<p>In an attempt to gain more insight on functional evolution of cAMP-PKA pathway I have taken a comparative approach and examined functional interactions of cAMP-PKA signaling in well-studied yeast developmental programs and closely related <italic>Saccharomyces sensu stricto<italic/>. species. I have shown that variation in cAMP-PKA signaling contributes significantly to variation in developmental responses in <italic>S cerevisiae. Variation in pseudohyphal growth and sporulation, two inversely correlated developmental strategies to nutrient limitation in yeast, proportional to variation in intracellular cAMP levels. <italic>S. cerevisiae strains proficient in pseudohyphal growth have higher intracellular cAMP concentrations relative to strains that sporulate efficiently. Phenotypic, genetic and signaling data presented here suggest that the cAMP-PKA signaling underlies a phenotypic trade-off between sporulation and pseudohyphal growth in <italic>S. cerevisiae<italic/>.</p><p>Further investigation into the role of cAMP-PKA signaling in closely related <italic>S paradoxus<italic/> and <italic>S bayanus revealed an antagonistic function of cAMP-PKA signaling for developmental responses in <italic>S. bayanus. Unlike in <italic>S. cerevisiae, increased cAMP concentrations surprisingly inhibit pseudohyphal response in <italic>S. bayanus<italic/>. Another unanticipated finding in this work is that in <italic>S. bayanus<italic/>. Flo11, required for pseudohyphal differentiation in S. cerevisiae, is dispensable. Additionally, interactions of cAMP-PKA signaling and the general-stress response mechanism appear reversed in <italic>S. bayanus<italic/>. As shown by deletion mutation, gene expression and pharmacological treatment data, altered interactions and alternative targets downstream of cAMP-PKA could critically contribute to alternative regulation of nutrient-induced development in <italic>S. bayanus<italic/>.</p><p>Intracellular cAMP concentrations show decaying oscillations upon glucose replenishment in derepressed yeast cells. The quantitative characteristics of oscillations are distinct within and between Saccharomyces species. Given the tight regulation of cAMP levels and its critical role, the variation in cAMP oscillatory dynamics could be reflective of differential interactions of cAMP-PKA signaling that also underlie induction of developmental programs to changing environments. As such, intracellular cAMP levels and dynamics could potentially be used as molecular phenotypes.</p> / Dissertation

Η cAMP αποτελεί ένα σημαντικό δεύτερο μήνυμα που ρυθμίζει την ανοσολογική απόκριση. Η αύξηση της ενδοκυττάριας cAMP αυξάνει την παραγωγή της IL-10 από μονοκύτταρα. Σκοπός της μελέτης είναι η αποσαφήνιση της συμμετοχής της cAMP στην παραγωγή της IL-10 από Τ-λεμφοκύτταρα όπου τα δεδομένα είναι ακόμα ασαφή. Ανθρώπινα Τ-λεμφοκύτταρα περιφερικού αίματος διεγέρθηκαν με anti-CD3/anti-CD28, anti-CD3 ή Ionomycin/PMA παρουσία ή απουσία παραγόντων που αυξάνουν την ενδοκυττάρια cAMP (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram και 10-6 Μ 8-Br-cAMP). Το πρωτεϊνικό προϊόν της IL-10 μετρήθηκε με ELISA ενώ η παραγωγή mRNA της IL-10 με Real Time PCR. Η ενεργότητα του υποκινητή της IL-10 ελέγχθηκε με διαμόλυνση των κυττάρων με πλασμίδια που φέρουν τον υποκινητή του γονιδίου (1327 bp) ή τμήματα αυτού (-1010, -500, -310, -235, -135 bp). Η δέσμευση των μεταγραφικών παραγόντων MEF-2 και CREB ελέγχτηκε σε πυρηνικά πρωτεϊνικά εκχυλίσματα με πειράματα EMSA, ενώ η ενεργότητα τους ελέγχθηκε με πειράματα διαμολύνσεως με πλασμίδια που ελέγχουν την ενεργότητα της λουσιφεράσης υπό τον έλεγχο των MEF-2 και CREB. Αύξηση της cAMP ελαττώνει την παραγωγή της IL-10 σε πρωτεϊνικό επίπεδο κατά 50-60% μετά από διέγερση με Ion/PMA, και κατά 80-90% με anti-CD3 ή με