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.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Psoriasis is a chronic inflammatory skin disease featuring abnormal keratinocyte proliferation and differentiation. A genetic risk factor for psoriasis (PSORS4) is a deletion of LCE3B and LCE3C genes encoding structural proteins in differentiated keratinocytes. Because analogs of 1,25-dihydroxyvitamin D3 (1,25D) are used in psoriasis treatment, we hypothesized that 1,25D and other VDR ligands act via the vitamin D receptor (VDR) to upregulate expression of LCE 3A/3D/3E genes, potentially mitigating the absence of LCE3B/LCE3C gene products. This hypothesis was pursued using cultured keratinocytes, quantitative real time PCR (qPCR) to assess LCE gene expression, competition binding assays to assess direct ligand binding to VDR, and reporter gene assays to assess the ability of VDR ligands to activate transcription in a VDR- and VDR response element-dependent fashion. qPCR results in a human keratinocyte line, HaCaT, suggested that 1,25D and selected alternate or candidate VDR ligands might upregulate LCE transcripts. Further experiments in primary human keratinocytes confirmed that 1,25D and 10 µM delphinidin do indeed upregulate all five LCE3 genes (LCE3A–E), especially in calcium-differentiated cultures. Further, competition binding assays revealed that delphinidin does in fact bind VDR, but only weakly (IC50 approximately 1 mM). Finally, 20 µM delphinidin was shown to be capable of upregulating a luciferase reporter gene linked to a vitamin D responsive element found near the LCE3 gene cluster. Taken together, these results are consistent with delphinidin (or a metabolite) stimulating LCE3 transcription in a VDR/VDRE-dependent manner. We propose that upregulation of LCE genes may be part of the therapeutic effect of 1,25D to ameliorate psoriasis by providing sufficient LCE proteins, especially in individuals missing the LCE3B and 3C genes. Results with delphinidin further suggest that this compound or its metabolite(s) might offer an alternative to 1,25D in psoriasis therapy.

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

When linear arrays of unrelated letters (e.g., 'sfdthnc') are presented tachistoscopically centred across a fixation point, letters presented at exterior positions (e.g., 's----c') aregenerally reported more accurately than letters presented in interior positions. This "exterior-letter advantage" suggests that processing is more efficient for exterior letters than for interior letters. Previous researchers have argued that the exterior-letter advantage can be fully accounted for by the influences of lateral interference and mask configuration. However, the processes responsible for the exterior-letter advantage are far from resolved, despite the robustness of the phenomenon and its occurrence in numerous investigations into visual information processing. The experiments reported in this study investigated the role of lateral interference and backward pattern masking in the exterior-letter advantage. To investigate the role of lateral interference, performance was compared across complete 7-letter arrays and arrays in which the presence and proximity of flanking letters was varied by (i) presenting only exterior letters and their immediately flanking interior letter, (ii) varying the number blank letter-spaces by which these letter-pairs were separated, (iii) varying the nature of the characters presented in these displays, and (iv) presenting each exterior/interior letter-pair in isolation. The role of backward pattern masking was investigated (i) using different mask configurations which either matched or exceeded the left and right boundaries of complete letter arrays, and (ii) using masks which overlay only the positions of each exterior/interior letter-pair. The findings indicate that while lateral interference and mask configuration each played a part, neither an imbalance in the number of immediately flanking letters for interior and exterior letters nor mask configuration can entirely account for the exterior-letter advantage.

