Darbo tikslas: sumodeliuoti apsauginio poveikio rozmarinų puskietes vaisto formas ir įvertinti jų kokybę. Darbo uždaviniai: Išnagrinėti mokslinėje literatūroje kvapniojo rozmarino (Rosmarinus officinalis L.) biologinį poveikį ir pritaikymo galimybes puskiečių preparatų gamyboje; parinkti rozmarinų ištraukų, kaip veikliosios medžiagos, sudėtį ir įvertinti jų kokybę bei ištirti jų biologinį aktyvumą tyrimais in vitro; sumodeluoti puskietes vaisto formas su rozmarinų ištraukomis ir ištirti jų kokybę; nustatyti pagrindo įtaką fenolinių junginių atpalaidavimui iš sumodeliuotų puskiečių vaisto formų su rozmarinų ištraukomis. Metodai: Rozmarinų ištraukų kokybė įvertinta nustatant spektrofotometriniu metodu fenolinių junginių kiekį ir antioksidacinį aktyvumą DPPH radikalų surišimo metodu. Antimikrobinis aktyvumas vertintas pagal Ph Eur. 01/2002, 2.6.12. Rozmarinų puskiečių preparatų kokybė įvertinta nustatant vienalytiškumą, pH reikšmę, antimikrobinį aktyvumą, fenolinių junginių atpalaidavimą iš puskiečių sistemų tyrimu in vitro. Rezultatai: Didžiausias fenolinių junginių kiekis (14,139±0,417mg/ml) nustatytas rozmarinų ištraukose 1:1, kai ekstrahentu naudotas 40 proc. (V/V) etanolis, o mažiausias (2,296±0,268mg/ml) – naudojant 96 proc. (V/V) etanolį. Tyrimų metu nustatyta, kad stipresnis antioksidacinis (EC50=9,63±0,34µg/ml) ir antimikrobinis aktyvumas būdingas rozmarinų ištraukoms 1:1, kai ekstrahentu naudojamas 40 proc. (V/V) etanolis. Rozmarinų puskiečių preparatų pH reikšmė -... [toliau žr. visą tekstą] / Work aim: to model protective effect rosemary semi-hard drug forms and evaluate their quality. Work objectives: To analyze biological effect of sweet rosemary (Rosmarinus officinalis L.) and application opportunities in the production of semi-hard preparations provided in scientific literature; to select the composition of rosemary extracts, as the active substance, and to evaluate their quality as well as analyze their biological activity by in vitro tests; to model semi-hard drug forms with rosemary extracts and to analyze their quality; to determine the influence of the basis on the release of phenolic compounds from the modelled semi-hard drug forms with rosemary extracts. Methods: The quality of rosemary extracts was evaluated spectrophonometrically determining the amount of phenolic compounds and antioxidant activity by DPPH radical bonding method. Antimicrobial activity was evaluated according to Ph Eur. 01/2002, 2.6.12. The quality of rosemary semi-hard preparations was evaluated by determining homogeneity, pH value, antimicrobial activity, release of phenolic compounds from semi-hard systems through in vitro test. Results: The largest amount of phenolic compounds (14,139±0,417mg/ml) was found in rosemary extracts 1:1, when 40 percent (V/V) ethanol was used as the extractant, and the least amount (2,296±0,268mg/ml) – using 96 percent (V/V) ethanol. During the tests it was found that stronger antioxidant (EC50=9,63±0,34µg/ml) and antimicrobial activity is typical to... [to full text]

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kristína Hudáková Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands I Vaccination against preventable infections prevents millions of deaths each year. Their immunity enhancing activity is strengthened by the presence of vaccine adjuvants. Development of vaccine adjuvants leads to improved safety profile and also can play a vital role in the research of new vaccines against pathogens against which the vaccines currently do not exist. The main aim of this diploma thesis was to verify the ability of rationally developed small molecule ligands to influence Toll-like receptors and thus their potential to be utilized as vaccine adjuvants. The assay was carried out using modified cell lines continually expressing the human TLR4 or TLR8 whose activation leads to production of secreted embryonic alkaline phosphatase. Ten analyzed substances labelled as DM 001 - DM 010 were examined for their agonistic and also antagonistic properties while interacting with the TLRs. Immunomodulatory activity of these tested samples was then determined by quantification of secreted alkaline phosphatase with the help of a colorimetric...