanog (pluripotency markers) and Ncadherin were assessed by real time PCR. Finally, the expression and cellular distribution of Oct4, Nanog and alpha-actin was assessed by fluorescence microscopy, using antibodies or labelled phalloidin, using a HCS platform (Operetta, Perkin Elmer) for the analysis of the results. The proliferation of cells undergoing differentiation was assessed by XTT assay. atRA inhibited proliferation and induced differentiation, as shown by the XTT assay results, and the decay of Oct4 and Nanog, and concomitant increase of N-cadherin levels over time, respectively. It was also observed spontaneous differentiation in the absence of atRA although in less extent. Finally, the HCS results showed that during the differentiation process, the loss of nuclear expression of Oct4 and Nanog is associated with alteration of cell phenotype, with redistribution of cortical actin and formation of stress fibers, characterizing the epithelialmesenchymal transition (EMT), an important mechanism involved in cell differentiation. The results of this study therefore demonstrate the feasibility of using the NTERA-2 cell line as a model for future HCS studies aiming identification of molecules that act in the modulation of fundamental properties of pluripotent stem cells.

Vitamin A metabolites and retinoids may slow the progression of breast cancer and elicit anti-neoplastic properties similar to those of omega-3 fatty acids. Studies using animal models show a decrease in the incidence, growth and metastisis of mammary tumors in the presence of specific fatty acids. This effect is also seen with use of retinoids, specifically all-trans retinoic acid (AtRA). Thus, fatty acids may also alter retinoid homeostasis in mammary carcinoma cells (MCF-7s). The potential for inter/co dependency among fatty acids and retinoids is considerable, and here it has been hypothesized that a decrease in cancer progression will occur in the presence of both compounds. MCF-7’s were seeded in a 48 well plate at 5,000 cells per well. After 24 hr, cells were treated with either 1 µM AtRA alone, fatty acids alone, or AtRA + fatty acids. Fatty acid treatments (Linoleic, and Linolenic) were administered at 2.5 uM concentrations. Each fatty acid treatment was also combined with 1 µM AtRA to determine if there is a synergistic effect on slowing cell growth. Both culture media and treatments were changed at 24 hour intervals over a 3 day trial. When compared to the controls, cells treated with 1 µM AtRA or 2.5 µM Linolenic acid both inhibited cell growth. Interestingly, when combined with Linolenic acid, AtRA treatment resulted in a significant (nearly 50%) additional growth inhibition when compared to treatment with AtRA alone. Our results suggest that AtRA and Linolenic acid have a inter/co dependency that significantly inhibits breast cancer cell growth in vitro by 73.4 % compared to control, and 49.7% compared to AtRA alone over 72 hours. We conclude that AtRA and linolenic acid have a combined effect in breast cancer cell proliferation in-vitro and their role in dietary prevention warrants further investigation.