Experiments were conducted on the cellular responses of monocotyledonous and dicotyledonous plants to sublethal and lethal temperatures. The study had four objectives: I. to compare temperatures that develop in stems and leaves of plants under flame, II. to determine the effect of heat on conduction in the phloem (of stems), III. to investigate some cellular responses to sublethal and lethal temperature treatments, and IV. to determine the effects of lethal temperatures on respiration and on the Hill reaction. When stems and leaves of plants were exposed to a flame at 1900°F. for 130 milliseconds, there was a reduction in amount of heat that penetrated into stems of monocotyledon plants as compared to that penetrating into stems of dicotyledon plants. This difference in penetration appeared to be related to the morphology of the stem at the time of measurement. There were no differences between species tested in leaf tissue injury as indicated by degree-second summations. Heat penetrated less into stems than into leaves, and less into older plants than into younger plants. The temperature was higher at the stem periphery than near the stem center. C¹⁴ activity was greatly reduced in the roots of flame treated soybean plants when compared to the non-flamed plants 24 hours after the photosynthesizing parts of the plants had been exposed to C¹⁴O₂. The function of the sieve elements in conduction of the C¹⁴ photosynthates may have been temporarily blocked by the heat treatment. Loss of chlorophyll and swelling of chloroplasts were observed in cells of elodea leaves exposed to sublethal temperature treatments. At the thermal death point of leaf cells of elodea, corn, soybeans, pigweed, and yellow foxtail there appears to be a disorganization of the tonoplasts, plasma membrane, chloroplast membranes, and an irreversible stoppage of cytoplasmic streaming in leaf cells of elodea. Approximately 40 percent of the cells in elodea leaves show criteria of cell death at a temperature treatment which results in necrotic leaf tissue. Plasmolysis was observed in leaf cells at the lethal temperature treatments but did not appear to be a primary event at the cellular thermal death point. Changes in leaf cells of elodea after death were observed on certain days after the leaves had been exposed to lethal temperature treatments. In plasmolyzed cells of elodea leaves, observed immediately after treatment, there appeared to be chlorophyll in the cytoplasms and the chloroplasts appeared shrunken. The chloroplasts stained a dark blue in these cells (when stained with toluidine blue) indicating a disorganization of the chloroplasts. Cells observed two days after lethal temperature treatments exhibited slightly bleached chloroplasts, the cytoplasms appeared to be coagulated, and the plasma membranes appeared to be breaking down. The nucleus appeared to be still intact in some of the cells. Cells observed four days after lethal temperature treatments exhibited bleached chloroplasts with only remnants of the chloroplasts being apparent in some cells. The protoplasm in all the cells show characteristics of coagulation and the plasma membrane and the nuclear membrane appeared to be broken-up. A sublethal temperature treatment, which had produced chlorosis in vivo, reduced the Hill reaction of isolated soybean chloroplasts but not corn chloroplasts. Lethal temperature treatments reduced the rate of respiration of corn and soybean leaf discs to less than 50 percent of the rate of the control treatment. / Ph. D.