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Vanda Machalová Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands II. The aim of this diploma thesis was to test the new substances at Toll-like receptors (TLR), namely TLR4 and TLR8 subtypes, as potential vaccine adjuvants. Adjuvants are required for new subunit vaccines to promote stimulation of a sufficient immune response. Stimulation of TLR receptors is safe and leads to the activation of both innate and adaptive immunity and to subsequent specific immune response. Testing was carried out on cell lines stable co-expressing the respective receptors, with colorimetric activity detection. In this work, both agonist and antagonist activity at TLR4 and TLR8 receptors was investigated and a half maximal effective concentration (EC50) of resiquimod was determined. Based on the results, the respective substances cannot be considered as potential adjuvants to the TLR8 receptor and DM014 exhibits the potential for agonist activity on the TLR4 receptor, whereas the substance DM015 can be used on further investigation of immunosuppression in autoimmune diseases and diseases associated with excessive activation of the...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Linda Tamášiová Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands III Research on new ligands with potential immunomodulatory activity is a tool for the development of new immunological adjuvants for use in vaccines, or as separate immunomodulators in the treatment of various diseases. The aim of this thesis was to analyze the ability of new ligands to stimulate toll-like receptors 4 (TLR4) and to assess the potential for further use of these substances based on established immunomodulatory activity. The analysis was performed on modified TLR4-expressing cell lines whose activation was subsequently detected using a colorimetric-enzymatic reaction. Sixteen new substances were tested for TLR4 receptor activation in comparison with a standard agonist. The results showed a significant TLR4 agonistic activity in several of the test substances, suggesting that they are activating ligands of the receptor tested. However, due to their low solubility, some of these substances are not suitable candidates for further use and testing. Taking all parameters into consideration, among of all of the evaluated substances that...

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hybrid composites with chitosan (CHI), fibroin (SF) and hydroxyapatite (HA) are biocompatible and attractive for bone engineering applications. The objective of this work was to prepare, to characterize and to evaluate in vitro cells behavior in contact with CHI, CHI/SF and CHI/SF/HA scaffolds. The biomaterials were produced from a CHI, CHI/SF, CHI/SF/HA solution in acetic acid and, after the production process, they were submitted the in vitro tests with STRO+1A, MC3T3-E1 and SaOS-2 cells for 07, 14 and 21 days. The scaffolds were characterized by X-ray diffraction, attenuated total reflection Fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy and energy dispersive spectroscopy. In addition, tests of porosity, pore size and contact angle were performed. Cell viability and activity were assessed by MTT reduction and alkaline phosphate activity detection. It was possible to prepare scaffolds 3D based on chitosan, fibroin and hydroxyapatite and the interaction between this materials were observed by physicochemical characterization. The composites were considered non-toxic and they were capable to promote cell adhesion, proliferation and differentiation. Additionally, they may be used in the therapy of bone defect repair. / Compósitos híbridos de quitosana (CHI), fibroína (SF) e hidroxiapatita (HA) são biocompatíveis e atrativos para aplicações na engenharia tecidual óssea. O objetivo desse trabalho foi preparar, caracterizar e avaliar o comportamento in vitro de compósitos 3D de CHI, CHI/SF, CHI/SF/HA. Os biomateriais foram produzidos a partir de uma solução de CHI, CHI/SF e CHI/SF/HA em ácido acético e após a realização dos processos de produção foram submetidos a testes in vitro com células STRO+1A, MC3T3-E1 e SaOS-2 por 07, 14 e 21 dias. Os arcabouços foram caracterizados por difração de raios-X, espectroscopia no infravermelho com transformada de Fourier com refletância total atenuada, análise termogravimétrica, calorimetria exploratória diferencial, microscopia eletrônica de varredura, espectroscopia de energia dispersiva, e submetidos a testes para verificação da porosidade, tamanho de poro e ângulo de contato. A viabilidade e a atividade celular foram avaliadas por redução de MTT e pela detecção da atividade de fosfatase alcalina. Foi possível preparar arcabouço 3D com matriz baseada em quitosana, fibroína e hidroxiapatita e houve interação entre esses materiais, sendo constatado através da caracterização físico-química. Ambos os compósitos foram considerados atóxicos e capazes de promover adesão, proliferação e diferenciação celular, podendo ser utilizados na terapia do reparo de defeito ósseo.

