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Há dados na literatura indicando que as células que saem do timo com o fenótipo CD4+CD25+ são desenvolvidas continuamente como uma linhagem independente e possuem um papel importante no processo de regulação da resposta imune. Essas células são chamadas células T reguladoras naturais. Várias questões sobre estas células permanecem em aberto, como por exemplo, como elas são geradas, o que é determinante na sua atividade reguladora e que marcadores específicos podem ser usados para identificá-las? Dentro deste contexto, o nosso objetivo neste trabalho foi identificar no timo e em timócitos CD4+/CD25+ humanos, novas moléculas potencialmente importantes no desenvolvimento e/ou na atividade supressora das células T reguladoras naturais. Para este objetivo, utilizamos a abordagem de phage display, com uma biblioteca de fagos de peptídeos, e timos humanos obtidos de pacientes portadores de cardiopatias congênitas, submetidos a cirurgias cardíacas realizadas no InCor. A busca dessas moléculas foi feita, separadamente, em 3 tipos de material biológico: timócitos totais, fragmento do tecido tímico e timócitos CD4+/CD25+. Antes da incubação da biblioteca de fagos com os timócitos totais e timócitos CD4+/CD25+ (separação em FACS), foi realizada uma etapa de preclearing, incubando-se a biblioteca de fagos com um pool de células mononucleares de sangue periférico (PBMC) ou timócitos CD4+/CD25-, respectivamente. Os fagos não ligantes, recuperados desta etapa, foram então incubados com as células de interesse. Para o tecido tímico não foi feita etapa de pre-clearing. Os fagos obtidos com os diferentes materiais biológicos foram recuperados em cultura de bactérias e usados em ciclos posteriores de seleção. Após três ciclos de seleção, os fagos foram seqüenciados e identificados quanto à expressão de peptídeos ligantes para timócitos totais, timo e timócitos CD4+/CD25+, e analisados em bancos de dados no BLAST. Os fagos selecionados para validação um ligante de tecido tímico: M2C e um ligante de timócitos CD4+/CD25+: R2A fazem similaridade a duas proteínas associadas ao metabolismo da Vitamina D3, molécula envolvida em imunorregulação e indução de tolerância, em diversos modelos experimentais. Porém, não há dados na literatura a respeito do seu papel em células T reg naturais. Na validação molecular desses fagos, apesar de certa variabilidade entre os diferentes ensaios, verificamos, por ELISA, que os fagos se ligam preferencialmente a 1,25 diidroxivitamina D3, forma ativa da Vitamina D3. Entretanto, nos ensaios de validação funcional, a influência da vitamina D na diferenciação dessas células não foi confirmada de forma consistente, uma vez que só tivemos aumento no número de células CD4+/CD25+, em cultura com Vitamina D, em poucos experimentos. As moléculas identificadas no presente estudo podem ter implicações relevantes no processo de diferenciação e na atividade de células T CD4+CD25+ reguladoras e serão mais investigadas na continuidade deste trabalho. / There are consistent data in literature indicating that thymic CD4+CD25+ cells play an important role in immune regulation and are continuously developed as an independent lineage in the thymus. These cells are known as natural regulatory T cells. Several questions about these cells remain unanswered, such as how they are generated, what is determinant in their regulatory function and which specific molecular markers can be used to identify them. Taking this into consideration, our aim was to identify new potentially important molecules in the development and/or supressive function of natural regulatory T cells, both in the thymus and in CD4+CD25+ thymocytes. For this, the phage display technique was employed, with a peptide phage library and thymic specimens obtained from children who underwent corrective cardiac surgery at the Heart Institute (InCor), in São Paulo. The search for these molecules was separately performed in 3 types of biological material: thymic tissue, thymocytes and CD4+CD25+ thymic cells. In the first stage, the phage peptide-library was incubated with a pool of PBMC (peripheral blood mononuclear cells). After the incubation, phages bound to PBMC were discarded (pre-clearing). In the second stage, unbound phages were incubated with either total thymocytes or CD4+CD25+ thymic cells. The pre-clearing stage was not perfomed in the thymic tissue. The phages obtained with after incubation with the different biological materials were recovered in E. coli culture and used in additional cycles of selection. After three rounds of selection, the recovered phages from the total thymocytes, from thymic tissue and thymocytes CD4+CD25+ were sequenced and their ligands identified. Among the phages selected for validation one ligand of thymic tissue: M2C and one ligand of CD4+CD25+ thymocytes: R2A present similarity to two proteins associated to the metabolism of Vitamin D3, a molecule involved in imunoregulation and toelrance induction in several experimental models. However, there are no data in the literature concerning the possible role of this moelcule in natural regulatory T cells. In the molecular validation of theses phages, although some variability between the diffeterent assays we have verified by ELISA, that the phages present preferential binding to the 1,25 dhydroxyvitamin D3, the active form of Vitamin D3. However, in the functional validation assays, the influence of the Vitamin D3 in the differentiation of these cells could not be consistently confirmed since we could observe an increase in the number of CD4+CD25+ cells cultured with vitamin D in only a few experiments. The ligand-receptor molecules we have defined in this study may have relevant implications in the development of CD4+CD25+ regulatory T cells in the thymus

