Orientador: André Luiz Barboza Formiga / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-25T06:44:24Z (GMT). No. of bitstreams: 1 Profirio_DanieldeMoraes_M.pdf: 7198649 bytes, checksum: 018efc71d59b6427976118e0cc5f772a (MD5) Previous issue date: 2014 / Resumo: Neste trabalho realizou-se a síntese de três ligantes contendo a unidade pirrolil-imina e de um complexo de Au(III). Os ligantes foram sintetizados partindo-se do pirrol-2-carboxaldeído e etilenodiamina ou orto-fenilenodiamina, obtendo-se assim o N,N'-bis(pirrol-2-il-metileno) etano-1,2-diamina (pyren), o N,N'-bis(pirrol-2-il-metileno)benzeno-1,2-diamina (bis-pyrophen), o N-(pirrol-2-il-metileno)benzeno-1,2-diamina (monopyrophen) e o complexo [Au(pyren)]PF6 como produtos. Os compostos foram caracterizados por análise elementar, MS, espectroscopia no IV e UV-Visível, RMN de H, C e HMBC H-N, cálculos teóricos por DFT e TD-DFT. Foram realizados também ensaios antibacterianos e citotóxicos com o complexo [Au(pyren)] e com o ligante pyren, sendo que o complexo apresentou maior atividade em ambos os ensaios comparado ao ligante. Com isso um mecanismo de ação via interação com o DNA foi investigado por estudos de fluorescência, ensaios de competição com brometo de etídio e espectroscopia de dicroísmo circular, revelando que o DNA é um possível alvo biológico do complexo / Abstract: In this work the syntheses of three ligands containing the pyrrolyl-imine unit and a Au(III) complex were performed. The ligands were synthesized starting from pyrrole-2-carboxaldehyde and ethylenediamine or ortho-phenylenediamine, to obtain N,N¿-bis (pyrrol-2-yl-methylene)ethane- 1,2-diamine (pyren), N,N¿-bis(pyrrol-2-yl-methylene)benzene-1,2-diamine (bis-pyrophen), N-(pyrrol-2-yl-methylene)benzene-1,2-diamine (mono-pyrophen) and the complex [Au(pyren)]PF6 as products. The compounds were characterized by elemental analysis, MS, IR, UV-Visible, H, C and (H-N) HMBC NMR spectroscopies and theoretical calculations by DFT and TD-DFT. Cytotoxic and antibacterial assays with [Au(pyren)] and the free ligand pyren were performed, and the complex showed a higher activity in both cases in comparison to the ligand. A mechanism of action via interaction with DNA was investigated by fluorescence techniques, competition studies with ethidium bromide and circular dichroism spectroscopy, showing that DNA is a potential biological target for the complex / Mestrado / Quimica Inorganica / Mestre em Química

Historiquement, les LT CD8 cytotoxiques ont été considérés comme la seule composante cellulaire du système immunitaire nécessaire et suffisante pour l’élimination de cellules infectées par des virus ou transformées, les LT CD4 ne jouant qu’un rôle auxiliaire, dans le développement et le maintien de la réponse immune effectrice, ou modulateur par la fonction suppressive des T-Reg. Aux côtés, de ces fonctions auxiliaires ou suppressive, nombre de données indiquaient que les LT CD4 pouvaient également exercer une activité cytotoxique directe. Nos travaux ont permis par l’analyse chez l’Homme, de l’expression des récepteurs α à l’IL-2 (CD25) et à l’IL-7 (CD127) à la surface des LT CD4 du sang périphérique d’identifier une population singulière de LT CD4 caractérisée par l’absence de ces deux molécules. Ces LT CD4, CD25-CD127-, faiblement représentées chez les sujets sains, entre 0,2-2% des LT CD4 totaux du sang périphérique, étaient fortement augmentées, entre 2-20% dans les infections chroniques VIH et Tuberculose, et notamment dans les cancers incluant les mélanomes uvéaux métastatiques (Mum) et les cancers du sein. Puisque prédominant dans des situations de stimulation chronique, ces LT CD4 ont été définis comme des LT CD4 chroniquement stimulés : chCD4. Dans le sang périphérique de patients atteints de cancer comme chez les sujets sains, la majorité de ces chCD4 arboraient un phénotype mémoire/effecteur (CD45RO+). Cependant, dans les Mum et les cancers du sein la proportion de chCD4 effecteurs (CD45RO+CD27-) était fortement augmentée. Par