Charakterisierung des Zytokinprofils in Erwachsenen mit einer latenten oder aktiven Tuberkulose in einem hoch endemischen Gebiet Die Tuberkulose (TB) stellt mit rund 2 Milliarden Infizierten weltweit ein globales gesundheitliches Problem dar. Während die große Mehrheit der infizierten Personen in der Lage sind die Krankheit zu kontrollieren, entwickelt sich bei ungefähr 10 % die aktive Form der TB aus. Der zugrunde liegende immunologische Prozess für diese Verteilung ist bis heute nicht bekannt und im Fokus dieser Arbeit. Das adaptive Immunsystem spielt eine entscheidende Rolle in der Immunabwehr gegen Mycobacterium tuberculosis (M. tuberculosis), dem Erreger der TB. Hierbei sind besonders CD4+ T-Zellen für die erfolgreiche Eingrenzung der Erkrankung verantwortlich. Im Vorfeld konnte bereits mehrmals eine Assoziation zwischen polyfunktionalen CD4+ T-Zellen und einem Schutz gegen verschiedenste Krankheitserreger gezeigt werden. Im Rahmen dieser Doktorarbeit wird versucht die Frage zu beantworten, ob eine erhöhte Frequenz von polyfunktionalen CD4+ T-Zellen auch gegen die Ausbildung einer aktiven TB schützen kann. Zur Bearbeitung dieser Fragestellung wurde das TH1 Zytokinprofil von Patienten mit aktiver TB untersucht und mit dem von gesunden latent infizierten Probanden (LTBI) verglichen. Desweiteren wurden die TB Patienten während der antimikrobiellen Therapie begleitet um Änderungen im Zytokinprofil von CD4+ T-Zellen beobachten zu können. Im Rahmen dieser Arbeit wurde zum ersten Mal die simultane Expression der vier TH1 Zytokine IFNg, TNFa, IL-2 und GM-CSF mit Hilfe der multifarben Durchflusszytometrie untersucht. Nach antigenspezifischer Stimulation konnten sowohl in unbehandelten und behandelten Patienten mit aktiver TB ein großer Anteil an multifunktionale Gedächtnis-T-Zellen nachgewiesen werden, die alle vier Zytokine gleichzeitig exprimierten. Bemerkenswerterweise konnte diese Population ebenfalls in LTBI gezeigt werden. Nach den ersten zwei Monaten der Therapie war der Anteil an multifunktionalen T-Zellen signifikant erhöht welches auf einen positiven Einfluss dieser Zellen auf die Behandlung hinweist. Um detaillierte Information über das Expressionspotential von CD4+ T-Zellen zu gewinnen wurden PBMCs mit einem Superantigen inkubiert. Hierbei unterschied sich das Zytokinprofil zwischen den beiden Studiengruppen signifikant und veränderte sich ebenfalls unter Therapie. Während die Expression von IFNg in TB Patienten niedriger war als in LTBI, war die Frequenz von TNFa, IL-2 und GM-CSF-positiver CD4+ T-Zellen signifikant höher in Patienten mit aktiver TB. Zusammenfassend ist zu sagen, dass sowohl in TB Patienten vor und nach Therapie, als auch in LTBI, multifunktionale CD4+ T-Zellen nachgewiesen werden können. Ein Unterschied in der Frequenz konnte dabei nicht festgestellt werden. Daher kann ein Zusammenhang zwischen der Existenz von multifunktionellen CD4+ T-Zellen und einem Schutz gegen eine mögliche Reaktivierung von der latenten zu der aktiven TB nicht beschrieben werden. / Characterization of the cytokine profile in adults with latent and active tuberculosis from a high endemic country Tuberculosis (TB) is a global health problem with ~2 billion infected people worldwide. The vast majority of infected individuals is able to control TB, while only ~10% develop active disease. The immunologic correlates determining the protection against reactivation of the latent form of active TB remain elusive. The adaptive immune system plays an important role in the response against Mycobacterium tuberculosis (M. tuberculosis), especially CD4+ T cells are crucial for efficient containment of the pathogen. Since polyfunctional CD4+ T cells have been associated with protection against various pathogens, the question was raised if higher frequencies of polyfunctional CD4+ T cells can be linked to protection against reactivation of active TB. To address this the TH1 T cell cytokine profile of active TB patients was analyzed and compared with healthy latently infected individuals (LTBI). Furthermore TB patients were followed up under anti-microbial therapy to monitor changes in the cytokine pattern expressed by CD4+ T cells. Hereby, for the first time, the simultaneous expression of four TH1 cytokines, IFNg, TNFa, IL-2 and GM-CSF, was investigated using multi color flow cytometry. After antigen-specific stimulation multifunctional memory T cells (CD45RO+) co-expressing IFNg, TNFa, IL-2 and GM-CSF were strongly represented in both treated and untreated TB patients. Interestingly, this proportion of polyfunctional memory T cells was also found in LTBI. After the first two months of drug treatment the proportion of antigen-specific polyfunctional T cells was significantly increased, indicating a positive impact of these cells during therapy. To gain detailed information about the potential of CD4+ T cells to produce cytokines we incubated PBMCs with a superantigen. In this case the profile was significantly different between these two groups and it changed during therapy. While the expression of IFNg was significantly lower in CD4+ T cell of TB patients in comparison to LTBI, the expression of TNFa, IL2 and GM-CSF showed significant higher frequencies in memory T cells of TB patients. To conclude, upon antigen stimulation, polyfunctional memory T cells are found in TB patients pre- and post therapy as well as in LTBI. A difference in the frequency between active TB patients and LTBI could not be detected and therefore a correlation with protection against reactivation from the latent to the active form of TB cannot be drawn.