Vitamin A and its metabolites retinal and retinoic acid are important molecules for the regulation of normal cellular growth, differentiation and other important functions. Retinoids are known to exert mutagenic as well as antimutagenic activity, although conflicting reports are known. All-trans retinoic acid (ATRA) is used in the treatment of many diseases such as acne, psoriasis and ichthyosis. It is also used in differentiated therapy of acute promyelocytic leukemia; however, it is frequently observed that relapses occur when ATRA is prescribed as maintenance therapy. Therefore, understanding the mechanism of action of ATRA in cells would be helpful in the development of high potent and low toxic chemotherapeutic agents. EA-4 is a newly synthesized steroidal analogue of ATRA and is considered as a promising agent for the inhibition of human leukemic cell growth. The study of genotoxicity is an important parameter for the design and development of new chemotherapeutic agents. Genotoxic effects of anticancer drugs in non-tumour cells are of special significance due to their possibility of inducing secondary tumours in cancer patients. Therefore, it is important to determine the genotoxic potential of a drug that will be used in chemotherapy, particularly in native human cells. Taking into consideration the above referred, it would be of interest to evaluate the genotoxic potential of EA-4 in comparison to ATRA, as to their ability to provoke micronucleus (MN) generation, due to both chromosome breakage and chromosome delay. Micronuclei originate from chromosome fragments or whole chromosomes, which lag behind at anaphase during nuclear division. According to our knowledge, there is no information on the ability of all-trans retinoic acid (ATRA) to induce micronucleus formation. To investigate the ability of ATRA and its steroidal analogue EA-4 to enhance micronucleation on human lymphocytes cultured in vitro, the Cytokinesis Block MicroNucleus (CBMN) assay was conducted. By this assay, the cytotoxic effect of the two retinoids was also estimated. To clarify the mechanism by which micronuclei are generated due to ATRA and EA-4 treatment, CBMN was combined with Fluorescence In Situ Hybridization (FISH) using an α-satellite pancentromeric probe to detect centromere inclusion and thus intact chromosome(s) in micronuclei or acentric chromosome fragments. ATRA and EA-4 were shown to be cytotoxic by decreasing CBPI (Cytokinesis Block Proliferation Index) to statistically significant levels in relation to untreated cells. A statistically significant increase in micronucleus frequency was also observed for both investigated compounds. ATRA generated micronuclei mainly via chromosome breakage while a mild effect on chromosome delay was also apparent. On the other hand, EA-4 generated micronuclei exclusively via chromosome breakage. To verify ATRA and EA-4 genotoxicity, micronucleation was investigated in a second biological system coming from a different organism, C2C12 mouse cells. Micronucleus analysis was achieved by α-tubulin/CREST immunostaining for the visualization of microtubules and the detection of kinetochore inside micronuclei and hence the inclusion of whole chromosome(s) or acentric chromosome fragments. Additionally the effect of ATRA and EA-4 on cell proliferation was investigated by the estimation of Mitotic Index (M.I.). We found that ATRA and EA-4 exerted cytotoxic activity in C2C12 mouse cells by reducing the cell proliferation rate at significant levels, as evaluated by the decrease of M.I. A statistically significant elevation in the frequency of interphase cells with micronuclei was shown. CREST analysis confirmed the