Zugl.: Heidelberg, University, Diss., 2004.

Düsseldorf, Universiẗat, Diss., 2000.

The 1£\, 25-dihydroxyvitamin D3 (1,25(OH)2D3) is involved in various physiological processes, including calcium/phosphorous homeostasis, cell growth, differentiation and apoptosis. 1,25(OH)2D3 induces the formation of VDR/RXR complex to up-regulate or down-regulate target gene expression. Recent studies find that VDR undergoes several post-translational modifications, such as phosphorylation and ubiquitination, which may regulate its transcriptional activity and/or stability. In this study, we identified VDR as a new target for small ubiquitin-related modifier (SUMO)-2 modification in vitro. In E. coli. SUMO-conjugation system, VDR is mainly sumoylated at Lys-103. SUMOylation of VDR enhanced VDR/RXR-mediated transcriptional activation as determined by promoter activity assay. In addition, 1,25(OH)2D3-induced expression of osteopontin was attenuated after mutation of VDR SUMOylation site. However, chromatin immunoprecipitation assay indicated that wild type and K103A mutant of VDR bound to the osteopontin promoter with similar affinity. Collectivity, our results suggest that SUMOylation of VDR may affect its transcriptional activity by modulating the interaction between VDR and co-activators.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1980. / Typescript. Vita. Includes bibliographical references.

In ständigem Kontakt mit zahlreichen Mikroorganismen benötigt unser Körper eine Vielzahl an Mechanismen zur Abwehr krankheitserregender Keime. Antimikrobielle Peptide unterstützen als Effektormoleküle die einzellige Epithelschicht des Darms, die als Mukosabarriere unseren Körper vor dem Eindringen pathogener Keime bewahrt. Die Expression des antimikrobiellen Peptids LL-37, auch Cathelicidin genannt, stellt eine Strategie des angeborenen Immunsystems zur Abwehr von Mikroorganismen im Kolon dar. Die Expression des Cathelicidin Gens camp kann, wie in der vorliegenden Arbeit untersucht wurde, durch exogene Faktoren, wie die biologisch aktive Form des Vitamin D3, das 1,25(OH)2D3, die kurzkettige Fettsäure Butyrat und das Ernährungssupplement Intestamin®, angeregt werden. Die Stimulation der Zellen mit den genannten Substanzen führte in allen gestesteten Zelllinien zeit- und dosisabhängig zu einer Steigerung der Expression des Cathelicidin Gens. Die Induktion der camp-Expression durch 1,25(OH)2D3 wird durch die Existenz eines Vitamin D Response Elements (VDRE) in der Promoterrregion des camp-Gens begründet, an das der Vitamin D-Rezeptor Komplex als ligandenabhängiger Transkriptionsfaktor bindet und die Genexpression vermittelt. Die Auswirkungen von Butyrat auf die Genexpression des Cathelicidins werden auf die Modifikation des Acetylierungsstatus der Histonproteine zurückgeführt. Butyrat bewirkt durch eine reversible Hemmung der Histondeacetylase die Hyperacetylierung bestimmter Kernhistone und greift auf diese Weise in die Regulation der Gentranskription ein. Das enterale Ernährungssupplement Intestamin®, das als Pharmakonutrition speziell für schwerkranke Patienten entwickelt wurde, ist reich an Glutamin-Dipeptiden, Tributyrin und Antioxidantien. Der Effekt, den Intestamin® auf die Expression des Cathelicidin Gens ausübt, ist wahrscheinlich auf Tributyrin zurückzuführen. Tributyrin, der Ester aus Glycerin und Butyrat, wird durch Hydrolyse zu Butyrat umgesetzt und bewirkt vermutlich die Steigerung der camp-Expression. Eine Co-Stimulation von GEKI 02 Zellen mit 1,25(OH)2D3 plus Butyrat erzielte nach 48 Stunden eine weitere Steigerung der Expression des Cathelicidin Gens gegenüber beiden Einzelsubstanzen. In allen anderen getesteten Zelllinien zeigte sich keine synergetische Wirkung der beiden Substanzen. Auch eine Co-Stimulation mit Intestamin® plus Butyrat konnte nicht zu einer stärkeren Zunahme der camp-Expression führen als eine Behandlung mit beiden Einzelsubstanzen. Eine synergetische Wirkung der Substanzen 1,25(OH)2D3 und Butyrat in GEKI 02 Zellen könnte durch die Acetylierung der Histone bedingt sein, die eine Auflockerung der Chromatinstruktur bewirkt, was wiederum die Bindung von Transkriptionsfaktoren wie dem Vitamin D-Rezeptor Komplex erleichtert. Die fehlende synergetische Wirkung in allen anderen getesteten Zelllinien könnte mit der Tatsache in Zusammenhang stehen, dass die Induktion der camp-Expression zeitabhängig ist: Vitamin D3 erzielte nach 24 Stunden, Butyrat nach 48 Stunden die deutlichsten Auswirkungen auf die Genexpression des Cathelicidins. Der Einfluss des MEK/ERK Signalweges auf die durch 