A resposta metabólica do bagre Genidens genidens de três estuários (rios Grande, Indaiá e Escuro) do município de Ubatuba SP foi estudada comparativamente em relação ao impacto da atividade humana na região. Encéfalo, fígado e músculo epaxial foram utilizados para determinar a atividade específica das enzimas lactato desidrogenase (LDH), malato desidrogenase (MDH) e arginase, como prováveis marcadoras desse impacto. As diferenças entre os níveis teciduais de LDH dos bagres coletados nos três estuários não foram significativamente diferentes, provavelmente devido a sua capacidade adaptativa, associada à sazonalidade das concentrações de oxigênio nas águas estuarinas. Os níveis de arginase hepática e muscular dos bagres coletados nos estuários dos rios Escuro e Indaiá, respectivamente, foram diferentes em relação aos dois outros estuários. Comportamento semelhante a esse foi constatado em relação aos níveis da enzima MDH, marcadora do potencial gerador de ATP desses tecidos. Nesse sentido, os níveis de arginase e MDH do fígado e músculo epaxial podem estar refletindo o esforço adaptativo metabólico desse bagre às condições ambientais dessas regiões estuarinas. / The metabolic response of catfish Genidens genidens of the three estuaries (Grande, Indaia and Escuro rivers) in the municipality of Ubatuba - SP was studied in comparison to the impact of human activity in the region. Brain, liver and epaxial muscle were used to determine the specific activity of the enzymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and arginase, as probable markers of that impact. The differences between the tissues levels of LDH of the catfishes collected in the three estuaries were not significantly different, probably due to its adaptive capacity, associated with the seasonality of the oxygen concentrations in estuarine waters. Arginase levels of liver and muscle of the catfishes collected in estuaries of rivers Escuro and Indaia, respectively, were different in the relation to the other two estuaries. Behavior was similar to that observed in the levels of the enzyme MDH, marker of the potential generator of ATP of those tissues. Accordingly, the levels of arginase and MDH of the liver and epaxial muscle might reflect the effort of that adaptive metabolic catfish to environmental conditions such estuarine areas.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-07T10:55:53Z No. of bitstreams: 2 Dissertação - Mirian de Sylvio - 2009.pdf: 865608 bytes, checksum: bba24a89ef1938c31376520a3eff1e60 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-11-07T11:29:38Z (GMT) No. of bitstreams: 2 Dissertação - Mirian de Sylvio - 2009.pdf: 865608 bytes, checksum: bba24a89ef1938c31376520a3eff1e60 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-07T11:29:38Z (GMT). No. of bitstreams: 2 Dissertação - Mirian de Sylvio - 2009.pdf: 865608 bytes, checksum: bba24a89ef1938c31376520a3eff1e60 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2009-12-15 / Infection with Toxoplasma gondii occurs throughout the globe, with a prevalence of up to 90% in the population. The physiological changes caused by this parasite are well studied in immunocompromised individuals and in cases of congenital transmission. In immunocompetent individuals the infection is usually asymptomatic and little explored by researchers. Experimental studies follow the pattern of human studies, and there fow mention about the biochemical changes (liver and kidney metabolisms) in the host infected by T. gondii. This study aimed the quantification of hepatic and kidney alterations caused by acute infections by T. gondii (non cystogenic strain – RH) and by chronic infections (cystogenic strain – ME-49). The control group was formed by mice without infection, only submitted to saline stress. Several enzymes were measured in serum and peritoneal exudate of mice infected and control such as: aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyltransferase (GGT), alkaline phosphatase (ALP), urea, creatinine and lactate dehydrogenase, using an automated methodology. AST and ALT presented a significative difference in the serum of mice infected with RH strain when compared to controls indicating a destruction of liver cells. The peritoneal exudates did not present significative changes in relation to controls nor did the urea and creatinine levels. The séric lactate dehydrogenase showed gradual changes in all days of the infection in mice peritoneal exudates as early as this change was evident only in the fifth day of infection. All samples of the group infected with ME-49 strain showed changes in serum and peritoneal exudate during all days of analysis. Only ALT peritoneal exudates showed no change