anti-CD3/anti-CD28. Η IL-10 παράγεται ακόμα και μετά από διέγερση μόνο με anti-CD3, εύρημα ειδικό για την IL-10, καθώς δεν παρατηρήθηκε αύξηση της παραγωγής άλλων κυτταροκινων (IL-2 & IL-4). Η ελάττωση της παραγωγής της IL-10 αντανακλάται και σε επίπεδο mRNA όπου οι αντίστοιχες μειώσεις είναι κατά 50% με όλους τους τρόπους διέγερσης. Η ενεργότητα του υποκινητή της IL-10 δεν επηρεάζεται από αλλαγές στα επίπεδα της cAMP όταν η διεγερση παρακάμπτει τον Τ κυτταρικό υποδοχέα. Ωστοσο, μειώνεται παρουσία αυξημένων συγκεντρώσεων cAMP όταν τα κύτταρα διεγείρονται μέσω του Τ κυτταρικού υποδοχέα. Το τμήμα του υποκινητή της IL-10 που επηρεάζεται από την ανασταλτική δραση της cAMP (50 % αναστολή) βρίσκεται στις πρώτες 500 bp πριν το TATA box, και περιέχει σημεία πρόσδεσης των μεταγραφικών παραγόντων MEF-2 και CREB, όπως ελέγχθηκε με το πρόγραμμα Consite. Η δέσμευση του MEF-2 σε πυρηνικά εκχυλίσματα διεγερμένων Τ-λεμφοκυττάρων μειώνεται κατά 70% παρουσία αυξημένης cAMP ενώ η ενεργότητα του δεν επηρεάζεται σημαντικά. Αντίθετα, η αύξηση της cAMP αυξάνει τόσο τη δέσμευση (x 2,5) όσο και την ενεργότητα του CREB (x 2). Η δράση της cAMP στην παραγωγή της IL-10 είναι ειδική για τα Τ-λεμφοκύτταρα και εξαρτάται από τον τρόπο διέγερσής τους. Η ρύθμισή της γίνεται τόσο σε μεταγραφικό όσο και σε μετά-μεταγραφικό επίπεδο. Η αύξηση της cAMP μπορεί να επηρεάσει την παραγωγή IL-10 από τα Τ-λεμφοκύτταρα παρεμβαίνοντας στην δέσμευση και την ενεργότητα των παραγόντων μεταγραφής MEF-2 και CREB. Ο τρόπος της αλληλεπίδρασης/συνεργασίας των MEF-2 και CREB παραμένει υπό διερεύνηση. / cAMP is a second messenger playing a crucial role in the signal transduction which controls the immune response, while IL-10 is considered to be an important regulator of this response. Elevation of intracellular concentration of cAMP has been shown to increase IL-10 production by monocytes. The aim of this study was the elucidation of the role of cAMP in IL-10 production by normal T lymphocytes, a mechanism that remains unclear. Fresh Human Τ-lymphocytes derived from PBMC of healthy donors where stimulated with anti-CD3/anti-CD28 or Ionomycin/PMA, in the presence or absence of cAMP elevating agents (10-6 Μ Forskolin, 10-6 Μ PGE2, 5x10-6 Μ Rolipram and 10-6 Μ 8-Br-cAMP). The protein product of IL-10 was measured by ELISA, the production of IL-10 mRNA by Real Time PCR and IL-10 mRNA stability was determined by the use of Actinomycin D (10 μM). The activity of IL-10 promoter was measured by luciferase reporter assay, after transfection of cells with plasmids carrying the wild type promoter (1037bp) or promoter fragments (constructs of -1010, -500, -310, -235, -135bp). PKA role was examined either by cotransfection experiments with a plasmid carrying a constitutively active mutant of the catalytic subunit of PKA-α isoform, or by the use of a specific PKA inhibitor Rp-8- Br-cAMP (10-50 μM). The presence of binding sites of transcription factors in the first 500bp of the IL-10 promoter, was validated using the web-based program CONSITE. Binding of the transcription factors MEF2 and CREB was investigated in nuclear extracts of stimulated human T cells with EMSA experiments. The activity of MEF2 and CREB was investigated independently with transfection experiments using plasmids containing the lusiferase reporter under the control of the transcription factors. Intracellular cAMP elevation, inhibits IL-10 protein production by 50-60%, when T cells are stimulated with Ionomycin/PMA, and by 80-90% after stimulation with anti-CD3 or anti-CD3/anti-CD28, while PKA blocking by Rp-8- Br-cAMP reversed cAMP mediated inhibition.. IL-10 steady state mRNA levels follow the same pattern of inhibition only after anti-CD3/anti-CD28 stimulation. cAMP elevation decreases IL-10 mRNA stability after I/PMA stimulation, whereas