1,25(OH)2D3, Butyrat und Intestamin® induzierte camp-Expression wurde in den durchgeführten Versuchen mittels des spezifischen MEK 1/2 Inhibitors U0126 untersucht. U0126 blockierte die Induktion des Cathelicidin Gens durch Intestamin® und Butyrat, was die Beteiligung des Signalweges MEK/ERK belegt. Vitamin D3 dagegen übt seinen Einfluss auf LL-37 nicht über den Signalweg MEK/ERK aus. Obwohl Vitamin D3 und Butyrat die Expression des Cathelicidin Gens camp induzieren, konnte die Inkubation der Zellen mit beiden Substanzen die antimikrobielle Aktivität der Kolonepithelzellen gegenüber E.coli im Vergleich zu unbehandelten Kontrollzellen nicht steigern. Ursächlich hierfür könnte neben der Wahl eines apathogenen Bakterienstammes das Mikromilieu der Umgebung sein. Außer der Konzentration des Peptids spielen insbesondere der pH-Wert und die Salzkonzentration des Kulturmediums eine wichtige Rolle, da sie die Ausbildung der Sekundärstruktur, nämlich der α-helikalen Konformation, des LL-37 beeinflussen, die für die Interaktion mit Biomembranen erforderlich ist. Aufgrund der zunehmenden Resistenzentwicklung von Bakterien gegenüber herkömmlichen Antibiotika, wächst das Interesse an der Erforschung antimikrobieller Peptide. Exogene Faktoren wie 1,25(OH)2D3, Butyrat und Intestamin® können eine Steigerung der Expression des Cathelicidin Gens erzielen. Ob sich dieser Effekt allerdings auch in-vivo zeigt und eventuell therapeutischen Einsatz finden könnte, müssen weitere Studien klären. / Defence of mucosal and epithelial surfaces against microbial pathogens involves the innate and adaptive immune response. The single cell layer of the colonic epithelium produces an array of immune modulators, including antimicrobial peptides, such as cathelicidins, that participate in the innate immune system. The aim of the present study was to analyse the effect of calcitriol, the histon-deacetylase inhibitor butyrate and the enteral pharmaconutrition supplement Intestamin® on the expression of the cathlicidin gene camp. After exposure to these stimuli a time and dose dependent induction of the expression of the cathelicidin gene was found in all investigated colorectal cell lines. The effect of calcitriol, 1,25(OH)2D3, is mediated by the vitamin D response element (VDRE) in the camp promoter that was bound by the vitamin D receptor (VDR). The induction of cathelicidin expression after treatment with butyrate is attributed to a reversible inhibition of histone deacetylases resulting in modulation of core histone and non-histone proteins and subsequent regulation of the gene transcription. The enteral pharmaconutrition supplement Intestamin® contains glutamine, antioxidant vitamins and tributyrin. The increased cathelicidin level after treatment with Intestamin® are ascribed to tributyrin because tributyrin is a novel structured lipid composed of three molecules of butyrate esterified with glycerol that induces the cathelicidin gene camp after hydrolysis to butyrate. When colorectal cells GEKI 02 were stimulated with 1,25(OH)2D3 plus butyrate, a further increase of cathelicidin expression was seen after 48 hours. This effect was not seen for the incubation with Intestamin® plus butyrate. Changes in the acetylation status of core histone and non-histone proteins caused by butyrate that enhance binding of the vitamin D receptor complex as a transcription factor might be responsible for the synergistic effect of butyrate and calcitriol. To determine the influence of the MEK/ERK signalling pathway in the investigated cell lines the specific MEK/ERK inhibitor U0126 was utilized. Inhibition of the MEK/ERK pathway blocked butyrate- and Intestamin®-induced cathelicidin expression while no effect was observed for calcitriol. Although incubation with vitamin D3 and butyrate increased cathelicidin expression, the antimicrobial activity against E.coli could not be enhanced in the investigated colorectal cells. This might be caused by selection of a commensal bacterium and the absence of relevant microenvironmental stimuli because the antibacterial activity correlates with the extent of α-helicity, which is influenced by ion composition, pH, and peptide concentration. Due to the fast development of bacterial resistance to traditional antibiotics, there is increasing interest in the research on antimicrobial peptides. Exogenic factors like 1,25(OH)2D3, butyrate and Intestamin® can enhance an expression of the cathelicidin gene. Further in vivo studies, however, are necessary to verify this effect for the potential future therapeutic application.