La dermatite de contact allergique (DCA) est une réaction exacerbée du système immunitaire cutané vis-à-vis d’un allergène de contact. La prévalence de la DCA étant de 20 % au sein de la population mondiale, il est important d’identifier les composés allergisants. Différentes réglementations européennes, telles que le règlement REACh ou la directive cosmétique, interdisent l’utilisation de test sur l’animal. C’est dans ce contexte que différentes méthodes alternatives ont été développées pour évaluer la sensibilisation cutanée. La stratégie actuelle d’évaluation du potentiel sensibilisant consiste à réaliser un ensemble de tests alternatifs, chacun mimant un évènement clé du mécanisme : l’hapténisation, l’activation des kératinocytes ou des cellules dendritiques.Cependant, ces tests utilisent principalement des monocultures et ne prennent donc pas en compte les interactions cellulaires qui peuvent avoir lieu in vivo. De plus, les évaluations de la pénétration et du métabolisme cutanés sont négligées dans les tests développés.Afin de mimer la fine orchestration des événements intervenant lors de la sensibilisation cutanée, nous proposons un modèle d’épiderme humain reconstruit (RhE) co-cultivé avec la lignée cellulaire THP-1, servant de substitut aux cellules dendritiques. Nous avons caractérisé, et étudié la pertinence de ce modèle à l’aide de molécules chimiques de référence. Ce travail a permis l’identification de biomarqueurs, tels que CD54, IL-8 et CCL3, spécifiques à l’évaluation in vitro de la sensibilisation cutanée des xénobiotiques. / Allergic contact dermatitis is an exacerbated reaction of skin immune system toward contact allergen. The prevalence of DCA being 20 % among the world population, it is important to identify allergens. Different European regulations such as the REACh regulation or the cosmetic directive prohibit the use of the test on animals. It is in this context that different methods have been developed to evaluate skin sensitization. The current sensitization potential assessment strategy consists of a set of alternative tests, each of which reproduce a key event of the mechanism: the haptenation, the activation of keratinocytes or dendritic cells.However, these tests are mainly based on monocultures and therefore do not account for the cellular crosstalk that happen in vivo. In addition, the evaluations of skin penetration and metabolism are neglected in the developed tests.In order to mimic the fine orchestration of the events involved in skin sensitization, we propose a model of reconstructed human epidermis (RhE) co-cultivated with the THP-1 cell line, as a substitute for dendritic cells. We have characterized and studied the relevance of this model using reference chemical molecules. This work has enabled the identification of biomarkers, such as CD54, IL-8 and CCL3, specific to the in vitro evaluation of skin sensitization to xenobiotics.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T12:17:35Z No. of bitstreams: 2 Dissertação - Lilian Cristina Morais de Andrade - 2013.pdf: 1070622 bytes, checksum: c44e7d6decc4c88ffa0d3bbacd190bc6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-09T12:19:51Z (GMT) No. of bitstreams: 2 Dissertação - Lilian Cristina Morais de Andrade - 2013.pdf: 1070622 bytes, checksum: c44e7d6decc4c88ffa0d3bbacd190bc6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-09T12:19:51Z (GMT). No. of bitstreams: 2 Dissertação - Lilian Cristina Morais de Andrade - 2013.pdf: 1070622 bytes, checksum: c44e7d6decc4c88ffa0d3bbacd190bc6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-09-06 / Taenia crassiceps cysticerci possess antigenic similarities to T. solium cysticerci and because of this and added to the relative easiness in its maintainance in laboratory they are used as an experimental model