ailleurs, si la plus part de ces cellules effectrices apparaissaient à un stade de différenciation terminale (CD57+), elles présentaient toutes les mêmes caractéristiques phénotypiques distinctes, définies par l’absence d’expression de la molécule de co-stimulation CD28 coordonnée à l’expression à leur surface de l’intégrine CD11b et du récepteur NK, 2B4. Dans les chCD4 effecteurs, nous avons également mis en évidence la présence spécifique de granules cytoplasmiques concentrant granzyme B et perforine, molécules impliquées dans la cytotoxicité directe de cellules cibles. Cette propriété fonctionnelle a été démontrée par des tests de cytotoxicité redirigée et était restreinte aux chCD4 effecteurs en comparaison aux autres sous-populations effectrices de LT CD4 conventionnels et T-Reg. L’analyse du profile de sécrétion de cytokines, révèle l’absence total de production d’IL-17 et un profile orienté Th1, soulevant la question du lignage de cette population particulière. L’absence d’expression de Ki67, marqueur des cellules en cycle, au sein de cette population de LT CD4 cytotoxiques, parallèlement à leur accumulation, suggérait qu’elles seraient capables de persister à l’état quiescent chez les patients. Par ailleurs, dans les Mum, nous avons mis en évidence que l’augmentation importante du nombre de chCD4 chez les patients, concordait avec la présence d’expansions oligoclonales au sien de cette population, et démontré une corrélation positive entre le pourcentage des cellules effectrices, chCD4 et LT CD8, LT CD8 parmi lesquels nous avons déterminé une fréquence élevée de cellules répondeuses spécifiques d’antigènes tumoraux associés à la tumeur. Nos travaux ont également permis d’évaluer l’impact de la chimiothérapie sur les populations lymphocytaires dans le sang périphérique. Chez les patientes atteintes de cancer du sein, traitées par chimiothérapie néo-adjuvante, c’est-à-dire préopératoire, nous avons constaté une augmentation du nombre de chCD4 chez 17/22 patientes. Nous avons mis en évidence que cette augmentation sous traitement était fortement corrélée au pourcentage de régression tumorale. L’ensemble de ces résultats apporte une nouvelle vision des LT CD4 dans l’immunité tumorale. (...) / Historically, CD8 positive Cytotoxic T Lymphocytes (CTL) have been associated with an effector immune response, while T cells with a CD4 phenotype where considered helper T cells. More recent data suggest that CD4 positive T cells are also capable of a direct cytotoxic activity. Through a systematic analysis of the IL-2 (CD25) as well as IL-7 (CD127) receptors α on the surface of CD4+ CTL in peripheral blood of patients before during and after treatment we were able to identify a specific CD4+ T cell population devoid of expression for these 2 molecules. These CD4+, CD25-CD127-, T cells only represent 0,2-2% of the total CD4+ pool in peripheral blood of healthy donors, while in the presence of a chronic infectious disease such as HIV or tuberculosis they were increased, representing up to 2-20% of all CD4+ T cells. Similarly, high numbers of CD4+, CD25-CD127-, T cells could be identified in the circulation in patients with metastatic uveal melanoma (muM) or with breast cancer. These chronically stimulated T cells (chCD4) demonstrate a memory effector phenotype (CD45RO+); while the majority shows a terminally differentiated phenotype (CD57+), these T cells all arbore distinct phenotypic characteristics as defined by the absence of expression of CD28 together with the presence of a surface expression of integrin CD11b and of the NK receptor, 2B4. The presence of cytoplasmic granules concentrating granzyme B and perforin, known to be responsible for T cell cytotoxicity, were identified in effector chCD4 while they were absent in conventional CD4+ T cells as well as in Tregs. This cytotoxic potential was demonstrated through redirected cytotoxicity assays that functionally confirm this feature of these chronically activated CD4+ T cells. The secretory cytokine profile showed