L’œdème cérébral est une complication associée à l’encéphalopathie hépatique (EH) lors d’une insuffisance hépatique chronique (cirrhose du foie). Présentement, l’origine de sa pathogenèse, vasogénique (rupture de la barrière hémato-encéphalique (BHE)) ou cytotoxique (prise anormale d’ions), n’a pas encore été déterminée. Il a été démontré que le co-transporteur Na-K-Cl (NKCC1) du côté luminal des microvaisseaux sanguins cérébraux (CMV) joue un rôle dans le développement de l’œdème cérébral dans des modèles d’ischémie où la bumetanide, un inhibiteur de NKCC, atténue l’œdème cérébral. Deux modèles d’EH ont été utilisés pour cette étude i) la ligature de la voie biliaire (BDL) qui présente l’hyperammoniémie chronique, l’œdème cérébral et le stress oxydatif systémique ; ii) l’anastomose portocave (PCA) qui présente de l’hyperammoniémie chronique seulement. Les buts du projet étaient de: i) définir l’origine du développement de l’œdème chez les rats BDL en étudiant l’extravasation de macromolécules, les jonctions serrées et l’activation des métalloprotéinases matricielles de la BHE; ii) observer les effets de l’hyperammoniémie chronique indépendamment sur la BHE chez les rats PCA; iii) évaluer le rôle de l’hyperammoniémie et du stress oxydatif et iv) étudier le rôle du NKCC1 dans les CMV dans la pathogenèse de l’œdème cérébral. Les résultats du projet démontrent que l’œdème est d’origine cytotoxique chez les rats BDL et que l’intégrité de la BHE est conservée chez les rats PCA malgré l’hyperammoniémie. L’expression génique du NKCC1 est associée à l’œdème mais pas son expression protéique et sa phosphorylation. Enfin, l’étude démontre que l’hyperammoniémie et le stress oxydatif indépendant ne jouent pas un rôle dans la pathogenèse de l’œdème mais suggère qu’ils y aient un effet synergique. / Brain edema is a complication associated with hepatic encephalopathy (HE) due to chronic liver failure (cirrhosis). It is unclear whether brain edema is of vasogenic (blood brain barrier (BBB) breakdown) or cytotoxic (abnormal cellular uptake of ions) origin. It has been demonstrated that the Na-K-Cl cotransporter (NKCC1) located on the luminal side of the cerebral microvessels (CMV) is implicated in the pathogenesis of brain edema in animal models of ischemia and that the administration of bumetanide, an inhibitor of NKCC, attenuates brain water increase. Two distinct animal models of chronic liver failure and HE are used in the present study; 1) bile duct ligation (BDL) where brain edema, chronic hyperammonemia and systemic oxidative stress are observed; 2) portacaval anastomosis (PCA) where only chronic hyperammonemia is observed. The aims of the study were to: i) determine the origin of brain edema in BDL rats measuring brain extravasation, tight junctions expression and matrix metalloproteinase activation; ii) observe the effects of chronic hyperammonemia on the BBB in PCA rats; iii) study the role of oxidative stress and hyperammonemia; iv) evaluate the role of NKCC in CMV in the pathogenesis of brain edema. The results of the study determined that brain edema in BDL rats is of cytotoxic origin and chronic hyperammonemia independently has no effect on the BBB. An increase of NKCC1 mRNA is associated with brain edema but protein expression and phosphorylation are not. Furthermore, hyperammonemia and oxidative stress independently are not implicated in the development of brain edema however a synergistic effect between the two pathogenic factors in BDL rats remains a possibility.