to T. solium studies. The objective of this study was to analyze the in vitro influence of glucose and insulin on the energetic and respiratory metabolism of T. crassiceps cysticerci exposed to low dosages of anti-helminthic drugs, albendazol and praziquantel, and also to different concentrations of glucose and insulin. Glucose is the main energy source to these parasites in their larval and adult stages and glycogen is their main energetic reserve. Glycogen may be converted into glucose in case of low glucose uptake and is stored in the cysticerci tegument. The cysticerci were collected from the peritoneal cavity of BALB/c female mice experimentally infected and maintained in the animal facilities of IPTSP/UFG. After 24hours of culture in RPMI culture medium supplemented and with glucose, glargine insulin (LANTUS®, Sanofi Aventis), albendazol and praziquantel, the cysticerci were removed and the medium was frozen in liquid nitrogen as to allow the metabolix stasis. Afterwards this culture medium was analyzes through HPLC as to permit the quantification of the following organic acids related to the carbohydrates metabolism: lactate and pyruvate, intermediary metabolism: citrate, α-ketoglutarate, succinate, fumarate, malate and oxaloacetate, fatty acids metabolism: β-hydroxibutyrate and propionate. It was possible to detect propionate and β-hydroxibutyrate secreted by T. crassiceps cysticerci which indicates the fatty acids oxidation as an alternative energy xv source used by the initial stage cysticerci which are in rapid growth. Also the detection of organic acids from the citric acid cycle indicates the in vitro aerobiosis performed by the initial stage cysticerci. / Os cisticercos de Taenia crassiceps possuem similaridades antigênicas com cisticercos de Taenia solium e por este motivo, associado ao fato de uma relativa facilidade de sua manutenção em animais de laboratório, são utilizados como modelo experimental para estudos da cisticercose por T. solium. O objetivo deste trabalho foi analisar in vitro a influência da glicose e da insulina no metabolismo energético e respiratório de cisticercos de T. crassiceps expostos aos fármacos anti-helmínticos albendazol e praziquantel em baixas doses, e diferentes concentrações de glicose e insulina. A glicose é a principal fonte de energia para estes parasitos tanto na fase larval quanto adultos e o glicogênio, que pode ser novamente convertido em glicose, é fonte de reserva energética, sendo encontrado armazenado em suas membranas. Os cisticercos foram provenientes da cavidade peritoneal de camundongos fêmeas Balb/c infectados experimentalmente e mantidos no biotério do IPTSP/UFG. Após 24 horas de cultivo em meio RPMI® suplementado e adicionado de glicose, insulina glargina (LANTUS®, Sanofi Aventis), albendazol e praziquantel, foram retirados da placa e o meio foi congelado em nitrogênio líquido para ocorrer estase metabólica. Posteriormente o meio de cultura passou por um processo de extração sendo analisado em Cromatografia Líquida de Alta Performance com detector UV (CLAE) dos seguintes ácidos orgânicos oriundos do metabolismo de carboidratos (lactato, piruvato), metabolismo intermediário (citrato, α-cetoglutarato succinato, fumarato, malato, oxaloacetato) e metabolismo de ácidos graxos (β-hidroxibutirato e propionato). Foram detectados propionato e β-hidroxibutirato secretados por cisticercos de T. crassiceps indicando a oxidação de ácidos graxos como fonte de energia alternativa, utilizada em especial nos estádios de crescimento e também em situações de baixas concentrações de substancias energéticas provenientes do hospedeiro. Em adição, a detecção de xiv ácidos orgânicos do ciclo do ácido cítrico nos cisticercos analisados confirma a aerobiose in vitro do estádio evolutivos inicial dos cisticercos.