in the anti-CD3/anti-CD28 stimulated cells, the mechanism of inhibition is mainly transcriptional. IL-10 promoter activity is reduced up to 60% when cells are stimulated with anti CD3/anti CD28 in the presence of cAMP elevating agents, but is not affected after stimulation with Ionomycin/PMA or cotransfection of the cells with constitutively active PKA mutant. Transfection assays with the different IL-10 promoter fragments revealed that the most responsible part of IL-10 promoter to cAMP mediated inhibition, is the first 500 bp after the TATA box. This part contains binding sites for the transcription factors MEF-2 and CREB, as validated by the web-based program Consite. Increased intracellular cAMP reduces the binding of MEF2 to nuclear extracts of stimulated T cells by 70 %, however its activity is not affected significantly. On the contrary, both the binding and the activity of CREB are increased in the presence of elevated cAMP. cAMP mediated inhibition of IL-10 production is PKA mediated and specific for T lymphocytes, depending on the nature/strength of stimulation. cAMP-dependent regulation of IL-10 production is controlled by transcriptional and/or post-transcriptional mechanisms depending on the nature of stimulus. Transcriptional mechanisms involve the transcription factors MEF2 and CREB, however the exact mechanisms of action of these factors deserves further elucidation. Cell and stimulus specific mechanism of regulation of IL-10 production is necessary for its immunoregulatory function.

Dovine River basin is a big contrast region. The south basin part falls into Suduva highlands and the north part falls into Uznemune lowland. This has very big influence to basin recreational distribution and development. The north basin part where is Zuvintas biosphere reserve with wetland complex and arable territory determine very low recreational activity and the south basin part with big lake and powerful landscape determine highly condition of recreation and tourism formation. Very important component of recreational potential of Dovine River basin is 33 lakes. Only big lakes Dusia, Simnas and Giluitis uses for recreation. The forested of Dovine River basin is only 23%, but these forest are very valuable. The biggest forest is Bukta, Kalniskes, Bijotai. There are 987 vegetal species, whereof 66 species are very protected and written into Lithuanian Red book and 5 species are European importance. There are 76 natural and cultural objects. Uneven recreational resources distribution, determine different basin tourism development. The north part of Dovine River basin with cultural heritage uses for cognitive tourism and the south basin part with big lakes and powerful landscape uses for rest tourism. The situation of tourist source of Dovine River basin is very good, because it situated between big Lithuanian cities Alytus and Marijampole. This influence very good condition of recreational facility. The recreation function of Dovine River basin is really versatile... [to full text]

This dissertation examines the experience of those involved in the typhoid fever outbreak at Camp Thomas, Chickamauga National Military Park, Georgia between April and August 1898. Among American volunteer soliders in the Spanish-American War, those stationed at this camp suffered the highest number of typhoid cases and deaths from typhoid. Treatments of the war have referred to the outbreak and some studies have examined it as part of wider subjects, but none from the standpoint of those involved, commanders, doctors, civilians, officers and enlisted men. The mobilized soldiers represented numerous states and reflected the disease experience of civilian society. The study considers the mobilization process, the disease outbreak and the aftermath.