<p>U radu je ostvarena sinteza četiri prirodna proizvoda, goniobutenolida A i B,<br />krasalaktona D i 3-deoksi-kardiobutanolida i 32 derivata i analoga polazeći iz<br />D-glukoze. Sinteza prirodnog stiril-laktona, kardiobutanolida&nbsp; i njegova 4<br />derivata je izvedena polazeći iz&nbsp; D-ksiloze.&nbsp; Ispitan je uticaj sintetizovanih<br />jedinjenja&nbsp; na inhibiciju rasta 10 tumorskih i jedne zdrave ćelijske linije.<br />Uspostavljene su i&nbsp; korelacije izmedju strukture i antiproliferativne aktivnosti&nbsp;(SAR) i ispitan je mehanzam antitumorskog dejstva.</p> / <p>Synthesis of four natural products: goniobutenolide A and B,&nbsp;crassalactone D, 3-deoxy-cardiobutanolide and their 32 analogues&nbsp;and derivatives is accomplished starting from&nbsp; D-glucose. Synthesis of natural styryl lactone cardiobutanolide and its four derivatives is &nbsp;achieved&nbsp; starting&nbsp; from&nbsp; D-xylose.&nbsp; Synthesized natural lactones, their analogues and derivatives were evaluated for their antiproliferative activity against ten malignant cell lines&nbsp; as well as against a single normal cell line. Influence of functional groups on antitumour activity was established by correlating structures of synthesized compounds&nbsp; with&nbsp; their&nbsp; biological&nbsp; activity (SAR).&nbsp; Also, mechanism of antitumour action was investigated.</p>

Na busca por espécies vegetais com atividade antileishmania, selecionaram-se, para o estudo, duas espécies bem conhecidas da família Asteraceae: Artemisia annua L. e Bidens pilosa L. Ambas são reconhecidamente utilizadas na medicina popular, como antiprotozoárias. Apesar de terem sido amplamente estudadas em diversos aspectos, alguns permaneceram inexplorados, até o momento, e foram abordados, neste trabalho. As duas espécies foram analisadas quanto aos aspectos químico e biológico de extratos (hidroetanólico e infuso) e frações orgânicas selecionados, em função da atividade antileishmania in vitro, frente às formas promastigotas de Leishmania amazonensis. Os extratos foram obtidos a partir de órgãos vegetais, em estados de conservação diferentes (in natura, droga) e coletados em fenofases distintas. Extratos e frações orgânicas das espécies estudadas mostraram promissora atividade antileishmania in vitro e baixo nível de citotoxicidade in vitro em células epiteliais humanas (HEP-2). No estudo químico dos extratos e frações bioativos, realizaram-se análises qualitativas e/ou quantitativas de terpenos, flavonóides e de marcadores específicos (artemisinina, quercetina e rutina), avaliando-se a variação da composição dos mesmos, nas diferentes fenofases consideradas. Discutiram-se as possíveis relações existentes entre a composição química e a atividade biológica verificada. Aspectos inéditos do estudo morfoanatômico de partes aéreas de A. annua foram descritos. / Plants are potential sources of new antileishmanial drugs. Two well-known antiprotozoal species were selected, from the Asteraceae family, for this study: Artemisia annua L and Bidens pilosa L. Despite the traditional and scientific accumulated knowledge, some aspects were not investigated before and were the subject of this work. Several extracts (infusions and ethanol 96 &#176;GL) and selected fractions from both species were evaluated according to the different parameters, such as: plant organs and/or parts, growth stages and drying state of starting materials (fresh, drug). Fractions were selected among those more active against promastigotes of Leishmania amazonensis. Ethanol extracts and their fractions showed a high level of in vitro antileishmanial activity and a low cytotoxicity on epithelial human cells (HEP-2). Qualitative and/or quantitative analysis of extracts and fractions were performed for terpenes, flavonoids and selected markers (artemisinin, quercetin and rutin) in order to characterize them and evaluate variations during the different growth stages. Correlations of the chemical composition and the biological activity were discussed. The main anatomical characters of the aerial parts of A. annua were described for the first time and illustrated by photomicrographs.