Universiẗat, Diss., 2000--Düsseldorf.

Das Secosterid Vitamin D3 wird durch die Nahrung aufgenommen oder im Organismus synthetisiert, wobei eine Reaktion in der Haut durch einen photochemischen Prozess katalysiert wird.Durch zwei Hydroxylierungsschritte in Leber und Niere wird Vitamin D3 über 25(OH) Vitamin D3 zum aktiven 1,25(OH)2 Vitamin D3-Hormon. 1,25(OH)2 Vitamin D3 hat eine wichtige Funktion im Knochenstoffwechsel, es reguliert die Ca2+-Resorption im Dünndarm. Die 1,25(OH)2 Vitamin D3-Synthese in der Niere wird durch Parathormon (PTH) kontrolliert. Ist die Serum Ca2+-Konzentration niedrig, wird PTH ausgeschüttet und die 1a-Hydroxylase, das 25(OH) Vitamin D3-aktivierende Enzym, stimuliert. Das Prinzip der (Seco)steroid-Aktivierung und -Inaktivierung in glandulären Organen, wie Leber und Niere mit anschließender Freisetzung der aktiven Hormone und Transport zu den jeweiligen Zielgeweben gilt heute nicht mehr uneingeschränkt. Auch Einzelzellen sind in der Lage Steroid-modifizierende Enzyme, die Hydroxylasen und Dehydrogenasen, zu exprimieren. Monozytäre Zellen exprimieren das 1,25(OH)2 Vitamin D3-aktivierende und das -inaktivierende Enzym, die 1a-Hydroxylase und die 24-Hydroxylase. Sie sind somit in der Lage, 1,25(OH)2 Vitamin D3 zu sezernieren, welches parakrin auf Nachbarzellen wirken kann. In diesem Zusammenhang wurde die Expression und Regulation der 1a-Hydroxylase in peripheren Blutmonozyten (PBM) und monozytären THP1-Zellen untersucht. Durch Supplementation der Zellen mit dem Substrat 25(OH) Vitamin D3 konnte die Produktion an aktivem 1,25(OH)2 Vitamin D3-Hormon in PBM signifikant gesteigert werden. In PBM konnte im Gegensatz zum systemischen Ca2+-Stoffwechsel nur ein geringer Einfluss auf die 1a-Hydroxylase-Aktivität beobachtet werden. Durch RT-PCR-Amplifikation konnte eine Expression des PTH Rezeptors Typ 1 (PTHR1) in PBM und Dendritischen Zellen nachgewiesen werden. Ein weiterer Ligand des PTHR1 ist PTH related Protein (PTHrP), ein Faktor der die Tumorhyperkalzämie propagiert. Durch Markierungsexperimente mit fluoreszenz-markiertem PTHrP konnte gezeigt werden, dass PTHrP an die Zellmembran von PBM und Dendritischen Zellen bindet und in den Zellkern von Dendritischen Zellen transportiert wird. Im Rahmen dieser Arbeit wurde die Expression 1,25(OH)2 Vitamin D3-responsive Gene in Monozyten/Makrophagen untersucht. Die Expression der 24-Hydroxylase wird innerhalb der Differenzierung von myeloischen THP1-Zellen zu Makrophagen- bzw. Osteoklasten-ähnlichen Zellen transient induziert. Als weiteres 1,25(OH)2 Vitamin D3-responsives Gen wurde die Expression von Osteopontin (OPN) untersucht. OPN ist ein vor allem in Knochen vorkommendes Matrixprotein, das wesentlich an der Zelladhäsion beteiligt ist. OPN wird in THP1-Zellen im Zuge der Differenzierung zunehmend exprimiert. Durch immunhistochemische Untersuchungen konnte OPN in Granulomen von Morbus Crohn- und Leberschnitten detektiert werden. Es spielt hier eine wesentliche Rolle bei der Granulomentstehung. Die Thioredoxin Reduktase 1 (TR1) ist ein Selenoenzym, welches maßgeblich an der Reduktion von Disulfidbindungen in Proteinen beteiligt ist. Es moduliert Protein/Protein- und Protein/DNA-Interaktionen wie die Bindung der