Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate: Pavel Hladký Consultant: PharmDr. Barbora Švecová, Ph.D. Title of Thesis: Formulation and (trans)dermal application of imiquimod Imiquimod (IMQ) is an active pharmaceutical substance which belongs to the group of heterocyclic imidazoquinolines. The mechanism of its effect is an induction of cellular immune response after topical administration, that is used for a treatment of tumors or viral diseases of the skin. In the Czech Republic it is available like a cream called Aldara® , the content of IMQ is 5 %. Although Aldara® is an effective medicine, many problems are associated with its use, especially high price, undesirable effects, disposable use, environmental pollution, etc. The aim of this work was to prepare new liposomes for topical administration containing lower ammount of IMQ (0.5 %) and evaluation of penetration of IMQ into human skin in vitro. To improve the entrance of the drug into the skin transdermal penetration enhancers were used. Permeation experiments were performed in Franz diffusion cells on human skin under conditions as close as possible to the physiological environment of the organism. Subsequently, the individual layers of the skin (stratum corneum, the epidermis,...

L’expression des immunoglobulines (Ig) par les cellules B est dictée par de multiples remaniements géniques appelées recombinaisons V(D)J, commutation de classe (CSR) et hypermutation somatique (SHM). Tous ces événements sont contrôlés par des éléments cis-régulateurs notamment au locus IgH. Les mieux décrits sont la région intronique Eµ et ses régions d’attachement à la matrice nucléaire MARsEµ et la région régulatrice en 3’ du locus IgH (3’RR), constituée de 4 éléments activateurs (hs3a ; hs1,2 ; hs3b ; hs4) et possédant une structure quasi-palindromique très conservée. Afin d’étudier ces régions régulatrices, nous avons analysé plusieurs modèles murins présentant la délétion de la totalité de la région Eµ, des MARsEµ, et de diverses parties de la 3’RR. Ces modèles ont mis au jour des fonctions insoupçonnées de ces régions régulatrices, à divers stades du développement B. Au delà de son rôle régulateur des réarrangements précoces de DH vers JH, nous avons précisé la fenêtre d’action de région Eµ et son rôle sur la transcription et l’expression de la chaîne lourde µ du stade pré-B jusqu’au stade B transitionnel. L’activateur Eµ module en effet l’expression du pré-BCR et du BCR et par conséquent conditionne l’orientation des cellules B matures vers les compartiments de la zone marginale et folliculaire. Notre étude indique également que Eµ n’influence pas le choix des segments VH au cours de la recombinaison VDJ. Nous montrons que les régions MARsEµ, sont impliquées dans le ciblage des gènes d’Ig par l’hypermutation somatique. Le mode d’action de ces régions MARsEµ reste à définir mais nous décrivons qu’il est découplé de la transcription et qu’il affecte également des loci situés sur des chromosomes différents : loci codant les chaînes légères d’Ig (Ig) et loci des cibles illégitimes de AID (Bcl6 et Cd83).Une étude comparant un nouveau modèle murin déficient pour la région quasi-palindromique 3’IgH (de hs3a à hs3b) à deux autres modèles de notre laboratoire (3’RR KO et hs3b-hs4 KO) nous a permis d’identifier deux modules fonctionnels complémentaires dans la région 3’RR. Le module distal (hs4) impliqué dans la régulation de l’expression de la chaîne lourde du stade B immature au stade B naïf et le module proximal (de hs3a à hs3b) nécessaire aux stades matures pour la SHM et la CSR vers la majorité des isotypes après activation, mais également pour la synthèse des anticorps par les plasmocytes. / Immunoglobulin (Ig) gene expression in B cells depends on multiple genetic rearrangements named V(D)J recombination, class switch recombination (CSR) and somatic hypermutation (SHM). These events are controlled by cis-regulatory elements which are, especially numerous within the IgH locus. Major IgH regulatory elements are Eµ composed of a core enhancer flanked by nuclear matrix attachment regions MARsEµ and the regulatory region located at the 3’end of