O citomegalovírus (CMV) é uma séria ameaça aos receptores de transplante de medula óssea. A reativação está associada com uma imunidade mediada por células TCD8+ defeituosa. Nosso objetivo foi correlacionar as diferentes subpopulações de células TCD8+ com a reconstituição imunológica dos pacientes, especificamente a imunidade anti-CMV, analisando as subpopulações de células T infundidas nas diferentes modalidades de transplante de medula óssea. Receptores de transplante alogênico de células tronco mobilizadas para o sangue periférico (n=16) ou coletadas diretamente da medula óssea (n=28) e receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico (n=22) foram avaliados. Verificamos que as transferências de células mobilizadas para o sangue periférico dos doadores, tanto nos transplantes alogênicos como autólogos, são proporcionalmente enriquecidas por subpopulações de células memória efetora e efetora, comparadas às transferências de células procedentes diretamente da medula óssea. Este enriquecimento por subpopulações de células TCD8+ mais diferenciadas foi também correlacionado com maior número de células contendo altos níveis de granzima B, considerado um marcador para linfócitos citotóxicos, sendo também encontrado em maior número nas transferências de células do sangue periférico. Entretanto, no pós-transplante, observou-se que somente os receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico, e não os das outras modalidades de transplante, exibiam números elevados de células T CD8+ de memória-efetora e efetora. Ao mesmo tempo, estes receptores apresentaram menos freqüentemente episódios de reativação pelo CMV, e mais freqüentemente produziram IFN-gama em resposta ao CMV. Portanto, a transferência de células do sangue periférico, desde que em ambiente autólogo, está associada não só com a transferência de células TCD8+ com um fenótipo mais maduro, mas também com uma persistência mais prolongada das mesmas, podendo proporcionar uma resposta imunológica antiviral mais rápida e eficiente, como esperado para as células de memória versus naïve. / Cytomegalovirus (CMV) is a serious threat to the recipients of bone marrow transplantation. Reactivation is associated with defective CD8+ T cell-mediated immunity. We aimed to correlate the different subsets of CD8+ T cells with the patients\' immune reconstitution, specifically anti CMV immunity, by analyzing the CD8+ T cell subsets infused in the different types of bone marrow transplantation. Recipients of allogeneic transplant of peripheral blood stem cells (n=16) or bone marrow (n=28) and recipients of autologous transplant of peripheral blood stem cells (n=22) were evaluated. We show that infusions of stem cells derived from donor\'s peripheral blood, either allogeneic or autologous, are proportionally enriched for the memory-effector and effector phenotypes, compared to the infusions of stem cells of bone marrow origin. This increased number of more differentiated subsets of CD8+ T cells was also correlated with an increased number of cells containing high levels of granzyme B, which is another reliable marker of cytotoxic lymphocyte, and which was also more evident in autologous recipients. However, post-transplant, we observed that only the recipients of autologous peripheral blood cells, and not the recipients of the other transplant modalities, exhibited very high numbers of memory-effector and effector TCD8+ cells. At the same time, they less frequently presented CMV reactivation, and more frequently produced IFN-gama in response to CMV antigens. Thus, transfer of stem cells from peripheral blood, provided in an autologous setting, is associated with transfer and prolonged survival of CD8+ T cells with a more mature phenotype, which may provide a more rapid and efficient anti-viral immune response, as expected for memory versus naïve cells.