Les rats GAERS (Genetic Absence Epilepsy Rats from Strasbourg) et NEC (Non-Epileptic-Control rats) dérivent de la lignée de rats Wistar-Hannover (WH). Chez les GAERS les premières crises d’épilepsies apparaissent vers l’âge de 25 jours puis leur nombre et leur durée augmentent pour se maintenir à l’âge adulte à raison d’une crise de 20 secondes en moyenne par minute lorsque l’animal est en veille calme. Ce développement spontané des crises d’épilepsies permet d’étudier la période silencieuse avant les premières crises ainsi que la survenue de ces premières crises (l’épileptogènese). De nombreuses études pharmacologiques et électrophysiologiques dans des modèles animaux d’épilepsie révèlent un rôle critique de la transmission dopaminergique dans la susceptibilité aux crises. Les GAERS adultes présentent une surexpression de l’ARNm des récepteurs dopaminergiques D3 comparé aux rats NEC. L’expression ainsi que la fonction du récepteur D3 et celles du transporteur de la dopamine (DAT) sont liées. Le but de ce travail a été de déterminer l’implication du RD3 et du DAT dans le développement de l’épilepsie en mesurant leur expression et fonction chez les GAERS avant l’apparition des premières crises. L’expression et la fonction du RD3 ont été mesurées par autoradiographie au [125I]-PIPAT et induction de bâillement par injection de quinpirole respectivement, dans les 3 lignées de rats (GAERS, NEC and WH) chez des rats adultes, à P14 et P21. L’expression du DAT a été analysée par imagerie SPECT à [123I]-Ioflupane chez les adultes et par autoradiographie au [3H]-GBR12935 chez les adultes, P14 et P21. Par ailleurs l’activité du DAT a été mesurée par capture de dopamine tritiée sur des synaptosomes de striatum, cortex et hippocampe de rats adultes des 3 lignées. La participation du RD3 dans les crises a été évaluée par EEG vidéo après l’injection d’agoniste D3 (quinpirole et PD128907) ou d’antagoniste (SB277011 et SR21502). Les expériences de radiographie ont révélé une surexpression des RD3 chez les GAERS dans les structures connues pour participer à l’initiation des crises (cortex somatosensoriel), à leur contrôle (noyaux accumbens) ainsi que dans d’autres structures (thalamus antérieur, tubercules olfactifs et ilots de calleja), à P14 et P21 comparé aux autres lignées. Ni le SPECT ni l’autoradiographie n’a relevé de modification d’expression du DAT chez les rats adultes, cependant à P14 et P21 le DAT est surexprimé au niveau du striatum. Ces résultats sont accompagnés d’une augmentation de la fonctionnalité du DAT aux 3 âges chez les GAERS compares aux autres lignées. Les agonistes D3 induisent une augmentation des décharges de pointes-ondes sans que les antagonistes aient un effet. De plus l’injection chronique d’aripiprazole (un neuroleptique connu pour sa capacité à stabiliser la libération de dopamine) chez les rats GAERS dès la naissance entraine une diminution des crises associée à une diminution d’expression des RD3. L’utilisation de lentivirus contenant une séquence shRNA anti-RD3 chez les GAERS adultes entraine aussi une diminution du nombre de crises d’épilepsie. Nos résultats suggèrent ainsi que l’augmentation du nombre de RD3 chez le GAERS est concomitante à une augmentation de la sensibilité du système dopaminergique, et cela avant l’apparition des premières crises épileptiques. Cette augmentation au cours de la maturation pourrait participer à l’aggravation progressive des crises d’épilepsie qui est la base de l’épileptogenèse et expliquer le déséquilibre de la tonicité dopaminergique que nous avons démontré chez l’adulte / Genetic Absence Epilepsy Rats from Strasbourg (GAERS) and Non-Epileptic-Control rats (NEC) derive from an original Wistar-Hannover rat strain (WH). The onset age of spike-and-waves discharges in GAERS is about 25 days post-natal (P25). In adult GAERS with fully developed epilepsy, dopamine plays a modulatory role in seizure expression. Adult GAERS display an over-expression of dopaminergic D3-receptors (D3R) mRNA as compared to NEC. Expression and function of D3R and dopamine transporter (DAT) are closely related. The aim of this work was to investigate the putative involvement of D3R and DAT during epileptogenesis by measuring their expression and functionality in GAERS before the onset of epilepsy (P25). D3R expression and functionality was investigated by [125I]-PIPAT autoradiography and quinpirole-induced yawning, respectively, in the three strains of rats (GAERS, NEC and WH) in adults, P14 and P21. DAT expression was investigated in GAERS and NEC by [123I]-Ioflupane SPECT imaging in adults and [3H]-GBR12935 autoradiography in adults, P14 and P21 rats. Furthermore, DAT activity was assessed by 3H-dopamine reuptake in synaptosomal living fractions of striatum, cortex and hippocampus of adult rats in the three strains. The involvement of the D3R was further investigated by video-EEG recording following systemic injections of either D3R agonist (quinpirole and PD128907) or antagonists (SB277011 and SR21502). Autoradiography showed an over-expression of D3R in GAERS in structures known to be involved in seizure initiation (somato-sensory cortex), seizure control (nucleus accumbens,) as well as in other structures (anterior thalamus, olfactory tubercles and islands of Calleja) at P14 and P21, as compared to age-matched NEC and WH. As in adults, this over-expression was associated with a higher number of quinpirole-induced yawns in GAERS at P14 and P21. Neither SPECT imaging nor autoradiographic data revealed any modification in DAT expression between the three strains in adults, however at P14 and P21 DAT is overexpressed in the striatum of GAERS rats. However, we found a consistent increase in 3H-dopamine reuptake in adult GAERS as compared to NEC and WH in the functional assay supporting an increase in dopamine translocation velocity. Administrations of D3R agonists increased spike-and-wave discharges, whereas antagonists had no effect. Furthermore, the chronic injection of aripiprazole (an atypical neuroleptic known to stabilize dopamine release) to GAERS pups reduced the number of seizures in adults along with a decreased expression of D3R. Preliminary data using lentiviral infection with shRNA anti-D3R also support reduced seizure number in adult GAERS rats. Our results suggest that an over-expression of functional D3R already exists before the onset of seizures in GAERS and that, despite a lack of changes in DAT expression, functional changes in this transporter occur in adults. They further support that a profound modification in basal ganglia function together with changes in D3R could be a conditional factor for epileptogenesis. The dopaminergic system appears persistently altered in spontaneous epileptic rats, which could contribute to the development of the chronic epileptic state and may represent a potential new target for antiepileptic therapies and/or improvement of quality of life of epileptic patients.

Detta dokument beskriver ett kandidatexamensarbete som genomförts av åtta civilingenjörsstudenter från Linköpings universitet. Examensarbetet bestod av att utveckla en webbapplikation för att möjliggöra navigation och visualisering av element ur den medicinska databasen SNOMED CT samt deras relationer. Den första delen av dokumentet består av gruppens gemensamma arbete och erfarenheter för att uppfylla kundens givna krav samt själva processen om hur det är att arbeta i ett större mjukvaruprojekt. Den andra delen består av studenternas individuella bidrag till examensarbetet som berör något spridda ämnen men som har en anknytning till mjukvaruutveckling och projektarbete.

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.