Submitted by Emanuel Varela Cardoso (emanuel.varela@ufcg.edu.br) on 2018-06-28T20:35:34Z No. of bitstreams: 1 JONEUSO TÉRCIO CAVALCANTI DA COSTA – DISSERTAÇÃO (UAEMa) 2015.pdf: 1493605 bytes, checksum: 64e995b963519b1073063638b52caeac (MD5) / Made available in DSpace on 2018-06-28T20:35:34Z (GMT). No. of bitstreams: 1 JONEUSO TÉRCIO CAVALCANTI DA COSTA – DISSERTAÇÃO (UAEMa) 2015.pdf: 1493605 bytes, checksum: 64e995b963519b1073063638b52caeac (MD5) Previous issue date: 2015-08-27 / As radiações não ionizantes (UV-A; UV-B e UV-C) do espectro eletromagnético do sol causam doenças aos seres humanos e precisam ser evitadas. O uso adequado do protetor solar minimiza tal risco. Atualmente, a tendência é introduzir materiais naturais, orgânicos ou inorgânicos, na composição dos protetores solares (fotoprotetores), no afã de neutralizar possíveis efeitos colaterais dos materiais sintéticos. Estudos mostraram que argilas (silicatos em camadas) incorporadas aos protetores solares podem bloquear radiação ultravioleta no comprimento de onda entre 250 a 400 nm dependendo da composição da argila. Acompanhando essa linha norteadora é que se baseou a elaboração deste trabalho onde duas argilas foram empregadas na preparação de fotoprotetores e aferida a eficácia em laboratório da Universidade Federal de Campina Grande (UFCG) através da avaliação in vitro por espectrofotometria. As argilas testadas foram a montmorilonita sódica comercial (Cloisite® Na + ) e a bentonítica Argel 35. As formulações foram preparadas em três farmácias de manipulação, em três cidades diferentes, Guarabira, João Pessoa e Campina Grande, no Estado da Paraíba. O método escolhido para avaliação do Fator de Proteção Solar (FPS) foi o método in vitro desenvolvido por Mansur em virtude da ótima correlação com método in vivo, além de apresentar outras vantagens a exemplo do baixo custo e o de se evitar exposição do ser humano a experimentos que possam causar danos à saúde. Antes de avaliar o FPS das formulações preparadas com as argilas foi feita a avaliação do FPS de protetores solares comerciais e foi confirmado que o método in vitro utilizado foi adequado na determinação do FPS de protetores solares. A introdução de argilas na composição dos protetores solares, como materiais naturais, em substituição ao material sintético TiO2 foi eficaz, especialmente para a argila bentonita Argel. A legitimidade em se estudar o efeito fotoprotetor dos materiais argilosos na composição dos protetores solares é justificada por grande relevância social, posto que são de baixo custo, e ainda por serem encontradas com facilidade na natureza. / Non-ionizing radiation (UV-A, UV-B and UV-C) of the sun's electromagnetic spectrum cause diseases to human and must be avoided. The proper use of sunscreen minimizes this problem. Currently, the trend is to introduce natural materials, organic or inorganic, in the composition of sunscreens, in order to neutralize possible side effects of synthetic materials. Studies have shown that clays (layered silicates) incorporated in sunscreens can block ultraviolet radiation in 250 to 400 nm wavelength range depending on the clay composition. Following this guideline, in this work, two clays were used in the preparation of sunscreen and its effectiveness was determined at Universidade Federal de Campina Grande (UFCG) laboratory through in vitro evaluation by spectrophotomery. The clays tested were Cloisite® Na + sodium montmorillonite and Argel 35 bentonite. The formulations were prepared in pharmacies in three different cities, Guarabira, Joao Pessoa and Campina Grande, in Paraíba state. The method chosen to evaluate the Sun Protection Factor (SPF) was in vitro method, developed by Mansur, due to the excellent correlation with in vivo method. Besides, there are other advantages like low cost and less damage to health since the volunteer is not exposed to the experiments. Before the evaluation of SPF of prepared formulations with the clay, the SPF of commercial sunscreens was determined and it was confirmed that the in vitro method was proper to define sunscreen SPF. The introduction of clay in the sunscreens composition, as natural materials, replacing TiO2 synthetic material was effective, especially for Argel bentonite clay. The legitimacy of studying the photoprotective effect of clay materials in the composition of sunscreens is justified by great social relevance, since they are inexpensive and found easily in nature.