absent IL-17 levels and a Th1 orientation, asking questions as regards to the lineage of this particular T cell population. Ki67 expression, a marker of cell proliferation, was absent, suggestive of their ability to persist quiescently in patients. However in muM patients we were able to demonstrate a vast oligoclonal increase in chCD4+ T cells, which correlated positively with CD8+ T cells. We were able to detect a high frequency of T cells responding against a specific tumor antigen among these CD8+ T cells. We furthermore studied the effects of chemotherapy on peripheral lymphocyte populations. In breast cancer patients who had been treated with preoperative (neoadjuvant) chemotherapy we detected high levels of effector chCD4 in 17/22 patients. Of particular interest was the fact that this increase through a course of chemotherapy treatment was strongly correlated to the percentage of regression of the original tumor. Together, these results cast new light on the role and function of CD4+ T cells in tumor immunity. Our observations show that CD4+ cytotoxic T lymphocytes do exist and suggest for the first time in human that they may have an important role in response to treatment and in particular in the establishment of a durable protective immune response.

Au cours de cette thèse nous avons étudié les propriétés anticancéreuses du pigment majoritaire de la betterave rouge : la bétanine, ainsi que son mode d’action. Nos travaux reposent sur une approche pluridisciplinaire. Nous avons tout d’abord mis au point un protocole d’extraction et de purification de la bétanine à partir de betteraves rouges fraîches. Plusieurs étapes de purification se terminant par la séparation des molécules d’intérêt sur HPLC semi-préparative sont nécessaires à l’obtention de la bétanine à un degré de pureté de 90 %, une qualité d’extrait jusqu’à présent inégalé. Nous avons ensuite évalué l’effet cytotoxique de notre extrait sur cellules cancéreuses. Nous avons pu démontrer son innocuité sur cellules non cancéreuses et identifier les voies de signalisation pouvant être impliquées. Nous avons ainsi pu avancer des pistes concernant le mode d’action de la bétanine sur les cellules, mais également soumettre pour la première fois l’idée d’une implication de l’autophagie dans la mort cellulaire induite par la bétanine. Enfin, nous avons montré, par des techniques d’analyse biophysique aux interfaces appliquées aux membranes cellulaires et biomimétiques, qu’indépendamment de son insertion jusqu’au cœur hydrophobe des membranes, la bétanine n’influait pas sur la fluidité et la perméabilité membranaire. Ce travail exploratoire confirme l’intérêt à porter à la bétanine qui, compte tenu de sa haute biodisponibilité, présente de nombreuses applications thérapeutiques potentielles. / During this thesis we studied the anticancer properties of the major beetroot’s pigment: betanin. Our work is based on a multidisciplinary approach.First we developed a protocol for the extraction and the purification of betanin from fresh beetroots. Several purification steps ended by separation in semi-preparative HPLC are required to obtain a betanin at 90 % pure, which is the highest purity ever recorded. Then we assessed the cytotoxic effect of our extract on cancer cells and its safety on non-cancer cells. By identifying the signaling pathways that might be involved in these effects, we were thus able to suggest ways concerning the mode of action of betanin on cells, but also propose, for the first time, the idea of an involvement of autophagy in cell death induced by betanin. Finally, we have shown by interfacial biophysical techniques applied on cell and biomimetic membranes that, regarless to its deep insertion in the hydrophobic core of the lipid bilayer, betanin did not affect the physical properties of the membrane such as its fluidity or its permability.This scoping study confirms the interest to bring to betanin which, given its high bioavailability, has many potential therapeutic applications.