Transkriptionsfaktoren AP1 und NFkB an DNA-responsive Elemente. Die Expression der TR1 wird in THP1-Zellen im Rahmen der Differenzierung induziert und ist in differenzierten Zellen maximal. Aktivitätsmessungen deckten sich mit dieser Beobachtung. In peripheren Blutmonozyten steigt die TR-Aktivität alleine durch Adhäsion der Zellen an das Kulturgefäß und nach Behandlung mit 1,25(OH)2 Vitamin D3. Die Untersuchungen der vorliegenden Arbeit zeigten eine Abhängigkeit der TR-Aktivität vom Differenzierungsgrad der Zellen und der Supplementation des Mediums mit dem Spurenelement Selen. Die Expression weiterer Selenoproteine in monozytären Zellen wurde nachgewiesen. So konnten durch 75Selenit-Markierungsexperimente neun Selenoproteine in THP1-Zellen detektiert werden, von denen fünf sezerniert werden. Ein weiteres, in monozytären Zellen charakterisiertes Selenoprotein ist die zelluläre Glutathionperoxidase. Ihre Aktivität konnte in Selenit-supplementierten Zellen um das 70fache gesteigert werden. Die Kultivierung monozytärer Zellen unter Selenit-Supplementation beeinflusst die Funktion dieser Zellen wesentlich. So konnte beobachtet werden, dass die Anzahl an phagozytierenden, zu Makrophagen differenzierten THP1-Zellen nach Selenit-Supplementation abnahm, während die Phagozytoserate der einzelnen Zellen anstieg. Die erzielten Ergebnisse zeigen, dass monozytäre Zellen mit Komponenten des 1,25(OH)2 Vitamin D3 Stoffwechsels ausgestattet sind und aktives 1,25(OH)2 Vitamin D3-Hormon produzieren, sezernieren und inaktivieren können. Die lokale Kontrolle der 1,25(OH)2 Vitamin D3 Stoffwechsels ausgestattet sind und aktives 1,25(OH)2 Vitamin D3-responsiver Gene, wie die Expression des Selenoproteins TR1, das einen direkten Einfluss auf den Redoxstatus und den Abbau reaktiver Sauerstoffverbindungen in diesen und Nachbarzellen ausübt. / The secosteroid 1,25(OH)2 vitamin D3 is either taken up by our daily diet or it is formed by a photochemical prosess in the skin. In liver and kidney vitamin D3 is hydroxylated in two steps to 25(OH) vitamin D3 and the active hormone 1,25(OH)2 vitamin D3. 1,25(OH)2 vitamin D3 plays an important role in bone metabolism. It is a key regulatorof the resorption of Ca2+ in the intestine. In the kidney 1,25(OH)2 vitamin D3 synthesis is controlled by parathyroid hormone (PTH). When the concentration of serum Ca2+ is low, PTH is secreted and 1a-hydroxylase, the 25(OH) vitamin D3 activating enzyme is induced in kidney. The picture of (seco)steroid activation and inactivation in glandular organs, like the liver and kidney, and the release and transport of the activated hormone to the target tissues has been modified recently. Single cells are also able to express steroid-modifying enzymes like hydroxylases and dehydrogenases. Monocytes express the 1,25(OH)2 vitamin D3-activating and the inactivating enzyme, i.e. the 1a-hydroxylase and the 24-hydroxylase. Thus they are able to build and secrete 1,25(OH)2 vitamin D3 which can act on neighbouring cells in a paracrine way. In this context the expression and regulation of the 1a-hydroxylase in peripheral blood monocytes (PBM) and THP1 cells was investigated. By supplementation of cells with the substrate 25(OH) vitamin D3 the