Der israelische Autor und Journalist Noah Klieger ist in der deutschsprachigen Forschung zur Holocaustliteratur, in deren Kontext theoretische Konzepte und Interpretationen zahlreicher Autoren (u.a. Ruth Klüger, Primo Levi) dieser Gattung vorliegen, bisher kaum beachtet worden. In der vorliegenden Arbeit steht seine 2010 erschienene Autobiographie „Zwölf Brötchen zum Frühstück“ im Zentrum. Innerhalb der Textanalyse wird der Frage nachgegangen, welche Bedeutung das Schreiben für Klieger hat und inwieweit seine als Reportage angelegte Autobiographie, die den sehr faktenbezogenen und dokumentarischen Stil des Journalisten widerspiegelt, den Rezipienten in der Interpretation lenkt und Authentizität erzeugt. Ausgehend von dieser Fragestellung werden für die Arbeit geführte Interviews mit Noah Klieger (oral history) einbezogen und der Erlebnisbericht „Ich habe den Todesengel überlebt“ von Eva Mozes-Kor, die das Konzept des Erlebnisberichtes mit all seinen Eigenschaften konstant bewahrt, zum Vergleich hinzugezogen. Im Fokus der Arbeit steht die Analyse der Autobiographie Kliegers, wobei auf das Genre Reportage, relevante Stilmittel, zentrale Begrifflichkeiten und Veröffentlichungskontexte sowie auf die Gedächtnistheorie von Maurice Halbwachs eingegangen werden. Abschließend wird die Thematik des Vergebens bei Klieger und Mozes-Kor erörtert. Die Forschungsergebnisse stellen den israelischen Holocaustüberlebenden Noah Klieger als Autor vor und verdeutlichen, dass die innerhalb der Gattung Holocaustliteratur gewählten Darstellungsweisen unterschiedliche Formen von Authentizität evozieren. / Israeli author and journalist Noah Klieger has been ignored by German scientists of Holocaust literature though there are many theoretical concepts and interpretations of other writers such as Ruth Klüger and Primo Levi. In this paper we will focus on his autobiography published in 2010 named “Zwölf Brötchen zum Frühstück”. In the text analysis we will discuss what the act of writing means to Klieger and to what extent his autobiography reflects relation to facts, documentary pen and influences recipients. In accordance with this leading question we will integrate interviews with Noah Klieger (oral history) and a report from Eva Mozes-Kor titled “Ich habe den Todesengel überlebt”. This report constantly maintains the characteristics of a report. The centre of attention is the analysis of Noah Klieger’s autobiography, the characteristics of a report, rhetorical devices, defining key terminology, the context of publishing as well as Maurice Halbwach’s memory research theories. Finally, we discuss forgiving based on texts by Klieger and Mozes-Kor is discussed. The results of the research introduce Israeli holocaust survivor Noah Klieger as an author and clarify that within holocaust literature the different forms of expression evoke many different facets of authenticity.