<p>U ovom radu prikazana je sinteza 11 tiazolnih izostera goniofufurona (1-11),<br />4 konformaciono kruta analoga goniofufurona (12-15) i jednog butadiolnog<br />derivata&nbsp; tiazofurina&nbsp; (16).&nbsp; Takođe,&nbsp; izvr&scaron;eno&nbsp; je&nbsp; ispitivanje&nbsp; i&nbsp; poređenje<br />biolo&scaron;kih&nbsp; aktivnosti&nbsp; sintetisanih&nbsp; analoga&nbsp; sa&nbsp; sa&nbsp; aktivno&scaron;ću&nbsp; i&nbsp; selektivno&scaron;ću<br />kako GF i TF tako i doksorubicina, jedinjenja &scaron;irokog spektra dejstva (DOX).<br />Hiralni&nbsp; prekursor&nbsp; novosintetisanih&nbsp; jedinjenja&nbsp; 1-15&nbsp; bila&nbsp; je&nbsp; D-glukoza,&nbsp; a<br />jedinjenja 16 D-arabinoza.</p> / <p>A multistep synthesis of 11 novel thiazole isosteres of goniofufurone (1-11), 4 novel conformationally constrained isosteres of goniofufurone (12-15), as well as&nbsp; one&nbsp; butanediole&nbsp; derivative&nbsp; of&nbsp; tiazofurin&nbsp; (16)&nbsp; has&nbsp; been&nbsp; achieved.&nbsp; In&nbsp; vitro cytotoxicity of newly synthetized derivatives has been evaluated and compared with the cytotoxicities of goniofufurone, tiazofurin and doxorubicin.</p>

Thesis (M.Sc.(Microbiology)) -- University of Limpopo, 2022 / This study aimed to investigate antimycobacterial and cytotoxic compounds from Cassia petersiana. Cassia petersiana was selected for the current study based on its traditional use for treating tuberculosis (TB) symptoms. Extraction is an important step in the use of medicinal plants; hence, solvents of varying polarity were employed to extract a wide range of compounds where chloroform was the best extractant (67 mg). As there is no relation between the amount of plant material extracted and the bioactivity of the extracts, standard tests were used to determine the presence of different phytochemical constituents from Cassia petersiana and the total phenolic, flavonoid, and tannin contents were quantified using colorimetric assays. It was revealed that all the tested phytochemical constituents were present, and it was proven that phenolic compounds were the most abundant, followed by the tannins, while the flavonoids were the least among the common phytochemical constituents quantified. The phytochemical compounds were further profiled on thin-layer chromatography (TLC) and developed in BEA, CEF, and EMW solvent systems. Colourful compounds which indicated diverse phytochemicals were visualised with both vanillin-sulphuric acid and ultraviolet light on the phytochemical chromatograms and good separation of the compounds was from the BEA solvent system. The qualitative and quantitative antioxidant activity and antimycobacterial activity assays were used to evaluate the extracts from Cassia petersiana. Minimal antioxidant activity was observed on the qualitative antioxidant activity profile. These findings correlated with the minimal quantity of antioxidants from extracts of Cassia petersiana from the quantitative antioxidant assays; ferric reducing power and DPPH scavenging activity assays. Cassia petersiana extracts had bioactivity against Mycobacterium smegmatis as indicated by the lowest MIC value. The cell viability effects of the acetone crude extract from Cassia petersiana were evaluated against the tryptophan hydroxylase-1 (TPH-1) macrophage cells. Large scale extraction procedure was employed to extract a sufficient amount of plant material in preparation for the isolation of the bioactive compound. Bioassay-guided fractionation combined with column chromatography and TLC were used to isolate and purify the bioactive compound from the n-hexane extract of Cassia petersiana. The purified isolated compound was elucidated as β-sitosterol, which showed remarkable bioactivity against Mycobacterium smegmatis only on the TLC-bioautographic assay, while the quantitative antimycobacterial activity was higher xx with the MIC value of 2.5 mg/mL. Although β-sitosterol is known as a good antioxidant, it showed no antioxidant activity on the qualitative antioxidant activity assay. Therefore, further studies, including in vivo assay, are recommended on the isolated compound to evaluate its biological activities before consideration of its use in the development of alternative drugs.