the locus (3’RR), composed of 4 enhancers (hs3a; hs1-2; hs3b; hs4) harbouring a highly conserved quasi-palindromic structure. To study these regulatory regions, we analyzed multiple mice models carrying endogenous deletions of either the entire Eµ region, MARsEµ only or various parts of the 3’RR. These models revealed unsuspected functions for all IgH regulatory regions, at various stages of B cell development. Beyond its role to control accessibility prior DH to JH rearrangements, we identified the window of activity for Eµ region and its particular role on transcription and expression of the µ heavy chain from pre-B to the transitional stages. Indeed, the Eµ enhancer modulates pre-BCR and BCR expression and consequently modulates the mature B cell fate toward marginal zone and follicular compartments. Our study also indicated that the absence of Eµ has no effect on VH segment usage during V to DJ recombination. We also showed that MARsEµ are implicated in the targeting of Ig genes by SHM. The mechanism by which MARsEµ enhances SHM remains to be clarified but we showed that the process does not depend on transcription. Surprisingly MARsEµ also modulates SHM occuring on genes located on different chromosomes: the Ig light chain loci (Igκ) and AID off-targets loci (Bcl6 and Cd83).In a study comparing a new mouse model devoid of the 3’ IgH quasi-palindromic region (hs3a to hs3b) to previous relevant models available in our laboratory (3’RR KO and hs3b-hs4 KO), we identified two complementary functional modules within the 3’RR. The distal module (hs4), which regulates Ig heavy chain expression (and therefore BCR expression) from the immature to mature naïve B cell stages and the proximal module (hs3a to hs3b and its quasi-palindromic structure) required at mature stages for SHM, CSR to most isotypes in activated B cells, and antibody production in plasma cells.

Orientador: Marise Aparecida Rodrigues Pollonio / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-17T20:58:01Z (GMT). No. of bitstreams: 1 Santos_BibianaAlvesdos_M.pdf: 679604 bytes, checksum: 8d581230bd1d2104faef357ea63607b6 (MD5) Previous issue date: 2011 / Resumo: A busca por alimentos com melhores propriedades nutricionais tem levado osegmentode processamento de produtos cárneos a rever suas formulações em relação a alguns componentes que podem aumentar o risco de doenças crônicas, tais como teores de gordura, sal, aditivos, entre outros. No entanto, a reformulação destes produtos deve manter as características de segurança, estabilidade, sabor e demais atributos característicos do produto. As carnes constituem-se em importante fonte de nutrientes importantes, como ácidos graxos, minerais, proteínas e peptídeos bioativos na dieta. Porém, produtos cárneos apresentam grande percentual de gordura saturada e colesterol além do elevado teor de sódio. Neste contexto, o objetivo do estudo foi substituir em 50% o percentual de gordura em produto fermentado cozido (pepperoni) por diferentes níveis de fibra prebiótica (3, 6 e 9% de frutooligossacarídeo ¿ FOS) e avaliar a qualidade global durante 60 dias de armazenamento refrigerado. E substituir em 50 e 75% o teor de NaCl por KCl em pepperoni ao utilizar realçadores de sabor (0,06% glutamato monossódico, 0,06% 5¿ribonucleotídeos- inosinato dissódico e guanilato dissódico) e aminoácidos (1% lisina e 0,075% taurina) com avaliação físico-química, cor, perfil de textura e sensorial. Pepperonis com teor reduzido de gordura adicionado de diferentes níveis de FOS mantiveram as características físico-químicas, microbiológicas, cor, perfil de textura e sensoriais comparadas à formulação controle (sem redução de gordura). Além disso, o frutooligossacarídeo adicionado não foi degradado durante todo o período de vida útil dos produtos. Os pepperonis com redução de cloreto de sódio em 50% foram similares ao produto controle (sem redução de NaCl) em relação a todos os parâmetros avaliados e a redução de sódio foi de 42,80%. No entanto, a redução de 75% NaCl proporcionou alterações na cor e perfil de textura. A aceitação sensorial também foi prejudicada na maioria das formulações estudadas porém a combinação