clastogenic activity of the studied retinoids that was indicated in human lymphocytes. Micronucleation due to ATRA was mediated mainly by chromosome breakage and in a lesser extent by chromosome delay. EA-4 was shown to induce chromosome breakage as well as chromosome delay, as opposed to human lymphocytes at which only clastogenic effect was shown. These observations suggest that, ATRA and EA-4 are able to provoke chromosome fragmentation, but additionally and in a lesser extent to disturb chromosome segregation at anaphase due to chromosome lagging. Cell cycle analysis showed that ATRA and EA-4 accumulated cells at ana-telophase. The analysis of ana-telophases revealed micronucleation, nucleoplasmic bridges and multinucleation, phenomena that may explain the dual genetic activity of ATRA and EA-4. Multinucleated and multimicronucleated interphase cells were also apparent, the second ones generated due to both chromosome delay and breakage. To further investigate the mechanism of genotoxic activity of ATRA and EA-4 we proceeded our research on two axes based on their aneugenicity and clastogenicity. Thus we studied the effect of ATRA and EA-4: i) on the integrity of mitotic spindle, as a target of aneugens by using double immunofluorescence staining of β- and γ-tubulin in C2C12 mouse cell line, which is a convenient system to apply this experimental procedure, and ii) to investigate the ability of the studied retinoids to induce double-strand breaks on DNA by using neutral Single Cell Gel Electrophoresis (SCGE assay-Comet assay) in two different cell lines, C2C12 mouse cells and HL-60 human leukemic cells. Analysis of mitotic spindle has shown that the studied retinoids affect chromosome orientation during metaphase by inducing bipolar metaphases with non-congressed genetic material due to abnormal microtubule network. In addition defects on centrosome duplication and/or separation were observed due to the presence of monopolar metaphases. Ana-telophases as well as interphases with supernumerary centrosomes were also apparent. Additionally, interphase cells with abnormal microtubule network were observed. The above findings may explain aneugenic as well as clastogenic activity of the studied retinoids. Comet assay revealed that ATRA and its steroidal analogue EA-4 provoke DNA migration due to double strand DNA fragmentation in both C2C12 mouse cells and HL-60 human leukemic cells. EA-4 was shown to be the stronger inducer of DNA fragmentation. These results confirm the findings from FISH and CREST analysis indicating that the studied retinoids show high clastogenic activity. . Taking into account the above, we may say that our findings clarify the cytotoxic and genotoxic activity of retinoic acid and the mechanism of its action by indicating its ability to induce chromosome breakage via double-strand DNA breaks and secondary its ability to provoke chromosome delay due to defects in microtubule network and mitotic spindle integrity. / Η βιταμίνη Α και οι μεταβολίτες της, ρετινόλη και ρετινοϊκό οξύ είναι ισχυροί παράγοντες για τη ρύθμιση σημαντικών λειτουργιών, όπως της κυτταρικής ανάπτυξης, διαφοροποίησης και άλλων. Τα ρετινοειδή είναι γνωστά για την μεταλλαξιγόνο αλλά και αντιμεταλλαξιγόνο δράση τους, αν και έχουν αναφερθεί αντικρουόμενα ευρήματα. Το all-trans ρετινοϊκό οξύ (ATRA) χρησιμοποιείται στη θεραπεία πολλών ασθενειών, όπως η ακμή, ψωρίαση, ιχθύωση, αλλά και