production of active 1,25(OH)2 vitamin D3 hormone could be enhanced significantly in PBM. In PBM only a slight influence of PTH on 1a-hydroxylase activity could be observed, in contrast to the regulation in systemic Ca2+-metabolism. An expression of PTH receptor type 1 (PTHR1) could be verified by RT-PCR from whole RNA isolated from PBM and dendritic cells. A further ligand of PTHR1 is PTH related protein (PTHrP), a factor which propagates the humoral hypercalcemia of malignancy. Labeling experiments with a fluorescently marked PTHrP showed clustered membrane staining of PBM and dendritic cells and a transport to the nucleus of dendritic cells. The expression of 1,25(OH)2 vitamin D3-responsive genes in monocytes/macrophages was investigated. 24-hydroxylase is induced transiently during the differentiation of myeloid THP1 cells to macrophages and osteoclast-like cells, respectively. Next, the expression of osteopontin (OPN), a further 1,25(OH)2 vitamin D3 responsive gene was studied. OPN is a matrix protein that is mainly found in bone, it carries a RGD-motive in its aminoacid sequence which can bind to integrins and is involved in cell adhesion. The expression of OPN is increased during differentiation of THP1 cells. By immunohistochemistry OPN could be detected in Crohn's disease and liver granulomas where it also plays an important role in granuloma formation. The thioredoxin reductase 1 (TR1) is a selenoenzyme that is mainly involved in the reduction of disulfide bonds of proteins. It modulates protein/protein and protein/DNA interactions like the binding of the transkription factors AP1 and NFkB to DNA-responsive elements. The expression of TR1 mRNA is induced during differentiation and is maximal in differentiated cells. Activity measurments parallel these observations. In PBM TR-activity is increased by the event of adhesion of cells to the culture dish and after treatment with 1,25(OH)2 vitamin D3. A dependence of TR-activity on the degree of differentiation of cells and the supplementation of the medium with the trace element selenium was observed. The expression of further selenoproteins in monocytic cells was investigated. In THP1 cells nine selenoproteins could be detected By labeling experiments with 75selenite. Five were found as secreted proteins in the culture supernatant. In monocytes cellular glutathione peroxidase (cGPx) is a well characterized selenoprotein. Activity could be increased 70fold by selenit supplementation. Under selenite supplementation the number of differentiated THP1 cells capable of phagocytosis was diminished while the rate of phagocytosis of single cells was enhanced. Taken together, the experiments clearly indicate that monocytic cells are equipped with the components of 1,25(OH)2 vitamin D3 metabolism and thus are capable of 1,25(OH)2 vitamin D3 hormone synthesis, secretion and turnover. Moreover, local control of 1,25(OH)2 vitamin D3 synthesis and inactivation directly regulates the expression of 1,25(OH)2 vitamin D3-responsive genes like the selenoprotein TR and thus even impacts on the cellular redox-status and defense against reactive oxygen species in these and neighbouring cells.

Zugl.: Berlin, Freie Universiẗat, Diss., 2009.