To what extent conformational preference of short peptide sequences within proteins determine their three-dimensional structure? Large-scale quantum chemical calculations coupled with modern solvation methods represent unique set of tools to elucidate key determinants of the biomolecular structure ab initio. Full conformational sampling was performed on model systems representing short peptide fragments. The computed data reveal some of the underlying physico-chemical principles determining the spatial structure of proteins, and provide very important data for finding and tuning the optimal algorithm that may provide a full coverage of (ideally all) low-energy conformers. Keywords: Conformational space, peptide fragments, protein structure, solvation methods, Ramachandran plot, DFT-D3 methods

Calcitriol [1α,25(OH)2D3], the hormonally active form of vitamin D3 (D3) is produced in both renal and extrarenal tissues. Our findings demonstrate that physiological doses of UVB radiation at 300 nm induce the conversion of 7-dehydrocholesterol (7-DHC) via preD3 and D3 into calcitriol in the pmol range in epidermal keratinocytes. The hydroxylation of photosynthesized D3 to calcitriol is strongly suppressed by ketoconazole, a known inhibitor of cytochrome P450 mixed function oxidases. The UVB-induced formation of calcitriol in human skin is demonstrable in vivo by the microdialysis technique. These results suggest that human skin is an autonomous source of hormonally active calcitriol. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Visualization of software systems is mainly used for understanding and modeling software architecture. Examples of what can be visualized are source code, systems during execution together with manipulated data or system architecture on either a detailed or a general level. The connection between all of these types of visualization is that they help computer engineers maintain or develop software systems. The largest challenge with visualizing software systems is when the systems in question are exceedingly large and containing an abundance of relations. A problem of this kind arise at the company DGC, where this study is performed. The company manages at present more than a couple hundred systems with over ten thousand relations between each other. The problem with the company’s existing visualization is that they are very difficult to follow and understand, since they mainly present raw data in the form of tables that do not show all relations optimally, and which can also easily distract the company stakeholders from their intended task. The aim of this study is to find a visualization method where a system and its underlying parts are shown together with their relations to other systems. The created visualization will be able to be used by several different stakeholders and will as clearly as possible present relevant information to them. Furthermore, it should also aim to contribute to a better understanding of the complex software systems and their relations. After the implementation of the chosen visualization method, a time-based study is performed to determine whether it poses an improvement of perception among the stakeholders or not. An interview is also conducted where these stakeholders provide their opinion on the created visualization and if they think it presents the necessary information in a pedagogical way. Analysis of the result of the timekeeping and the interview answers indicate that the aim of the study is met. The majority of the resulting times show that the created visualization is more efficient than the original ones, and the interview answers furthermore show how different stakeholders all together agree that the visualization can be used in both future development and maintenance. / Visualisering av mjukvarusystem används i stor utsträckning för att förstå och modellera mjukvaruarkitektur. Ett par exempel på vad som kan visualiseras är: källkod, system under exekvering tillsammans med manipulerat data eller systemarkitektur på detaljerad eller övergripande nivå. Sambandet mellan alla dessa typer av visualiseringar är att de hjälper dataingenjörer att underhålla och utveckla mjukvarusystem. Den största utmaningen i att visualisera mjukvarusystem är när systemen i fråga är väldigt stora och har många beroenden. Ett sådant problem uppstår på företaget DGC där denna undersökning utförs. Företaget hanterar i dagsläge mer än hundratals mjukvarusystem med över tiotusentals beroenden sinsemellan. Problemet med företagets befintliga visualiseringar är att dessa är väldigt svåra att följa eftersom de oftast presenterar rådata i form av tabeller som inte visar alla relationer på ett optimalt sätt och distraherar lätt intressenter på företaget från sin avsedda uppgift. Undersökningen syftar på att finna en visualiseringsmetod där ett system och dess underliggande systemdelar visas tillsammans med deras beroenden till andra. Den framtagna visualiseringen skall kunna användas av flera olika intressenter, och i högsta möjliga mån presentera relevant information för dem på ett tydligt sätt. Vidare ska den skapade visualiseringen också bidra till bättre förståelse för komplexa mjukvarusystem och deras beroenden. Efter framtagandet av visualiseringen utförs en tidsundersökning, för att avgöra huruvida den skapade visualiseringen stimulerar förståelse hos de olika intressenterna på företaget. Det utförs även en intervju där intressenterna på företaget lämnar sina synpunkter angående den skapade visualiseringen och om den kan presentera nödvändig information på ett pedagogiskt sätt. Analys av resultaten av tidtagningarna och svaren på intervjuerna visar tydligt att undersökningens syften nåtts. Majoriteten av tidtagningarna visade att den skapade visualiseringen är effektivare än de ursprungliga och intervjusvaren uppvisar hur olika intressenter ser att den kan användas vid vidareutvecklingen eller underhåll, vilket alla intressenter höll med om.