Previous studies have suggested that phosphodiesterase (PDE) hydrolysis of cyclic adenosine monophosphate (cAMP) is important in the generation of specific and segregated cAMP signals within cells. The purpose of this study was to determine if PDE compartmentation was important in cardiac ventricular myocytes. Therefore, we investigated the effects of β-adrenergic (β-AD) stimulation with isoproterenol in the presence of cilostamide, a PDE3 inhibitor, or Ro 20-1724, a PDE4 inhibitor, on unloaded cell shortening, L-type calcium currents and intracellular calcium levels in freshly dissociated adult rat ventricular myocytes. PDE3 inhibition resulted in a 216 ± 17 % (n=8) increase in unloaded cell shortening after ten minutes of isoproterenol exposure, whereas isoproterenol produced a statistically smaller increase of 155 ± 12 % (n=8) in the presence of PDE4 inhibition. There was a non-significant trend for PDE4 inhibition to produce larger increases in calcium currents (179 ± 17 % (n=4) of controls) than PDE3 inhibition (155 ± 10 % (n=6) of controls). Both PDE3 and PDE4 inhibitors had similar effects on isoproterenol-stimulated increases of calcium transient amplitude with values of 209 ± 14 % (n=8) and 185 ± 12 % (n=8), respectively. Determination of sarcoplasmic reticulum (SR) calcium load using caffeine pulse experiments demonstrated that PDE4 inhibition and isoproterenol superfusion produced a statistically larger increase in SR-calcium loading (139 ± 9 % (n=6)) than PDE3 inhibition and isoproterenol superfusion (113 ± 9 % (n=6)). These results suggest that PDE3 may be active in proximity to the contractile apparatus of cardiac myocytes, whereas PDE4 may be localized in a domain consisting of the L-type calcium channel and junctional SR. Consequently, our study provides functional evidence for differential localization of PDE isoforms in cardiac myocytes. / Thesis (Master, Physiology) -- Queen's University, 2008-01-18 10:14:29.671 / CIHR OGS OGSST

The ubiquitous second messenger cAMP acts to integrate and translate information encoded by extracellular messenger molecules, including hormones and neurotransmitters. Intracellular cAMP concentrations are regulated through coordinated changes in the activities of adenylyl cyclases (ACs) and cyclic nucleotide phosphodiesterases (PDEs). Freely diffusing cAMP can reach concentrations sufficient to activate cAMP effector proteins, such as protein kinase A (PKA) or the exchange protein activated by cAMP (EPAC), except in defined compartments where PDEs are localized which allows for spatial and temporal control of the cAMP signal. In human aortic vascular endothelial cells (HAECs) and HEK293T cells we recently identified a macromolecular complex consisting of PDE3B and EPAC and showed that this complex coordinated cAMP-induced effects on adhesion of these cells to fibronectin-coated surfaces. Using “pull-down” assays and peptide array-based approaches we have identified the molecular determinants which coordinate the formation of this complex. Our evidence suggests that the extreme N-terminal 13 amino acids of PDE3B represent the portion of PDE3B that interacts with EPAC. In addition, although several EPAC-encoded peptides were shown to bind PDE3B, immunoprecipitation-based studies identified a region proximal to the cAMP-binding domains as likely to have a dominant role in this binding. Of functional relevance, a cell permeable peptide containing these amino-terminal 13 amino acids of PDE3B antagonizes PDE3B-EPAC interactions in cells. In addition, this peptide impacted the ability of cAMP-elevating agents to coordinate EPAC-dependent cell adhesions. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-10-06 19:07:12.01

Darbo objektas: jaunimo vasaros stovyklos „Merkurijus“ teikiamos paslaugos. Darbo tikslas: įvertinti jaunimo vasaros stovyklos „Merkurijus“ teikiamas paslaugas Darbo problema: Nepakankama paslaugų pasiūla jaunimui vasaros stovyklose? Darbo uždaviniai: 1. Atskleisti vaikų vasaros poilsio ir užimtumo organizavimo ypatumus. 2. Apžvelgti stovyklų pasiūlą pagal veiklos rūšis vasaros sezono metu Lietuvoje. 