στη θεραπεία κακοηθειών όπως η μυελογενής λευχαιμία. Συχνά σε περιπτώσεις όπου το ATRA αποτελεί τη βασική θεραπεία παρατηρούνται υποτροπιάσεις Έτσι, η κατανόηση του μηχανισμού δράσης του ATRA στα κύτταρα θα αποτελέσει χρήσιμο εργαλείο για την ανάπτυξη νέων, ισχυρών και μη-τοξικών θεραπευτικών παραγόντων προερχόμενων από αυτό. Το EA-4 είναι ένα πρόσφατα συντεθέν στεροειδικό ανάλογο του ATRA, που θεωρείται υποσχόμενος παράγοντας για την αναστολή της ανάπτυξης ανθρώπινων λευχαιμικών κυττάρων. Η μελέτη της γονιδιοτοξικότητας αποτελεί σημαντική παράμετρο για το σχεδιασμό και την ανάπτυξη νέων θεραπευτικών παραγόντων. Οι γονιδιοτοξικές επιπτώσεις αντικαρκινικών φαρμάκων σε μη-καρκινικά κύτταρα είναι ιδιαίτερης σημασίας, και αποτελούν πιθανή αιτία εμφάνισης δευτερογενών όγκων σε ασθενείς. Έτσι, είναι σημαντικό να μελετηθεί η γονιδιοτοξική δράση ενός φαρμάκου που θα χρησιμοποιηθεί στη χημειοθεραπεία. Λαμβάνοντας υπόψη όλα τα παραπάνω, θεωρήθηκε ενδιαφέρον να εκτιμηθεί η γονιδιοτοξικότητα του EA-4 σε σύγκριση με το ATRA ως προς την ικανότητά τους να προκαλούν την εμφάνιση μικροπυρήνων (MN) είτε μέσω της χρωμοσωματικής θραύσης είτε μέσω της χρωμοσωματικής καθυστέρησης. Οι μικροπυρήνες προέρχονται από χρωμοσωματικά θραύσματα ή ολόκληρα χρωμοσώματα, τα οποία καθυστερούν κατά την ανάφαση της μείωσης ή της μίτωσης. Σύμφωνα με όσα μέχρι σήμερα γνωρίζουμε, δεν φαίνεται να υπάρχουν στοιχεία που αφορούν την ικανότητα του all-trans ρετινοϊκού οξέος (ATRA) να επάγει το σχηματισμό μικροπυρήνων. Για τη διερεύνηση της ικανότητας του ATRA και του στεροειδικού αναλόγου του EA-4 να επάγει την εμφάνιση μικροπυρήνων, πραγματοποιήθηκε η μέθοδος αναστολής της κυτταροκίνησης (CBMN assay) σε ανθρώπινα λεμφοκύτταρα in vitro. Με την ίδια μέθοδο εκτιμήθηκε και η κυτταροτοξικότητα των δύο ρετινοειδών. Για την διευκρίνιση του μηχανισμού δημιουργίας των μικροπυρήνων από τη δράση των ATRA και EA-4, η μέθοδος CBMN συνδυάστηκε με την in situ υβριδιποίηση με φθοροχρώματα (FISH) και χρήση α-δορυφορικού (α-satellite) πανκεντρομερικού ανιχνευτή για την επισήμανση του κεντρομέρους και την ανίχνευσή του σε μικροπυρήνες. Η παρουσία σήματος υβριδοποίησης στους μικροπυρήνες υποδηλώνει την ύπαρξη άθικτου χρωμοσώματος στο εσωτερικό τους. Το αντίθετο υποδεικνύει την παρουσία άκεντρου χρωμοσωματικού θραύσματος. Τα αποτελέσματα έδειξαν ότι και οι δύο χημικές ενώσεις προκαλούν στατιστικά σημαντική αύξηση της συχνότητας των μικροπυρήνων Το ATRA οδηγεί στην δημιουργία μικροπυρήνων κυρίως μέσω χρωμοσωματικής θραύσης, και σε ηπιότερο βαθμό μέσω χρωμοσωματικής καθυστέρησης. Αντίθετα, το EA-4 επάγει το σχηματισμό μικροπυρήνων αποκλειστικά μέσω χρωμοσωματικής θραύσης. Επίσης το ATRA και το EA-4 παρουσάζουν ισχυρή κυτταροτοξικότητα, όπως φάνηκε από τη στατιστικά σημαντική μείωση του κυτταρικού δείκτη πολλαπλασιασμού (CBPI), σε σύγκριση με τις καλλιέργειες του μάρτυρα. Προκειμένου να επιβεβαιωθεί η γονιδιοτοξικότητα του ATRA και του EA-4, διερευνήθηκε η ικανότητά τους να προκαλούν αυξημένες συχνότητες μικροπυρήνων σε ένα δεύτερο βιολογικό σύστημα, την κυτταρική σειρά ποντικού C2C12. Η ανάλυση των MN πραγματοποιήθηκε με τη μέθοδο διπλού ανοσοφθορισμού α-τουμπουλίνης/CREST, για την ανίχνευση σήματος κινητοχώρου στο εσωτερικό του μικροπυρήνα κι έτσι την παρουσία ολόκληρου χρωμοσώματος. Επίσης,η κυτταροτοξικότητα τους διερευνήθηκε με την εκτίμηση του μιτωτικού δείκτη. Με τη ίδια μέθοδο αναλύθηκε η πρόοδος του κυτταρικού κύκλου. Παρατηρήθηκε