Achieving low unemployment in an environment of weak growth is a major policy challenge; a more egalitarian distribution of hours worked could be the key to solving it. Whether worksharing actually increases employment, however, has been debated controversially. In this article we present stylized facts on the distribution of hours worked and discuss the role of work-sharing for a sustainable economy. Building on recent developments in labor market theory we review the determinants of working long hours and its effect on well-being. Finally, we survey work-sharing reforms in the past. While there seems to be a consensus that worksharing in the Great Depression in the U.S. and in the Great Recession in Europe was successful in reducing employment losses, perceptions of the work-sharing reforms implemented between the 1980s and early 2000s are more ambivalent. However, even the most critical evaluations of these reforms provide no credible evidence of negative employment effects; instead, the overall success of the policy seems to depend on the economic and institutional setting, as well as the specific details of its implementation. (authors' abstract) / Series: Ecological Economic Papers

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D3, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D3 also enhanced radiosensitivity and promoted autophagy in MCF7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D3 failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D3 was not attenuated by either a pharmacological or genetic block to autophagy; this was due largely to the promotion of apoptosis via the suppression of protective autophagy that occurs in response to radiation alone. Moreover, pharmacological blockade of autophagy did not sensitize noncancerous MCF10a cells to radiation; conversely, 4T1 mouse mammary tumors were highly sensitive to pharmacological inhibition of autophagy, suggesting selective radiosensitization against cancer cell lines. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D3). In addition, this work highlights the technical challenge of establishing the potential cytotoxic function of autophagy in an experimental system where the cytoprotective function may be concurrently expressed.