Natural products are acknowledged sources of novel compounds for use in the treatment of diseases such as cancer, malaria, and human African trypanosomiasis. However, health burdens of such diseases still remain high, with drug resistance leading to failure of current medication. Therefore, there is a need for new treatments, and this project considers the potential of Nigerian ethno-medicinal plants and their products. Firstly, the aims were to isolate cytotoxic compounds through bio-guided evaluation and fractionation from 3 medicinal plants; Vitellaria paradoxa, Cyperus articulatus and Securidaca longepedunculata used traditionally in the treatment of cancer in North-East Nigeria. Extracts from S. longepedunculata were the most active when assessed in a panel of cancer cell lines, with IC50 values below 10 µg/ml, whilst fractions isolated from V. paradoxa and C. articulatus were moderately cytotoxic and able to overcome drug resistance mechanisms in drug resistant cell lines. In the second part of the thesis, novel cryptolepine analogues were semi-synthesized using environmentally friendly methods and evaluated for cytotoxic, anti-plasmodial and anti-trypanosomal activity. The compounds were found to be highly cytotoxic in cancer cell lines with the ability to overcome drug resistant mechanisms, with sub-µM IC50 values, and were also active against drug resistant strains of Plasmodium parasites in addition to Trypanosoma brucei, with IC50 values below 500 nM, and 300 pM respectively. / Schlumberger Faculty for the Future Foundation

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Opportunistic bacterial pathogens are present in the intestines of all mammals. These bacteria are symbionts to a certain extent, but under certain conditions these organisms can be deadly. Intestinal opportunistic pathogens encompass many genera and include organisms such as those in the Bacteroides fragilis group (i.e. B. fragilis and B. thetaiotaomicron), Escherichia coli, and Clostridium perfringens, resulting in an array of diseases and serious health risks. Typically these diseases affect individuals in poor or weakened health (elderly, immuno-compromised, neonates, etc.) but can affect healthy individuals as well. The intestinal tract is the main area of infection for these bacteria, however some of these organisms can be involved in wound infections, septicemia, urinary tract infections, and meningitis. This study focused on three areas: 1) Analysis of differences in gene expression between Bacteroides and Escherichia coli, in order to learn more about promoter structure, 2) Establishment of a diabetic mouse model for use in examining bacterial synergy during a polymicrobial infection, and 3) Characterization of Escherichia coli 360A and evaluation of the role of several virulence factors and environmental modulators in the pathogenesis of this strain. We used a newly developed lux gene reporter to evaluate gene expression in Bacteroides. We observed that there are barriers in both transcription and translation initiation that appear to limit the expression of foreign genes in Bacteroides. We were able to establish a mouse model for studying synergy during a polymicrobial infection and observed that E. coli 360A provided synergy towards B. fragilis NCTC 9343. These experiments also showed that the longer a mouse is afflicted with the complications of diabetes the more susceptible it is to polymicrobial infections. Systemic infections were used to evaluate the contribution of several virulence factors and environmental modulators in the pathogenesis of E. coli 360A. The results showed that a strain lacking both virulence factors CNF1 and HlyA, the terminal oxidase cytochrome o, or a double cyo/cyd mutant were, deficient in survival in the spleen, but not the liver of BALB/c mice. / Ph. D.

No / Buddlejol (1), a new sterol, has been isolated from the ethyl acetate soluble fraction of the antispasmodic plant Buddleja asiatica along with stigmasterol (2), lignoceric acid (3), taraxerol (4) and alpha-amyrin (5), respectively. The structure of Buddlejol (1) was established as (24S)-stigmast-5,22-diene-7 beta-ethoxy-3 beta-ol by spectral analysis and comparison with closely related structures. Buddlejol revealed to be a competitive inhibitor of chymotrypsin with the Ki value of 10.60 A mu M as indicated by Lineweaver-Burk and Dixon plots and their re-plots against its chymotrypsin inhibition assay, while the other compounds showed less inhibitory potential. The bioassay-guided isolation was stimulated by the preliminary cytotoxic screening of various fractions of B. asiatica.