3. Ištirti vasaros stovyklos veiklą „Merkurijaus“ pavyzdžiu. Darbo išvados: Stovyklų tikslai ir uždaviniai orientuoti į optimalų stovyklautojų užimtumą, kad jaunimas ne tik atvažiavęs į stovyklą pailsėtų, bet ir išmoktų bendrauti, bendradarbiauti, sužinotų daug naujo ir kitą vasarą norėtų ten sugrįžti. Vaikams ir jaunimui siūlomos vasaros stovyklos ne tik Lietuvoje, bet ir užsienyje. Veikia daug stovyklų, orientuotų į vieną veiklą (sporto, kalbų, jaunųjų kariūnų, meno, pilietiškumo-tautiškumo, sveikatos, kultūrinio-etninio paveldo, lyderystės-pilietiškumo, pakantumo-tolerancijos, gamtosaugumo-ekologiškumo ir daugiau). Lyginant vasaros stovyklos „Merkurijus“ veiklą su kitomis Lietuvos stovyklomis galima teigti, kad visos Lietuvos stovyklos turi tuos pačius siekius: kad atostogaujantis jaunimas patirtų kuo daugiau teigiamų įspūdžių, kad sužinotų kažką naujo, kad vėl norėtų sugrįžti. Skatinti vaikus vykti į stovyklą, pasakoti daugiau apie stovyklų naudą ir jose vykdomus užsiėmimus. Mokyklose vaikams pravesti vasaros laisvalaikio leidimo pamokėles apie galimybės išvykti į stovyklas... [toliau žr. visą tekstą] / Object: youth summer camp „Merkurijus“ activity. Objectives: of the thesis: To appraise youth summer camp „Merkurijus“ activity. Problem: underexpose supply to the youth of a summer camp complexion activities? Aims: 1) to reveal children summer recreation and occupation organisation singularities. 2) to review the camps supply, according of activity types at the summer season in Lithuania 3) analyse the type of a summer camp activities accordind „Merkurijus“ example and relatively with the other camps. Conclusions: the camps objetives and aims are focused to the optimal campers occupation, that it wouldn‘t be the reason for the youth just to relax, also to learn how to socializme, collaborate, to know a lot of new and want to come back next summer. There are a lot of working camps, which are focused to the one activity ( sport, languages, art, tolerancy, young-warrior, citizion-nationality, ecology and others). To comparable summer camp „Merkurijus“ activity with Lithuania camps, may to propose that all Liethuania camps have one and the same aims: that youth which are in holiday, expereance more positive impressions, to know something new and to reach them to retrieve back again. To stimulate the children go to the camps, to tell them more about the camps benefits and activities, which are pursued. In the schools for the children to give the lessons about recreation, opportunaties to go to other camps, to give them notelets and to argument why is useful and litting there to... [to full text]

The inability to tan properly after sun exposure strongly correlates with increased incidence of skin cancer. The melanocortin 1 receptor (MC1R) is a transmembrane Gs-coupled cell surface receptor found on epidermal melanocytes that transmits pro-survival and pro-differentiation signals mediated by the second messenger cAMP. Humans carrying loss-of-function polymorphisms in MC1R signaling exhibit higher incidences of skin cancers including melanoma. This study focused on the physiologic effects of topical application of forskolin, an adenylate cyclase activator, in extension (Mc1re/e) K14-SCF animals, which model the fair-skinned UV-sensitive human. Twice daily application of the drug promoted accelerated pigmentation, increased skin darkening due to epidermal deposition of melanin pigment, and induced epidermal melanin, which protected the skin against UV injury as judged by “minimal erythematous dose” (MED). Moreover, MC1R signaling regulated the expression of antioxidant enzymes at the transcriptional level. The human melanoma cell line A375, known to harbor a loss-of-function signaling mutation in MC1R, was used to determine effects of cAMP stimulation on the expression of antioxidant enzymes. We observed increases in expression of genes that control the biosynthesis and regulation of glutathione including the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), glutathione peroxidase, GPX, and glutathione reductase GSR. In addition, there is an increase in manganese superoxide dismutase (MnSOD) at the protein level. There was accumulation of MnSOD in the mitochondria after pharmacologic induction of cAMP with forskolin. Addition of the oxidative agent H2O2 enhanced the expression of MnSOD at the protein level as early as one hour after MC1R stimulation. Oxygen consumption rate on mitochondria was measured using Seahorse analysis; pharmacologic activation of MC1R/cAMP signaling did not affect mitochondrial metabolism. In addition, topical application of a crude extract of Solidago inhibited UV-induced inflammation in K14-SCF mice. Several UV-induced cytokines, including TNF-α, were down-regulated at the transcriptional level after topical application of Solidago extract. Together, these results indicate that MC1R signaling protects melanocytes from UV damage by regulating antioxidant enzyme expression and suggest that pharmacologic cAMP induction may be a useful preventive mechanism against UV-mediated skin sunburn and oxidative injury.

No description available.