Master of Science / Department of Animal Sciences and Industry / Jim Nelssen / Seven experiments using a total of 3,251 preweaned pigs, nursery pigs, and sows were used to determine the effects of: 1) supplemental vitamin D[subscript]3 on suckling and nursery pig growth, and maternal performance, and 2) high sulfate water, dietary zeolite and humic substance on nursery pig performance. Also, a web-based survey was developed to question pork producers and advisors of the swine industry on their knowledge of feed efficiency. Experiment 1 tested an oral dose of either; none, 40,000 or 80,000 IU vitamin D[subscript]3 given to pigs 24 to 48 h after farrowing. No differences in growth performance or bone mineralization were observed, but vitamin D[subscript]3 supplementation increased serum 25(OH)D[subscript]3 on d 10, 20, and 30, but returned to control values by d 52. Experiments 2 and 3 evaluated an oral dose of vitamin D[subscript]3 to pigs just before weaning, as well as added D[subscript]3 in nursery diets and in drinking water. There were no effects on growth performance; however, serum 25(OH)D[subscript]3 increased with all sources of vitamin D[subscript]3 supplementation. Experiment 4 evaluated if pigs had a preference to 1 of 3 dietary concentrations of vitamin D[subscript]3. Pigs ate less feed from diets containing very high levels of vitamin D[subscript]3 compared to commonly supplemented levels. Experiment 5 evaluated 3 levels of vitamin D[subscript]3 in sow diets. There were no effects on sow productivity, subsequent pig performance, or piglet bone ash content. However, increasing vitamin D[subscript]3 increased sow serum 25(OH)D[subscript]3, milk vitamin D, and pig serum 25(OH)D[subscript]3. Experiment 6 and 7 evaluated the effects of dietary zeolite and humic substances in nursery pigs drinking high sulfate water. Ultimately, pigs drinking high sulfate water had increased fecal moisture content and decreased growth performance, and feed additives evaluated were ineffective in ameliorating these negative effects. Finally, data collected from the feed efficiency survey suggest that there are knowledge gaps about practices that effect feed efficiency. Results from this survey will help extension educators better target specific industry segments with current information and provide more specific areas of future research where lack of information has been identified.

This paper constitutes a first attempt to shed light on the role of income distribution in household debt, macrofinancial stability and financial market access in Central, Eastern and Southeastern Europe (CESEE). This issue has not been adequately addressed so far. Using data from the OeNB Euro Survey for the period from 2009 to 2017, we explore the question whether interpersonal comparisons affect a household's probability of having a loan. We use multilevel probit modeling to take into account the hierarchical structure of the data. Our results support the notion that the relative income position, along with absolute income, has an impact on households' likelihood of having a loan, but this is valid mainly for households above the median of the income distribution. We show this impact for almost all components of household debt, but evidence is strongest for mortgage and car loans. Interpersonal comparisons turn out to be a weaker predictor of a household's propensity to have a loan in CESEE countries with a more equal income distribution.

Sufficient vitamin D is important for overall health. However, cutaneous production of vitamin D is limited by season and little vitamin D naturally occurs in food. Therefore, vitamin D supplementation is necessary. Vitamin D is available in pharmacies as vitamin D2 and vitamin D3, and can also be obtained by irradiating mushrooms to produce vitamin D2. Types of vitamin D supplementation were tested to compare their ability to increase vitamin D status and their effect on broad gene expression in human white blood cells. 2000 IU of vitamin D2, vitamin D3 or vitamin D2 in irradiated mushroom powder were given to subjects daily for twelve weeks. A placebo mushroom powder group was included in the second half of the study. To determine the effect of different supplementation on vitamin D status, whole blood was obtained weekly and serum was assayed for 25(OH)D2 and 25(OH)D3. Change in total 25(OH)D was determined from baseline to twelve weeks; 25(OH)D levels in the placebo mushroom powder group did not change significantly at 1.8 ± 1.8 ng/ml (9.6 ± 9.6%), the mushroom D2 group increased by 10.9 ± 10.2 ng/ml (53.2 ± 49.8%), the supplemental D2 group increased by 11.8 ± 7.4 ng/ml (60.2 ± 37.8%) and the supplemental D3 group increased by 21.7 ± 8.9 ng/ml (114.2 ± 46.8%). As expected, the total active form of vitamin D (1,25-dihydroxyvitamin D) showed no change in all groups because of its tight regulation. To determine the potential influence of vitamin D supplementation on differential gene expression in the immune system, white blood cells were isolated from whole blood samples taken before and after supplementation. RNA was extracted, and microarray assays were performed. Gene Set Enrichment Analysis was completed to determine strongly influenced pathways. However, due to the numerous variables between halves of the study, gene expression data was treated as separate studies. Even so, pathways involving RNA activation and degradation were significant between mushroom powder and mushroom D2 supplementation in both halves of the study, indicating the influence of compounds in mushrooms on RNA metabolism pathways. Supplemental vitamin D2 affected gene expression, though only two pathways showed significant change. Supplemental vitamin D3 was found to influence pathways involved in replication, transcription, and translation in both halves of the study. In conclusion, mushrooms powder, mushroom vitamin D2, supplemental vitamin D2, and supplemental vitamin D3 all influence differential gene expression in human white blood cells.