<p>&nbsp;&nbsp; Cilj ove doktorske disertacije predstavljao je ispitivanje fitohemijskog sastava i biolo&scaron;ke aktivnosti vodenih i metanolnih ekstrakata svežih i suvih plodova, kao&nbsp; i pekmeza&nbsp; pripremljenog&nbsp; po tradicionalnoj recepturi od plodova&nbsp; četiri (od kojih se jedna javlja u dve forme)&nbsp; samonike&nbsp; vrsta&nbsp; roda&nbsp; <em>Sorbus</em>&nbsp; L.:&nbsp; S.<em> aucuparia</em>, S.<em> domestica</em>, S. <em>torminalis</em>&nbsp; f.&nbsp; t<em>orminalis</em>, S. <em>torminalis</em>&nbsp; f. <em>semitorminalis</em>&nbsp; i&nbsp; S. intermedia Ispitivanje fitohemijskog sastava obuhvatalo je LC-MS/MS analizu&nbsp; 44 odabrana fenolna jedinjenja&nbsp; i&nbsp; hinske kiseline (organska&nbsp; kiselina). Takođe, spektrofotometrijski je&nbsp; određen&nbsp; sadržaj&nbsp; ukupnih fenolnih i flavonoidnih jedinjenja, kao i&nbsp; sadržaj askorbinske kiseline. Evaluacija biolo&scaron;ke aktivnosti obuhvatala je&nbsp; <em>in vitro&nbsp; </em>ispitivanja antioksidantne aktivnosti, kao i ispitivanje uticaja ekstrakata odabranih vrsta&nbsp; roda&nbsp;<em> Sorbus</em>&nbsp; na aktivnost enzima acetilholinesteraze, antimikrobni, kao i antiproliferativni potencijal.</p><p>&nbsp;&nbsp; Sumiranjem dobijenih rezultata može se zaključiti da sveži i suvi plodovi ispitivanih vrsta&nbsp; <em>Sorbus</em>,&nbsp; kao i&nbsp; pekmezi&nbsp; predstavljaju&nbsp; umeren izvor&nbsp; fenolnih jedinjenja.&nbsp; Kao najzastupljenije fenolne kiseline izdvojile su se protokatehinska i&nbsp; ferulna, a među flavonoidima amentoflavon i kvercetin-3<em>-O-</em>glukozid.&nbsp; Pored toga, hinska kiselina je zabeležena u značajnoj količini u svim analiziranim ekstraktima.</p><p>&nbsp;&nbsp;&nbsp; Ekstrakti ispitivanih vrsta pokazali su&nbsp; umeren&nbsp; antioksidantni potencijal koji se ogleda u njihovoj sposobnosti neutralizacije nekoliko radikalskih vrsta, redukcionom potencijalu i sposobnosti inhibicije lipidne peroksidacije.&nbsp; Ispitivanjem uticaja ekstrakata odabranih vrsta&nbsp;<em> Sorbus</em>&nbsp; naaktivnost&nbsp; enzima&nbsp; acetilholinesteraze&nbsp; dokazana&nbsp; je&nbsp; jedino&nbsp; umerena aktivnost&nbsp; ekstrakata vrste&nbsp; <em>S. aucuparia</em>.&nbsp; Takođe, ispitivani ekstrakti&nbsp; vrsta roda&nbsp; <em>Sorbus</em>&nbsp; ispoljili su umerenu&nbsp; antimikrobnu&nbsp; aktivnost u pogledu inhibicije&nbsp; rasta Gram pozitivne bakterije,&nbsp; <em>Staphylococcus aureus</em>, i Gram negativne bakterije,&nbsp;<em> Escherichia coli</em>.&nbsp; Vodeni i metanolni ekstrakti svežih i suvih plodova vrste&nbsp; <em>S. aucuparia</em>&nbsp; pokazali su umeren i inhibitorni potencijal prema rastu tumorskih (HeLa, MCF7, HT-29), ali i netumorskih ćelijskih linija (MRC-5).&nbsp; Rezultati dobijeni u ovoj doktorskoj distertaciji&nbsp; ukazuju na značajan&nbsp; biopotencijal plodova i pekmeza&nbsp; ispitivanih vrsta&nbsp;<em> Sorbus</em>&nbsp; i ukazuju na njihovu primenu u prehrambenoj industriji u vidu funkcionalne hrane.</p> / <p>The aim of presented&nbsp; PhD&nbsp; thesis was investigation of phytochemical composition and biological activity of water and methanol extracts of fresh and air-dried&nbsp; fruits, as&nbsp;&nbsp; well&nbsp; as&nbsp; jam,&nbsp; made according to traditional recipe,&nbsp; of fruits&nbsp; of four&nbsp; (one of them occurs in two forms)&nbsp; wild growing&nbsp; <em>Sorbus</em>&nbsp; L. species:&nbsp; <em>S. aucuparia, S. domestica, S. torminalis&nbsp; f. torminalis, S. torminalis f. semitorminalis</em> and <em>S. intermedia</em>. Examination of phytochemical composition included LC-MS/MS analysis of 44 selected phenolic compounds&nbsp; and&nbsp; quinic acid (organic acid).&nbsp; Also, total phenolic and flavonoid contents, as well as&nbsp; ascorbic acid&nbsp; content,&nbsp; were determined spectrophotometrically. Biological activity evaluation of extracts of<em>&nbsp; Sorbus</em>&nbsp; species included&nbsp;<em> in vitro&nbsp;</em> investigation of antioxidant, anti-acetylcholinesterase, antimicrobial&nbsp; and cytotoxic activity.</p><p>&nbsp;&nbsp; According to obtained results, fresh and air-dried&nbsp; fruits, as well as jam&nbsp; present&nbsp; moderate&nbsp; source of phenolic compounds. Amongs examined phenolic acids protocatechuic&nbsp; and ferulic acids were the most abundant, and amongs investigated flavonoids amentoflavone and quercetin-3-<em>O</em>-glucoside wete present in noticeable amount.&nbsp; Furthermore, high concentration of quinic acid was present in all examined extracts.</p><p>&nbsp;&nbsp; Extracts of all examined species showed&nbsp; moderate&nbsp;&nbsp; antioxidant activity in terms of radical scavenging ability, reduction potential and inhibition of lipid peroxidation.&nbsp; Also, investigation of&nbsp; anti-acetylcholinesterase activity revealed moderate activity only of extracts of <em>S. aucuparia</em>. Furthermore, examinated extracts of<em> Sorbus</em> species showedmoderate antimicrobial activity against Gram&ndash;positive bacteria, <em>Staphylococcus aureus</em>, and Gram-negative bacteria,&nbsp;<em> Escherichia coli</em>.&nbsp; In addition, water&nbsp; and methanol&nbsp; extracts of fresh and air-dried&nbsp; fruits of&nbsp;<em> S.aucuparia</em>&nbsp; showed&nbsp; inhibitory activity toward&nbsp; tumor (HeLa, MCF7, HT-29), and also non-tumor (MRC-5) cell lines. Presented results indicate significant&nbsp; biopotential of examined&nbsp; fruits of&nbsp; <em>Sorbus&nbsp;</em> species&nbsp; and&nbsp; support their use in food industry as functional food.</p>