ότι το ATRA και το EA-4 παρουσιάζουν κυτταροτοξική δράση στα κύτταρα C2C12 μειώνοντας το ρυθμό κυτταρικού πολλαπλασιασμού σε στατιστικά σημαντικά επίπεδα. Επιπλέον αποκαλύφθηκε στατιστικά σημαντική αύξηση της συχνότητας κυττάρων με μικροπυρήνες. Η επισήμανση του κινητοχώρου επιβεβαίωσε τη θραυσματογόνο δράση των υπό μελέτη ρετινοειδών που παρατηρήθηκε στα ανθρώπινα λεμφοκύτταρα. Η δημιουργία μικροπυρήνων μέσω του ATRA ήταν αποτέλεσμα κυρίως χρωμοσωματικής θραύσης και σε μικρότερη έκταση χρωμοσωματικής καθυστέρησης, σε συμφωνία με τα ευρήματα από τα πειράματα στις καλλιέργειες ανθρώπινων λεμφοκυττάρων. Αντίθετα, παρατηρήθηκε ότι το EA-4, πλην της ισχυρής θραυσματογόνου δράσης, προκαλεί και χρωμοσωματική καθυστέρηση. Οι παρατηρήσεις αυτές υποδεικνύουν ότι το ATRA και το EA-4 είναι ισχυροί θραυσματογόνοι παράγοντες, αλλά σε μικρότερο βαθμό είναι ικανοί να διαταράξουν και τον χρωμοσωματικό αποχωρισμό κατά την πυρηνική διαίρεση. Η μελέτη του κυτταρικού κύκλου έδειξε ότι τόσο το ATRA και όσο και το EA-4 προκαλούν καθυστέρηση συσσωρεύοντας τα κύτταρα στα στάδια ανάφασης και τελόφασης της πυρηνικής διαίρεσης. Κύτταρα που συσσωρεύονται στα παραπάνω στάδια χαρακτηρίζονται από την εμφάνιση πυρηνοπλασματικών γεφυρών, την παρουσία περισσότερων του ενός πυρήνων, αλλά και την παρουσία μικροπυρήνων, φαινόμενα τα οποία είναι σύμφωνα με τη διττή γενετική δράση των ATRA και EA-4. Επίσης, παρατηρήθηκαν πολυπύρηνα μεσοφασικά κύτταρα και μεσοφασικά κύτταρα με πολλαπλούς μικροπυρήνες, με τον δεύτερο τύπο κυττάρων να προέρχεται τόσο από χρωμοσωματική θραύση όσο και από χρωμοσωματική καθυστέρηση. Έτσι, φαίνεται ότι τα δύο υπό μελέτη ρετινοειδή μπορούν να χαρακτηρισθούν μόρια με θραυσματογόνες αλλά και ανευπλοειδογόνες ιδιότητες. Για τη λεπτομερέστερη ανάλυση του μηχανισμού δράσης του ATRA και του EA-4 σχεδιάσθηκαν πειράματα σε δύο βασικούς άξονες που αφορούσαν την περαιτέρω μελέτη τόσο της ανευπλοειδογόνου όσο και της θραυσματογόνου δράσης τους. Έτσι, μελετήθηκε η επίδραση του ATRA και του EA-4 αντίστοιχα ως προς: α) την ακεραιότητα της μιτωτικής συσκευής, η οποία αποτελεί κυτταρικό στόχο ανευπλοειδογόνων ενώσεων. Η μελέτη πραγματοποιήθηκε στην κυτταρική σειρά C2C12, μέσω της μεθόδου διπλού ανοσοφθορισμού για τη β- και γ-τουμπουλίνη, δομικά στοιχεία των μικροσωληνίσκων και του κεντροσώματος, και β) την δημιουργία δίκλωνων ρηγμάτων στο DNA μέσω της μεθόδου ηλεκτροφόρησης μοναδιαίων κυττάρων (SCGE assay-Comet assay) σε δύο διαφορετικές κυτταρικές σειρές, στα κύτταρα ποντικού C2C12 και στα λευχαιμικά κύτταρα ανθρώπου HL-60. Τα αποτελέσματα μας έδειξαν ότι τα υπό εξέταση ρετινοειδή επηρεάζουν τον χρωμοσωματικό προσανατολισμό κατά τη μετάφαση με την εμφάνιση διπολικών μεταφάσεων με τα χρωμοσώματα μη-διατεταγμένα στο ισημερινό πεδίο, λόγω ανωμαλιών του δικτύου των μικροσωληνίσκων. Επίσης, φάνηκε ότι προκαλούν ανωμαλία στον πολλαπλασιασμό και πιθανόι στον αποχωρισμό των κεντροσωμάτων, παρατήρηση που δικαιολογείται από την παρουσία μονοπολικών μεταφάσεων, καθώς και ανάτελοφάσεων αλλά και μεσοφασικών κύττάρων με υπεράριθμο κεντροσωματικό αριθμό. Επιβεβαιώθηκε επίσης η επίδρασή τους στην πορεία του κυτταρικού κύκλου με συσσώρευση των κυττάρων στα στάδια ανάφασης-τελόφασης. Επιπρόσθετα, φάνηκε ότι το ΕΑ-4, στη μεγαλύτερη συγκέντρωση, διακόπτει τον κυτταρικό κύκλο στο στάδιο της μετάφασης. Παράλληλα, παρατηρήθηκε διαταραχή στη δομή του δικτύου των μικροσωληνίσκων. Όλα τα παραπάνω ευρήματα