Submitted by CARINA GUIDI PINTO null (carina_guidi@hotmail.com) on 2016-02-26T12:56:51Z No. of bitstreams: 1 Dissertação Carina Guidi Pinto.pdf: 2764188 bytes, checksum: 30bbdd1a9fc0615c8dae9f321f8a83a1 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-02-26T18:59:28Z (GMT) No. of bitstreams: 1 pinto_cg_me_bot.pdf: 2764188 bytes, checksum: 30bbdd1a9fc0615c8dae9f321f8a83a1 (MD5) / Made available in DSpace on 2016-02-26T18:59:28Z (GMT). No. of bitstreams: 1 pinto_cg_me_bot.pdf: 2764188 bytes, checksum: 30bbdd1a9fc0615c8dae9f321f8a83a1 (MD5) Previous issue date: 2016-01-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Pró-Reitoria de Pesquisa (PROPe UNESP) / A ingestão de etanol compromete a estrutura do cérebro, apresentar efeitos bifásicos sobre a atividade motora, agindo como um estimulante ou depressor dependendo da dose ou a duração de utilização. Ele interfere na absorção e metabolismo de vitamina D3, o que se correlaciona com alguns distúrbios neurológicos e neuropsiquiátricos. Há relatos sobre a associação de etanol com alterações ósseas, incluindo baixos níveis de vitamina D3. Com base nisso, o objetivo deste estudo foi avaliar os efeitos em testes de comportamento da administração isolada de vitamina D3 ou a sua administração em associação com etanol, durante alcoolismo crônico. A fim de conseguir isso, foram utilizados dois grupos experimentais: ratos Wistar machos (n = 20) e ratos UChB linhagem machos (n = 20) (bebedores voluntários de etanol). Ambos os grupos foram divididos em dois subgrupos: Vitamina D3 - 12.5μg / kg / dia (500 UI) de colecalciferol (WV, n = 10, e UV, n = 10), e de controle (CC, n = 10, e UC, n = 10), durante um período de 75 dias. O peso corporal análises e testes de comportamento (reflexo de sobressalto acústico e campo aberto) foram realizados em 90 e 165 dias de idade. Além disso, os níveis de plasma de corticosterona foram medidos a 165 dias, sem diferença estatística entre os grupos experimentais. O grupo Wistar apresentou valores mais baixos ASR no momento final (Controle e completada), enquanto as percentagens PPI foram maiores no grupo inicial. No grupo UChB houve nenhuma diferença em percentagens PPI com o pré-estímulos utilizados. Quando as respostas ASR foram comparados entre os grupos (Wistar e UChB), o grupo UChB apresentaram menores amplitudes de ASR quando comparados com os animais do grupo de ratos Wistar. Em relação à PPI percentagens dos três estímulos pré utilizados no grupo UChB, as percentagens foram maiores em comparação com os animais do grupo de ratos Wistar, os valores ASR foi menor no grupo UChB. A presença de fezes e urina foi semelhante tanto nos momentos iniciais e finais, no teste de campo aberto, para todos os grupos experimentais. Considerando-se os outros parâmetros de campo aberto, diminuição da locomoção foi observado no grupo UChB no subgrupo vitamina D3, bem como no grupo de controlo final, em comparação com o controlo inicial. Quando os grupos Wistar e UChB foram comparados, este último apresentado taxas mais elevadas na locomoção. Os resultados obtidos estão em consonância com o efeito sedativo / depressivo de etanol sobre o sistema nervoso central, reduzindo a ASR de amplitude, com um aumento provável ao longo dos circuitos inibitórios que medeiam a PPI, a determinação do aumento na percentagem de inibição no grupo UChB, bem como um aumento na locomoção apresentada pelo grupo UChB no teste de campo aberto, caracterizada por uma diminuição no efeito aversivo ao novo ambiente. Além disso ressaltamos que a dose de vitamina D3 usado não teve efeito sobre o parâmetro de comportamento analisados, diferenças observadas neste grupo parecendo estar relacionada a se acostumar com a sonda gástrica. Este estudo descreve pela primeira vez na literatura as diferenças nas respostas ao teste acústico de sobressalto reflexo entre ratos Wistar e UChB, este último geneticamente predispostos a ingestão de etanol, uma linhagem adequada para ser usada em experiências compreendendo análises de comportamento em face da ingestão de etanol. / Ethanol intake compromises brain structure, presenting biphasic effects over motor activity, acting as a stimulant or depressor depending on the dose or duration of use. It interferes in vitamin D3 absorption and metabolism, what correlates to some neurologic and neuropsychiatric disorders. There are reports on the association of ethanol with bone alterations, including low levels of vitamin D3. Based on that, the objective of this study was to evaluate the effects in behavior tests of isolated administration of vitamin D3 or its administration in association with ethanol, during chronic alcoholism. In