Το καρκίνωμα εκ πλακώδους επιθηλίου ή πλακώδες καρκίνωμα (ΠΚ) του δέρματος είναι ο δεύτερος πιο συχνός καρκίνος του δέρματος και εμφανίζεται συνήθως σε έδαφος ακτινικής υπερκεράτωσης (ΑΚ). Τα νεοπλασματικά κύτταρα εκφράζουν μια ποικιλία αντιγόνων προσελκύοντας με αυτό τον τρόπο λεμφοκύτταρα, τα διηθούντα τον όγκο λεμφοκύτταρα (Tumor Ιnfiltrating Lymphocytes-TILs) στο μικροπεριβάλλον του όγκου. Με βάση το ανοσοφαινοτυπικό τους προφίλ η πλειοψηφία των TILs εκφράζει το μόριο CD3 και αφορά σε Τ-λεμφοκύτταρα. Αυτά με την σειρά τους διαχωρίζονται σε CD8+ Τ-κυτταροτοξικά λεμφοκύτταρα και CD4+ Τ-λεμφοκύτταρα. Η έκφραση του δείκτη CD25+ επιτρέπει τον διαχωρισμό των CD4+ λεμφοκυττάρων σε δύο επιπλέον υποομάδες, τα T-επικουρικά (CD4+/CD25-) και τα Τ-ρυθμιστικά (CD4+/CD25+) κύτταρα. Ο πιο αξιόπιστος δείκτης για την ανίχνευση των Τ-ρυθμιστικών κυττάρων (Tregs) θεωρείται ο Forkhead box P3 (Foxp3). Ο αυξημένος αριθμός των TILs έχει συσχετισθεί με την πρόγνωση και τη θεραπευτική αντιμετώπιση σε μια ποικιλία νεοπλασμάτων, περιλαμβανομένων και καρκίνων του δέρματος όπως το μελάνωμα. Η μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης, ωστόσο, δεν έχει ακόμα αποσαφηνισθεί και ποικίλλει στα διάφορα είδη νεοπλασμάτων. Στα πλαίσια της παρούσας διπλωματικής εργασίας η ανίχνευση των Τ-λεμφοκυττάρων έγινε με ανοσοϊστοχημική μέθοδο με τη χρήση των κατάλληλων αντισωμάτων (CD3, CD4, CD8, Foxp3). Η παρουσία των κυττάρων αξιολογήθηκε με τη χρήση φωτονικού μικροσκοπίου με δυο διαφορετικά ημιποσοτικά συστήματα βαθμολόγησης για όλους τους μελετηθέντες Τ-κυτταρικούς υποπληθυσμούς καθώς και με ένα τρίτο σύστημα ποσοτικής καταμέτρησης για τα Τregs. Σκοπός ήταν να γίνει σύγκριση των μεθόδων ώστε να βρεθεί η καταλληλότερη μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης στο πλακώδες καρκίνωμα του δέρματος, να υπολογιστεί ο αριθμός των Tregs στις μελετούμενες οντότητες χρησιμοποιώντας το βέλτιστο σύστημα βαθμολόγησης και να αναζητηθούν διαφορές στην πυκνότητα των λεμφοκυτταρικών υποπληθυσμών μεταξύ των οντοτήτων, οι οποίες μπορεί να έχουν παθογενετική ή θεραπευτική κλινική σημασία. / Squamous cell carcinoma (SCC) of the skin is the second most common skin cancer. It usually develops in a background of actinic keratosis (AK). Neoplastic cells express a variety of antigens, attracting lymphocytes in the tumor microenvironment (Tumor Ιnfiltrating Lymphocytes-TILs). Based on the immunophenotypic profile, the majority of TILs expresses the CD3 molecule, so they are T-lymphocytes. These in turn are divided into CD8+ cytotoxic T-lymphocytes and CD4+ T-lymphocytes. The expression of CD25 enables the separation of CD4+ lymphocytes in two further subgroups, the T-helper (CD4+/CD25-) and T-regulatory (CD4+/CD25+) cells. The most reliable marker for the detection of T-regulatory cells (Tregs) is considered to be the Forkhead box P3 (Foxp3). The increased number of TILs is associated with prognosis and treatment in a variety of tumors, including skin cancers, such as melanoma. The evaluation methology of lymphocytic infiltration, however, is not yet clarified and varies in different types of tumors. In the context of this thesis, the detection of T-lymphocytes was performed by immunohistochemistry using the appropriate antibodies (CD3, CD4, CD8, Foxp3). The presence of the cells was evaluated using light microscopy with two different semiquantitative scoring systems, for all the T-lymphocyte subpopulations, as well as with a third system of quantification of Tregs. The purpose was to compare the methods in order to find the most suitable methodology for the evaluation of the lymphocytic infiltration in squamous cell carcinoma of the skin, to evaluate the number of Tregs in the studied entities using the best method and examine for differences in the density of lymphocyte subpopulations.