ερμηνεύουν τόσο την ανευπλοειδογόνο όσο και τη θραυσματογόνο δράση των δύο ρετινοειδών. Με τη μέθοδο ηλεκτροφόρησης μοναδιαίων κυττάρων δείχθηκε ότι το ATRA και το στεροειδικό του ανάλογο EA-4 προκάλεσαν τη δημιουργία «κομητών», δηλαδή πυρήνων με ανώμαλη μορφολογία μέσω του σχηματισμού δίκλωνων θραυσμάτων DNA. Το φαινόμενο αυτό παρατηρήθηκε τόσο στα κύτταρα ποντικού C2C12 όσο και στα λευχαιμικά κύτταρα ανθρώπου HL-60, με το EA-4 να παρουσιάζει ισχυρότερη επαγωγή θραύσης του DNA. Τα αποτελέσματα αυτά επιβεβαιώνουν τα ευρήματα των μεθόδων FISH και CREST, υποδεικνύοντας ότι τα υπό εξέταση ρετινοειδή παρουσιάζουν ισχυρή θραυσματογόνο δράση. Λαμβάνοντας υπόψη όλα τα παραπάνω, μπορούμε να ισχυριστούμε ότι τα ευρήματά μας διευκρινίζουν την κυτταροτοξική και γονιδιοτοξική δράση του ρετινοϊκού οξέος. Υποδεικνύουν ιδιότητες ισχυρώς θραυσματογόνων παραγόντων μέσω δημιουργίας δίκλωνων ρηγμάτων στο DNA των κυττάρων. Δευτερογενώς μπορούν να χαρακτηρισθούν ως ήπιες ανευπλοειδογόνες ενώσεις που προκαλούν ανώμαλο χρωμοσωματικό αποχωρισμό μέσω ανωμαλιών τόσο του δικτύου των μικροσωληνίσκων όσο και της ακεραιότητα της μιτωτικής συσκευής.

Στην παρούσα εργασία πραγματοποιήθηκε γονιδιωματική ανάλυση της επίδρασης αντιψωριασικών φαρμάκων σε καλλιέργειες ανθρώπινων κερατινοκυττάρων με τη χρήση μικροσυστοιχιών DNA. Σε καλλιέργειες κυττάρων HaCaT χορηγήθηκαν τα παράγωγα του ρετινοϊκού οξέος all-trans retinoic acid (ATRA) και acitretin, σε διαβαθμισμένες δόσεις, προκειμένου να διαπιστωθεί η επίδραση τους στα κύτταρα. Μελετήθηκε η βιωσιμότητα των κυττάρων με τις δοκιμασίες της χρωστικής Trypan Blue και ΜΤΤ. Επιλέχθηκαν δύο συγκεντρώσεις (10^-6 και 10^-8 Μ) των φαρμάκων που αντιστοιχούσαν σε βιωσιμότητα κυττάρων περίπου 80%, οι οποίες χορηγήθηκαν εκ νέου σε καλλιέργειες κυττάρων HaCaT. Τα κύτταρα συλλέχθηκαν, έγινε εκχύλιση του RNA και έλεγχος της ποιότητας του με ηλεκτροφόρηση (Bioanalyzer). Το RNA χρησιμοποιήθηκε για την in vitro μεταγραφή cDNA που σημάνθηκε με φθοριοχρώματα και άμεσα ακολούθησε υβριδισμός σε πλακίδιο μικροσυστοιχιών (OneArray) το οποίο περιείχε ανιχνευτές για όλο το ανθρώπινο γονιδίωμα μαζί με τους κατάλληλους μάρτυρες. Σε κάθε πλακίδιο υβριδίστηκαν ταυτόχρονα cDNA από κύτταρα στα οποία είχε χορηγηθεί ρετινοειδές και κύτταρα στα οποία δεν είχε χορηγηθεί. Η επεξεργασία των δεδομένων της σαρώσεως με ειδικό λογισμικό ανέδειξε 700 περίπου γονίδια που ρυθμίζονται θετικά ή αρνητικά σε στατιστικά σημαντικό βαθμό. Για την επαλήθευση των αποτελεσμάτων που προέκυψαν από τις μικροσυστοιχίες, επιλέχθηκαν 34 γονίδια τα οποία συμμετέχουν σε βασικές βιολογικές διεργασίες όπως πρωτεϊνοσύνθεση, κυτταρική σηματοδότηση, πολλαπλασιασμός, κυτταρική διαφοροποίηση, κυτταρικός θάνατος, φλεγμονή. Επιπρόσθετα, επιλέχθηκαν 22 γονίδια τα οποία έχουν επίσης κομβικό ρόλο σε σηματοδοτικά μονοπάτια και κυτταρικές λειτουργίες. Η επιλογή αυτή έγινε για να μελετηθεί πιο σφαιρικά η επίδραση των φαρμάκων σε βασικούς κυτταρικούς μοριακούς μηχανισμούς. Στο σύνολο των επιλεγμένων γονιδίων έγινε ποσοτική Real-Time PCR και για το σκοπό αυτό έγινε σχεδιασμός ειδικών εκκινητών. Η qRT-PCR εν τέλει, επιβεβαίωσε τα αρ-χικά αποτελέσματα από τα microarrays. Διαπιστώθηκε ότι η κυτταρική απόκριση στη χορήγηση των ρετινοειδών, εξειδικευμένα για κάθε δραστική ουσία και για κάθε δόση δεν είναι μονοσήμαντη, αλλά ότι ταυτόχρονα επάγονται λειτουργικά μονοπάτια με διαφορετικούς ρόλους. Επίσης διαπιστώθηκε ότι η μεταβολή κατά δύο