de glutamato monossódico com inosinato dissódico/guanilato dissódico, lisina e taurina resultou em formulações com notas semelhantes à formulação controle. Neste experimento, a redução de sódio foi de 68,10%. Deste modo, o estudo demonstra que a redução de gordura e cloreto e sódio em produto cárneo fermentado cozido é possível e traz benefícios à qualidade nutricional destes produtos / Abstract: The demand for food with improved nutritional properties has led the meat products industry to re-evaluate their formulations. However, the reformulation of these products must preserve the food safety, stability, taste and other functional properties. The meat will constitute an important source of nutrients such as fatty acids, minerals, proteins and bioactive peptides in the diet. On the other hand, meat products have a high content of saturated fat and cholesterol, besides the high sodium content and very low levels of fibers. In this context, the aim of this study was: replace 50% of the total fat in cooked fermented product (pepperoni) by different levels of prebiotic fibre (3, 6 and 9% of fructooligosaccharides - FOS) and to evaluate the overall quality during 60 days of in refrigerated storage. And to replace 50 and 75% of NaCl content by using KCl in the pepperoni using flavor enhancers (06% monosodium glutamate, 06% 5'ribonucleotídeos-inosinate disodium and guanylate disodium) and amino acids (1% lysine and 075% taurine) with physico-chemical evaluation, color, texture profile and sensory analysis compared to a control formulation (no fat reduction). Pepperoni with reduced fat supplemented with different levels of FOS maintain the physico-chemical and microbiological properaties, color, texture profile and sensory characteristics when compared to the control formulation (no fat reduction). Moreover, the fructooligosaccharides were not degraded during the whole store period of the products. Pepperonis with 50% sodium chloride reduction were similar to the control (no reduction of NaCl) for all evaluated parameters, and the sodium reduction was 42.80%. However, the reduction of 75% NaCl resulted in changes in color and texture profile. The sensorial acceptance was also impaired in most of the formulations of this study, although the combination of monosodium glutamate with disodium inosinate / disodium guanylate, lysine and taurine resulted in formulations with a similar score when compare to the control formulation. In this experiment, sodium reduction was 68.10%. Thus, the study demonstrates that reducing fat and sodium chloride in fermented cooked meat products is possible and can bring benefits in the nutritional quality of these products / Mestrado / Tecnologia de Alimentos / Mestre em Tecnologia de Alimentos

Orientador: Marise Aparecida Rodrigues Pollonio / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T08:45:12Z (GMT). No. of bitstreams: 1 Campagnol_PauloCezarBastianello_D.pdf: 1477397 bytes, checksum: 5c8de3ec7acc19e6ae4f318371d09efe (MD5) Previous issue date: 2011 / Resumo: É crescente a importância que os consumidores atribuem aos aspectos que melhoram a sua qualidade de vida. A dieta não é o único fator que afeta o bem-estar e a saúde, mas é um dos mais importantes. O objetivo dos consumidores é ter uma dieta balanceada, com alimentos saudáveis, porém, sensorialmente agradáveis. Os embutidos fermentados, bem como os produtos cárneos em geral, tradicionalmente apontados como os vilões da dieta saudável devido ao alto teor de gordura, colesterol, sódio e aditivos químicos, podem pertencer a esse novo segmento de mercado. Para isto, esforços devem ser dirigidos para a redução desses componentes na formulação, melhorando assim a imagem dos produtos cárneos frente aos consumidores. Desta forma, este trabalho teve como objetivos estudar a influência da redução de sódio e gordura na qualidade de embutidos fermentados. Para reduzir a quantidade de sódio, embutidos fermentados foram produzidos com substituição de cloreto de sódio (NaCl) por cloreto de potássio (KCl) e se utilizaram aminoácidos (lisina e taurina) e realçadores de sabor (extrato de levedura, inosinato dissódico e