L’œdème cérébral est une complication associée à l’encéphalopathie hépatique (EH) lors d’une insuffisance hépatique chronique (cirrhose du foie). Présentement, l’origine de sa pathogenèse, vasogénique (rupture de la barrière hémato-encéphalique (BHE)) ou cytotoxique (prise anormale d’ions), n’a pas encore été déterminée. Il a été démontré que le co-transporteur Na-K-Cl (NKCC1) du côté luminal des microvaisseaux sanguins cérébraux (CMV) joue un rôle dans le développement de l’œdème cérébral dans des modèles d’ischémie où la bumetanide, un inhibiteur de NKCC, atténue l’œdème cérébral. Deux modèles d’EH ont été utilisés pour cette étude i) la ligature de la voie biliaire (BDL) qui présente l’hyperammoniémie chronique, l’œdème cérébral et le stress oxydatif systémique ; ii) l’anastomose portocave (PCA) qui présente de l’hyperammoniémie chronique seulement. Les buts du projet étaient de: i) définir l’origine du développement de l’œdème chez les rats BDL en étudiant l’extravasation de macromolécules, les jonctions serrées et l’activation des métalloprotéinases matricielles de la BHE; ii) observer les effets de l’hyperammoniémie chronique indépendamment sur la BHE chez les rats PCA; iii) évaluer le rôle de l’hyperammoniémie et du stress oxydatif et iv) étudier le rôle du NKCC1 dans les CMV dans la pathogenèse de l’œdème cérébral. Les résultats du projet démontrent que l’œdème est d’origine cytotoxique chez les rats BDL et que l’intégrité de la BHE est conservée chez les rats PCA malgré l’hyperammoniémie. L’expression génique du NKCC1 est associée à l’œdème mais pas son expression protéique et sa phosphorylation. Enfin, l’étude démontre que l’hyperammoniémie et le stress oxydatif indépendant ne jouent pas un rôle dans la pathogenèse de l’œdème mais suggère qu’ils y aient un effet synergique. / Brain edema is a complication associated with hepatic encephalopathy (HE) due to chronic liver failure (cirrhosis). It is unclear whether brain edema is of vasogenic (blood brain barrier (BBB) breakdown) or cytotoxic (abnormal cellular uptake of ions) origin. It has been demonstrated that the Na-K-Cl cotransporter (NKCC1) located on the luminal side of the cerebral microvessels (CMV) is implicated in the pathogenesis of brain edema in animal models of ischemia and that the administration of bumetanide, an inhibitor of NKCC, attenuates brain water increase. Two distinct animal models of chronic liver failure and HE are used in the present study; 1) bile duct ligation (BDL) where brain edema, chronic hyperammonemia and systemic oxidative stress are observed; 2) portacaval anastomosis (PCA) where only chronic hyperammonemia is observed. The aims of the study were to: i) determine the origin of brain edema in BDL rats measuring brain extravasation, tight junctions expression and matrix metalloproteinase activation; ii) observe the effects of chronic hyperammonemia on the BBB in PCA rats; iii) study the role of oxidative stress and hyperammonemia; iv) evaluate the role of NKCC in CMV in the pathogenesis of brain edema. The results of the study determined that brain edema in BDL rats is of cytotoxic origin and chronic hyperammonemia independently has no effect on the BBB. An increase of NKCC1 mRNA is associated with brain edema but protein expression and phosphorylation are not. Furthermore, hyperammonemia and oxidative stress independently are not implicated in the development of brain edema however a synergistic effect between the two pathogenic factors in BDL rats remains a possibility.