during all days of analysis. An increase in urea at all doses was observed, however, creatinine showed a change only within 120 days of infection. The LDH was altered in the serum in all days of analysis. In conclusion, the T. gondii infection may cause hepatic and kidney injuries either when caused by non-cystogenic as by cystogenic strains of the parasite. / A infecção pelo Toxoplasma gondii ocorre em todo o mundo, com prevalência de até 90% na população conforme seus hábitos culturais e condições socioeconômicas. As alterações fisiopatológicas provocadas por este parasito são muito estudadas nos indivíduos imunocomprometidos, nos casos de transmissão congênita, e nos indivíduos imunocompetentes a infecção é, geralmente, assintomática e pouco explorada pelos pesquisadores. Experimentalmente, os estudos seguem o padrão dos estudos humanos, e há pouca referência sobre as alterações bioquímicas (hepáticas e renais) no hospedeiro infectado pelo T. gondii. Este trabalho objetivou avaliar as alterações hepáticas e renais causadas por esse parasito em camundongos na fase aguda, usando a cepa não cistogênica (RH), e na fase crônica, com a cepa cistogênica (ME-49), tendo como controles camundongos sem infecção, somente submetidos ao estresse de inoculação com salina. Foram dosadas no soro e no exsudato peritoneal dos camundongos infectados e controles os níveis das enzimas Aspartato aminotransferase (AST), Alanina aminotransferase (ALT), Gamaglutamiltransferase (GGT), Fosfatase alcalina (FAL), desidrogenase lática (DHL) e dos seguintes compostos: uréia e creatinina, por metodologia automatizada. As enzimas AST e ALT apresentaram diferença significativa no soro de camundongos infectados com cepa RH, demonstraram alterações em relação aos controles indicando uma destruição das células hepáticas. No exsudato peritoneal não foram demonstradas alterações em relação aos controles. A uréia e creatinina dosadas não demonstraram alteração significativa. A enzima lactato desidrogenase sérica apresentou alterações gradativas em todos os dias de infecção do camundongo no soro, já no exsudato peritoneal essa alteração foi evidenciada somente no quinto dia da infecção, mostrando que com o aumento de parasitos e a destruição celular causada por esse, essa enzima presente em várias células é responsável por demonstrar aumentos consideráveis. Todas as amostras de soro analisadas do grupo infectado com a cepa ME-49 demonstraram alterações durante todo período de acompanhamento. Enquanto que no exsudato peritoneal não mostrou nenhuma alteração durante todo período analisado. Houve aumento crescente na uréia em todos os dias de analises, porém, a creatinina não apresentou nenhuma alteração. A LDH mostrou-se alterada no soro em todos os dias de analisado. Conclui-se que a infecção pelo T. gondii pode provocar alterações hepáticas e renais ao longo do curso de infecção, tanto em infecções com cepa cistogênica quanto com cepa não cistogênica.

Les matériaux hydroxydes doubles lamellaires (HDL) sont une classe d'argiles anioniques synthétiques dont la structure est basée sur celle du brucite Mg(OH)2 dans lesquelles une partie des cations métalliques divalents sont été remplacés par des ions trivalents donnant ainsi des feuillets chargés positive. Cette charge est équilibrée par l'intercalation d'anions dans la région interlamellaire hydratée. Les identités et les rapports des cations di- et trivalents et l'anion interlamellaire peuvent être varié sur une large gamme, donnant lieu à une large classe de matériaux isostructurales. Le matériau d’origine de cette classe est l’hydrotalcite (HT) et les HDL sont par conséquent également connus comme des matériaux de type hydrotalcite. Bien que les caractéristiques de base de la structure soient bien comprises, des aspects structurels détaillés ont fait l'objet de certaine controverse dans la littérature afin de maîtriser leurs propriétés et leurs applications potentielles. Dans ce travail de thèse nous avons retenu deux types de HDL MgAl et ZnAl qui ont été largement introduits dans diverses applications, tels que la sorption des molécules d'intérêt biologique (enzyme et médicament) et l'élaboration d'électrodes. La spécificité de ce travail repose sur l’immobilisation d’une enzyme modèle, la lactate déshydrogénase dans ces deux matrices ainsi qu’un médicament anti-bactérien, la berbérine, afin d’étudier les interactions entre ces deux biomolécules et la phase HDL introduite et de répondre à leurs exigences d'applications dans le domaine médical. Dans un second temps nous avons tenté d’étudier les deux phases mentionnées de plus en plus fine en termes de structure, morphologie et profil électrochimique en vue de les