τάξεις μεγέθους της δόσης που προσλαμβάνουν τα κύτταρα επάγει αντίρροπες κυτταρικές αποκρίσεις. Συγκεκριμένα, η ολιστική προσέγγιση της μεταβολής της γονιδιακής έκφρασης ανέδειξε ότι η χορήγηση ATRA σε συγκέντρωση 10^-6Μ στις κυτταροκαλλιέργειες ευνοεί την πρωτεϊνοσύνθεση και την διαφοροποίηση των κυττάρων ενώ ασκεί αντιφλεγμονώδη δράση. Η χορήγηση της δραστικής ουσίας ATRA στη δόση 10^-8Μ ευνοεί τη διαφοροποίηση των κυττάρων HaCaT σε μεγαλύτερο βαθμό από τον πολλαπλασιασμό τους. Επιπλέον, φαίνεται ότι σε αντίθεση με τη μεγαλύτερη δόση, ευνοείται η σύνθεση μορίων που επάγουν την φλεγμονή. Παρόμοια, η ασιτρετίνη στη δόση 10^-6Μ ευνοεί τη διαφοροποίηση των κυττάρων και την σύνθεση μορίων που επάγουν τη φλεγμονή. Η ασιτρετίνη στην μικρότερη δόση (10^-8 Μ) ευνοεί κύρια την διαφοροποίηση, λιγότερο τον πολλαπλασιασμό των κυττάρων και φαίνεται ότι προάγει σε σημαντικό βαθμό την απόπτωση. Οι μεγαλύτερες δόσεις των ρετινοειδών που μελετήθηκαν φαίνεται ότι είναι απαγορευτικές για τον πολλαπλασιασμό των κυττάρων σε αντίθεση με τις μικρότερες δόσεις. Επισημαίνεται ότι για τη θεραπεία της ψωρίασης όπου χρησιμοποιούνται, επιθυμητές δράσεις είναι η παραγωγή μορίων με αντιφλεγμονώδη δράση, ο περιορισμός του αυξημένου κυτταρικού πολλαπλασιασμού, αύξηση της κυτταρικής απόπτωσης και τέλος πολύ ση-μαντικό είναι η επιτυχής περάτωση της διαφοροποίησης των κυττάρων. Συμπερασματικά, η χρήση τεχνικών υψηλής απόδοσης, κύρια των μικροσυστοιχιών cDNA που επιτρέπουν την εκτεταμένη μελέτη του γονιδιώματος και της qRT-PCR για πιο στοχευμένη μελέτη, μπορούν να διαδραματίσουν σημαντικό ρόλο στην εξακρίβωση μοριακών μηχανισμών. / In the current project we performed gene expression profiling, using cDNA microarrays, when specific doses of derivatives of retinoic acid were applied in HaCaT cell culture. These specific drugs are used in the treatment of psoriasis but their exact effect in molecular level remains elusive. All-trans retinoic acid and acitretin were applied in gradient doses. Cell viability was monitored using MTT and Trypan blue assays. Two specific doses (10^-6 & 10^-8 M), in which cell viability was approximately 80%, were chosen for the treatment of HaCaT cells. Subsequently, the treated cells were collected and RNA was extracted using standard methods. At a next step, RNA quality was examined by electrophoresis (Bioanalyzer) and spectrometry. High quality RNA showing no traces of degradation was used as template for in vitro transcription. Finally, synthesis of fluoro-labeled cDNA was performed from RNA derived from both treated and untreated samples and was immediately hybridized to DNA microarray slides (OneArray). Analysis of the hybridization data was performed using specific software. As a result, 700 genes (both up-regulated and down-regulated) were chosen for further analysis. Among them, 34 genes were chosen to validate the microarrays results by the use of quantitative Real-Time PCR. These genes appeared to play crucial role in basic cellular functions like protein synthesis, signal transduction, cell death, cell differentiation and proliferation. Furthermore, 22 additional essential genes that are related to the above processes were chosen in aim to examine drugs effects. The data processing revealed that the 10^-6 M dose of ATRA has a positive effect in protein synthesis, cell differentiation and anti-inflammatory action. Moreover ATRA at a concentration of 10^-8 M