guanilato dissódico). Para reduzir a quantidade de gordura, embutidos fermentados foram produzidos utilizando-se gel de celulose amorfa como substituto de gordura e também se avaliou a utilização de fibra de soja em embutidos fermentados com reduzido teor de gordura. Os resultados mostraram que a utilização de KCl como substituto de NaCl resultou numa redução de sódio próxima ao nível de substituição de NaCl por KCl, no entanto, foram observados defeitos sensoriais em um nível de substituição de 50%. Os aminoácidos (lisina e taurina) e os realçadores de sabor (inosinato dissódico, guanilato dissódico e extrato de levedura) reduziram os defeitos sensoriais causados pela substituição de 50% do teor de NaCl por KCl, permitindo a elaboração de embutidos fermentados sensorialmente aceitáveis com aproximadamente metade da quantidade de sódio. Já com relação aos embutidos fermentados produzidos com redução de gordura, observou-se que o gel de celulose amorfa foi capaz de substituir até 50% do teor de gordura da formulação sem comprometer a qualidade do produto. Este nível de substituição possibilitou a produção de embutidos fermentados com uma redução dos teores de gordura e colesterol de aproximadamente 45 e 15%, respectivamente. A utilização de 1% de fibra de soja em embutidos fermentados produzidos com uma redução de 15 para 10% de toucinho na formulação não alterou os aspectos físico-químicos, microbiológicos e sensoriais e proporcionou uma redução de aproximadamente 40% do teor de gordura final / Abstract: The importance that consumers attach to aspects which improve their quality of life has been increasing steadily. Diet is not the only factor affecting well-being and health, but it is one of the most important. Throughout the developed world, more people are seeking a balanced diet consisting of healthy foods that appeal to their senses. Fermented sausages and meat products in general, have traditionally been described as the villains in the diet because of their high fat, cholesterol and salt (or sodium) content, along with the frequent presence of chemical additives in them. However, these foods have recently become a part of the new balanced-diet-seeking market. To create truly healthy meats, efforts are needed to reduce less healthy components in the formulation and thereby improving the image of meat products among the consumers. Therefore, the aim of this work was to study the effect of sodium and fat reduction on the quality of fermented sausages. In order to reduce the amount of sodium, fermented sausages were produced by replacing sodium chloride (NaCl) with potassium chloride (KCl), and amino acids (lysine and taurine) and flavor enhancers (yeast extract, disodium guanylate and disodium inosinate) were used as well. In order to reduce the amount of fat, fermented sausages were produced using amorphous cellulose gel as a fat substitute; in addition the use of soy fiber in low-fat fermented sausages was evaluated. The study?s results showed that the use of KCl as a substitute for NaCl resulted in a reduction of sodium to a similar level of substitution of NaCl by KCl, however, sensory defects were observed in a substitution level of 50%. The addition of the amino acids (lysine and taurine) and the flavor enhancers (inosinate disodium, disodium guanylate and yeast extract) reduced the sensory defects caused by the replacement of 50% NaCl by KCl and enabled the production of fermented sausages that had acceptable sensory qualities with half as much sodium content. As for those fermented sausages made with fat reduction, the results suggested that the substitution of up to 50% of the pork back fat content by amorphous cellulose gel did not lessen the product quality. The levels of fat and cholesterol in these sausages were decreased by approximately 45% and 15%, respectively. The addition of 1% soy fiber in fermented sausages produced with a reduction from 15 to 10% of pork back fat in the formulation did not alter the physical-chemical, microbiological and sensory aspects of the sausages but did reduce their fat content by approximately 40% / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos

T lymphocytes can be rendered specific against a wide range of antigens by the genetic transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has shown promising results for cancer therapy. Previous experiments in our laboratory have shown low rates of gene transfer using retroviral vectors. In this study, investigations have been done to increase the number of genetically modified cells. Different enhancers such as PLL and polybrene have previously been used in combination with retroviral transduction. The optimal retroviral protocol in this study showed to be the use of retrovectors produced with twice the normal concentration of the plasmids encoding env and gag-pol rather than the use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together with a centrifugation step increased the rate of modified cells even further. Alternative approaches of gene transfer were also investigated, including plasmid transfection and adenoviral transduction. While transfection protocols yielded low numbers of modified cells, adenoviral vectors showed the highest rate of gene transfer. / Cancer är den sjukdom som idag, efter hjärt-kärl-sjukdomar, kräver flest dödsfall i i-länder. Som en alternativ behandlingsmetod mot cancer pågår just nu forskning om genetiskt förbättrade immunceller, s.k. chimära T lymfocyter, skulle kunna användas för att döda tumörceller. De chimära cellerna är utrustade med en konstgjord receptor som är en fusion av en antikropp och en signalkedja. Det gör att cellerna kan riktas mot ett brett urval av cancertyper. Att få cellerna att ta upp generna som behövs för den konstgjorda receptorn har visats sig vara problematiskt. Den här studien har därför som mål att förbättra cellernas förmåga att ta upp gener. För detta har vi använt oss av retrovirus- och adenovirus-system tillsammans med försök att få cellerna att spontant ta upp generna, sk. plasmid-transfektion. Studien har visat att de båda virussystemen ger högst antal modifierade celler. Olika substanser som tidigare har visat sig förhöja graden av gentillförsel har testats, men vår studie har visat att tillverkningen av virusvektorerna har större påverkan på resultaten än någon av de olika hjälpmedlen.

The need for alternative cognitive enhancers has risen due to the fact that clinical trial results of the drugs currently approved for treating these disorders have not been satisfactory. IRAP has become a possible drug target for treating cognitive impairment brought about by Alzheimer’s disease, head trauma or cerebral ischemia, among others. This came after the revelation that Angiotensin IV enhances memory and learning. Angiotensin IV, the endogenous ligand of IRAP has been structurally modified with the aim of producing potent IRAP inhibitors. However, the peptidic nature of these inhibitors restricts their use; they are not likely to cross the blood brain barrier. Other strategies for generating IRAP inhibitors have been through structure-based design and receptor based virtual screening. These drug-like molecules have exhibited positive results in animal studies. IRAP inhibitors have been identified via a HTS of 10500 low-molecular weight compounds to give the hit based on a spirooxindole dihydroquinazolinone scaffold, with an IC50 value of 1.5 µM. In this project, some analogues to this hit compound have successfully been synthesized using a known method, whereas others have been synthesized after additional method development. The application of the developed method was found to be limited, because poor yield was obtained when a compound with an electron withdrawing substituent on the aniline was synthesized. As a result of this, modification of this method may be required or new methods may have to be developed to synthesize these types of analogues. Inhibition capability of 5 new spirooxindole dihydroquinazolinones was tested through a biochemical assay. Compound 6e emerged as the most potent inhibitor in the series, with an IC50 value of 0.2 µM. This compound will now serve as a lead compound and should be used as a starting point for future optimization in order to generate more potent IRAP inhibitors.