order to achieve that, two experimental groups were used: male Wistar rats (n=20), and UChB lineage male rats (n=20) (volunteer ethanol drinkers). Both groups were divided in two subgroups: Vitamin D3 – 12.5µg/kg/day (500 UI) of cholecalciferol (WV, n=10, and UV, n=10), and Control (WC, n=10, and UC, n=10), for a period of 75 days. Body weight analyses and behavior tests (acoustic startle reflex and open field) were conducted at 90 and 165 days of age. In addition to that, corticosterone plasma levels were measured at 165 days, with no statistical difference between the experimental groups. The Wistar group presented lower ASR values in the final moment (Control and supplemented), while the PPI percentages were higher in the initial group. In the UChB group there was no difference in PPI percentages with the pre-stimuli used. When the ASR responses were compared between groups (Wistar and UChB), the UChB group presented lower ASR amplitudes as compared to the animals of the Wistar group. In relation to PPI percentages in the three pre stimuli used in the UChB group, the percentages were higher as compared to the animals in the Wistar group, the ASR values been smaller in the UChB group. The presence of feces and urine was similar both in the initial and final moments, in the open field test, for all the experimental groups. Considering the other parameters of open field, decreased locomotion was noted in the UChB group in the vitamin D3 subgroup, as well as in the final control group as compared to the initial control. When the Wistar and UChB groups were compared, the latter presented higher rates in locomotion. The results obtained are consonant to the depressive/sedative effect of ethanol over the CNS, reducing ASR amplitude, with a likely enhancement over the inhibitory circuits which mediate PPI, determining the increase in inhibition percentage in the UChB group, as well as an increase in locomotion presented by the UChB group in the open field test, characterized by a decrease in the aversive effect to the new environment. In addition we emphasize that the dose of vitamin D3 used did not have effect over the behavior parameter analyzed, differences observed in this group seeming to be related to getting used to the gavage. This study describes for the first time in literature the differences in responses to the acoustic startle reflex test between Wistar and UChB rats, the latter genetically predisposed to ethanol intake, a lineage adequate to be used in experiments comprising analyses of behavior in face of ethanol intake. / PROPe/RENOVE: 0252/010/14

Vitamin D deficiency is an epidemic issue in all age groups in Western countries and that affects both skeletal and non-skeletal functions. Even with the wide application of food fortification, vitamin D deficiency tends to increase continuously. Being hydrophobic in nature, vitamin D has poor solubility; thereby it negatively affects its absorption and bioavailability when compared to other hydrophilic dietary compounds. The need to develop a novel strategy is of greater importance to enhance its bioavailability and thereby improving vitamin D level in the body. In this study, lipid-based delivery of oil-in-water nanoemulsion (diameter < 200nm) was utilized to improve the bioaccessibility and oral bioavailability of vitamin D3. First, we examined the in vitro relative bioaccessibility of nanoencapsulated vitamin D3 using a simulated gastrointestinal system. The study results showed that nanoemulsion-based delivery system significantly increased the relative bioaccessibility by 3.94 fold when compared to the coarse emulsion (diameter >200nm), as indicated by the concentration of vitamin D3 in the mixed micelles. To evaluate the in vivo bioavailability of vitamin D3 an animal study was conducted. Mice were assigned randomly to three groups: vitamin D3 nanoemulsion (n=6), coarse emulsion (diameter > 200nm) (n=6) and vehicle (nanoemulsion without vitamin D3) (n=3), which is the control group. After 3-days of feeding emulsion by mixing in drinking water, the serum 25(OH)D3, a biomarker of vitamin D availability, was measured using immunoassay. We found that serum 25(OH)D3 level in animals fed with vitamin D3 nanoemulsion was significantly higher than in those animals fed with coarse emulsion (22.7 ± 1.10 ngmL-1 vs 17.92 ± 2.82 ngmL-1). It indicated that nanoemulsion improved the in vivo bioavailability by 28%. These results showed that the nano-based delivery systems can be utilized to improve vitamin D level, and further human studies are warranted for its application to the human population in order to improve the vitamin D status.