promotes differentiation more than proliferation and it has inflammatory effect as well as acitretin has in 10^-6 M dose. On the other, hand acitretin in 10^-8 M dose facilitates differentiation more than proliferation but mainly induces cell death. Generally, high doses (10-6 M) of the drugs inhibit cell proliferation more efficient than low doses (10-8 M). In fact, during psoriasis treatment, the anti-inflammatory action, inhibition of cell proliferation, induction of cell differentiation and cell death are considered desirable drug effects. Our study shows that cDNA microarray analysis represents a powerful tool that can be used for extended genomic studies and the results that are obtained can be validated and used for the elucidation of several molecular mechanisms.

Metabolic syndrome is a prevalent disease characterized by concurrent manifestation of insulin resistance, obesity, dyslipidemia, hypertension and other hemodynamic and metabolic disorders. It has multifactorial type of inheritance and its resultant phenotype is determined by both environmental and genetic factors as well as their interactions. That is the main reason why comprehensive analysis of the genetic component of this syndrome is complicated in human population. Genetically designed experimental animal models are significant tools for analysis of genetic architecture of human complex conditions including the metabolic syndrome. The aim of this Thesis is utilization of functional and comparative genomic tools to uncover pathogenesis of metabolic syndrome aspects and their genetic determinants. We also studied pharmacogenetic interactions of these genetic determinants with drugs affecting particular components of the metabolic syndrome. Establishing and utilizing several genetically designed congenic rat strains, we undertook four different research projects focusing on pharmacogenetic interaction of all-trans retinoic acid and ondansetron with differential segment of rat chromosome 8, pharmacogenetic interaction of differential segment of rat chromosome 4 and dexamethasone, determining Plzf...

Metabolic syndrome is a prevalent disease characterized by concurrent manifestation of insulin resistance, obesity, dyslipidemia, hypertension and other hemodynamic and metabolic disorders. It has multifactorial type of inheritance and its resultant phenotype is determined by both environmental and genetic factors as well as their interactions. That is the main reason why comprehensive analysis of the genetic component of this syndrome is complicated in human population. Genetically designed experimental animal models are significant tools for analysis of genetic architecture of human complex conditions including the metabolic syndrome. The aim of this Thesis is utilization of functional and comparative genomic tools to uncover pathogenesis of metabolic syndrome aspects and their genetic determinants. We also studied pharmacogenetic interactions of these genetic determinants with drugs affecting particular components of the metabolic syndrome. Establishing and utilizing several genetically designed congenic rat strains, we undertook four different research projects focusing on pharmacogenetic interaction of all-trans retinoic acid and ondansetron with differential segment of rat chromosome 8, pharmacogenetic interaction of differential segment of rat chromosome 4 and dexamethasone, determining Plzf...