The interest of drug delivery via the skin is progressively increasing due to its convenience and affordability. However, the skin has an effective barrier function which hinders drugs to penetrate. This can be overcome, using topical formulations, which contain vehicles optimized for penetration into skin or permeation through skin into underlying tissues or blood stream. In development of transdermal drugs, ex-vivo skin models are frequently used for permeability studies. However, these models may suffer from limited reproducibility, due to their biological variability, and are related to ethical issues. This has led to development of in-vitro skin models. These new skin models need to be verified by comparison to the existing models before applied in drug studies. In this project, penetration and permeation of proteins and small molecules was studied using an ex-vivo pig skin model. The effect of topical formulations and penetration enhancers was also investigated. The results showed a significant increase in penetration and permeation in the absence of stratum corneum, proving its barrier effect. While no significant differences could be seen regarding molecule size in the presence of stratum corneum (intact skin model), there was significantly less permeation measured for large protein molecules tested than for smaller tested compounds in the absence of the stratum corneum (damaged skin model). The permeation and penetration were slightly increased in presence of the penetration enhancing limonene oil. / Intresset för läkemedel som tas upp genom huden ökar ständigt eftersom administreringen är både bekväm och lättillgänglig. Däremot har huden en effektiv barriärfunktion, vilket hindrar penetration av läkemedlen. För att lösa detta problem kan läkemedlen presenteras i olika formuleringar, vilka innehåller transportmedel anpassade för att öka penetrationen in i huden eller permeationen genom huden, till underliggande vävnad samt blodomloppet. Vid utvecklandet av läkemedel som ska tas upp genom huden används ofta ex-vivo hudmodeller i penetrations- och permeationsstudier. Dessa modeller kan emellertid ha otillräcklig reproducerbarhet, på grund av biologisk variation, samt är kopplade till etiska problem. Detta har lett till utvecklandet av hudmodeller av in-vitro typ. Dessa nya hudmodeller måste verifieras genom jämförelse med existerande modeller, innan de används i läkemedelsstudier. I det här projektet studerades penetration och permeation av proteiner och små molekyler i en ex-vivo hudmodell från gris. Effekten av olika formuleringar och penetrationsförstärkare undersöktes också. Resultaten visade en signifikant ökning av penetration och permeation vid avsaknad av stratum corneum, vilket påvisar dess barriärfunktion. Samtidigt som ingen märkbar skillnad gällande molekylstorlek kunde urskiljas vid närvaro av stratum corneum (intakt hudmodell), var permeationen av större molekyler tydligt minskad i jämförelse med små molekyler vid avsaknad av stratum corneum (skadad hudmodell). Både penetrationen och permeationen var svagt förhöjda vid närvaro av penetrationsförstärkaren limonen.

No description available.

The stem cell-like seam cells of the nematode, Caenorhabditis elegans, represent a tractable and powerful model for studying stem cell biology. rnt-1, the worm homologue of the mammalian RUNX family of transcription factors, together with the CBFβ homologue bro-1, is essential for the proliferation of the seam cells. RUNX genes and CBFβ are important regulators of stem cell development in mammals, and are associated with a variety of cancers. The worm seam cell model offers an opportunity to examine how these genes function in stem cell biology. The aim of this work was to shed light on the genetic network in which bro-1 and rnt-1 function, and to reveal the identity of regulators of these genes as well the downstream targets of the bro-1/rnt-1 pathway. Here, a number of genes that interact with bro-1 and rnt-1 have been identified. ELT-1, a GATA transcription factor, is shown to be a direct regulator of bro-1. Findings which show that the MEIS gene unc-62 acts upstream of bro-1/rnt-1 and regulates the symmetry of seam cell divisions are also presented. The seam cell marker, scm::gfp, is widely used in studies of the seam cells; here the results of an investigation into its identity and functional links are described. In addition, the mechanism underlying spatial regulation of rnt-1 was examined; this led to the discovery of distinct tissue-specific enhancer modules within an intron of this gene. Finally, interactions between pal-1 and bro-1/rnt-1 are reported and described. Together, these findings provide a framework for furthering our understanding of the mechanisms and genes associated with the functioning of bro-1 and rnt-1 in the worm.

The major physiological function of hemoglobin (Hb) is to bind, transport and deliver oxygen to tissues; made efficient by endogenous effectors, such as protons and 2,3-diphosphoglycerate. Synthetic allosteric effectors of Hb (AEHs) are also known to modulate Hb oxygen affinity, showing potential for the treatment of sickle cell disease (SCD) and ischemic-related diseases. In this project, AEHs which increase Hb affinity for oxygen, including derivatives of the anti-sickling compounds, 5HMF and benzaldehydes, as well as an AEH that decreases Hb affinity for oxygen, RSR-13, were synthesized for their effects on Hb oxygen binding property and their capability to release NO from substituted nitrate ester moieties. Compounds that were found to increase Hb affinity for oxygen were further tested for their anti-sickling activities. Structural studies were carried out to gain insight into the compound’s mode of action. Development of these agents could be a therapeutic strategy for SCD or ischemic-related diseases.