Malondialdehyde (MDA) is a mutagenic and carcinogenic product of lipid peroxidation which has also been found at elevated levels in smokers. MDA reacts with nucleic acid bases to form pyrimidopurinone DNA adducts, of which 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimidol[1,2-α]purin-10(3H)-one (M1dG) is the most abundant and has been linked to smoking. Mutations in the TP53 tumour suppressor gene are associated with half of all cancers. This research applied a multidisciplinary approach to investigate the toxic effects of MDA on the human lung fibroblasts MRC5, which have an intact p53 response, and their SV40 transformed counterpart, MRC5 SV2, which have a sequestered p53 response. Both cell lines were treated with MDA (0-1000 µM) for 24 and 48 h and subjected to a variety of analyses to examine cell proliferation, cell viability, cellular and nuclear morphology, apoptosis, p53 protein expression, DNA topography and M1dG adduct detection. For the first time, mutation sequencing of the 5’ untranslated region (UTR) of the TP53 gene in response to MDA treatment was carried out. The main findings were that both cell lines showed reduced proliferation and viability with increasing concentrations of MDA, the cell surface and nuclear morphology were altered, and levels of apoptosis and p53 protein expression appeared to increase. A LC MS-MS method for detection of M1dG adducts was developed and adducts were detected in CT-DNA treated with MDA in a dose-dependent manner. DNA appeared to become more fragmented with increasing MDA concentration, and the number of mutations in the 5’ UTR region of the TP53 gene also increased. The majority of mutations observed were insertions, compared to lung cancer mutation data where the majority were G to T transversions. This was unexpected, suggesting that tobacco smoke compounds have a different role in mutagenesis than endogenous lipid peroxidation. Thus, MDA has been found to have a clear effect on human lung fibroblasts at both the cellular and DNA level.

Na busca por espécies vegetais com atividade antileishmania, selecionaram-se, para o estudo, duas espécies bem conhecidas da família Asteraceae: Artemisia annua L. e Bidens pilosa L. Ambas são reconhecidamente utilizadas na medicina popular, como antiprotozoárias. Apesar de terem sido amplamente estudadas em diversos aspectos, alguns permaneceram inexplorados, até o momento, e foram abordados, neste trabalho. As duas espécies foram analisadas quanto aos aspectos químico e biológico de extratos (hidroetanólico e infuso) e frações orgânicas selecionados, em função da atividade antileishmania in vitro, frente às formas promastigotas de Leishmania amazonensis. Os extratos foram obtidos a partir de órgãos vegetais, em estados de conservação diferentes (in natura, droga) e coletados em fenofases distintas. Extratos e frações orgânicas das espécies estudadas mostraram promissora atividade antileishmania in vitro e baixo nível de citotoxicidade in vitro em células epiteliais humanas (HEP-2). No estudo químico dos extratos e frações bioativos, realizaram-se análises qualitativas e/ou quantitativas de terpenos, flavonóides e de marcadores específicos (artemisinina, quercetina e rutina), avaliando-se a variação da composição dos mesmos, nas diferentes fenofases consideradas. Discutiram-se as possíveis relações existentes entre a composição química e a atividade biológica verificada. Aspectos inéditos do estudo morfoanatômico de partes aéreas de A. annua foram descritos. / Plants are potential sources of new antileishmanial drugs. Two well-known antiprotozoal species were selected, from the Asteraceae family, for this study: Artemisia annua L and Bidens pilosa L. Despite the traditional and scientific accumulated knowledge, some aspects were not investigated before and were the subject of this work. Several extracts (infusions and ethanol 96 &#176;GL) and selected fractions from both species were evaluated according to the different parameters, such as: plant organs and/or parts, growth stages and drying state of starting materials (fresh, drug). Fractions were selected among those more active against promastigotes of Leishmania amazonensis. Ethanol extracts and their fractions showed a high level of in vitro antileishmanial activity and a low cytotoxicity on epithelial human cells (HEP-2). Qualitative and/or quantitative analysis of extracts and fractions were performed for terpenes, flavonoids and selected markers (artemisinin, quercetin and rutin) in order to characterize them and evaluate variations during the different growth stages. Correlations of the chemical composition and the biological activity were discussed. The main anatomical characters of the aerial parts of A. annua were described for the first time and illustrated by photomicrographs.