employer en tant que matériaux d’électrode pour le développement de biopile / DHs are a class of synthetic anionic clays whose structure is based on brucite-like layers Mg(OH)2 inwhich some of the divalent cations have been replaced by trivalent ions giving positively-charged sheets.This charge is balanced by intercalation of anions in the hydrated interlayer regions. The identities andratios of the di- and trivalent cations and the interlayer anion may be varied over a wide range, giving rise toa large class of isostructural materials. The parent material of this class is the naturally occurring mineralhydrotalcite and LDHs are consequently also known as hydrotalcite-like materials. Although the basicfeatures of the structure are well understood, detailed structural aspects have been the subject of somecontroversy in the literature. In this thesis, we have selected two types of LDH, MgAl and ZnAl, which havebeen widely introduced in various applications, such as sorption of molecules of biological interest (enzymeand drug) and the development of electrodes. The specificity of this work lies on the immobilization of amodel enzyme, lactate dehydrogenase in both matrices as well as an anti-bacterial drug, berberine, inorder to study the interactions between these two biomolecules and the introduced LDH phase and tobetter address their challenges of applications in the medical field. Second, we have tried to study the twophases mentioned above more and more accurately in terms of structure, morphology and electrochemicalprofile in order to use them as electrode materials for microbial fuel cell device

L’objectif de cette thèse est de développer une nouvelle souche de champignon filamenteux pour la production d’acide lactique. Pour répondre à cet objectif, nous avons tout d’abord d’exploré la biodiversité fongique à la recherche de champignons filamenteux capables de produire de l’acide lactique ou présentant de bonnes prédispositions pour la production d’acides organiques sans neutralisation du pH. Grâce à ce criblage, une souche sauvage d’Aspergillus brasiliensis a été sélectionnée et utilisée pour construire, grâce à l’ingénieure métabolique, les premières souches d’Aspergillus capables de produire de l’acide lactique à pH acide. Ces souches ont ensuite été caractérisées et les conditions de culture en fioles et fermenteurs volumes ont été étudiées. Cette étude des conditions de culture donne des résultats prometteurs et dévoile de nombreuses voies possibles pour continuer à améliorer la production d’acide lactique par ces organismes. / The objective of this thesis is to develop a new filamentous fungal strain for the production of lactic acid. To meet this goal, we first explored the fungal biodiversity in order to find filamentous fungi able to produce lactic acid or having good predispositions for the production of organic acids without pH neutralization. Through this screening, a wild type strain of Aspergillus brasiliensis was selected and used to construct, using the metabolic engineer, new strains capable of producing lactic acid at an acidic pH. These strains were then characterized and culture conditions in flasks and bioreactors were studied. The study of culture conditions shows promising results and reveals many possible ways to further improve the production of lactic acid by these organisms.

PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal Universtiy of Technology / The genus Tulbaghia has been used in traditional medicine to treat various ailments such as fever, earache, tuberculosis and esophageal cancer. However, there is limited scientific evidence to support its use. Therefore the objectives of this study were to perform phytochemical analysis, investigate the antioxidant, antimicrobial, anticancer, immunomodulatory activities and toxicity of crude acetone and water extracts from selected Tulbaghia species. Standard methods were used for preliminary phytochemical analysis. The total phenolic content of the plant extracts was determined using the folin ciocalteu method whereas the total flavonoids were determined by using the aluminium chloride colorimetric method. DPPH and ABTS assays were used to evaluate the antioxidant activity. The antimicrobial activity was assessed by agar well diffusion, microtiter dilution and time kill assays. For anticancer studies, the antiproliferative activity of the extracts was evaluated using the MTT assay on Hkesc-1 and KB cells. Morphological changes of the cancer cells treated with extracts were examined using light microscopy. Induction of apoptosis was assessed using fluorescence microscopy and acridine orange/ethidium bromide staining. Flow cytometry analysis was conducted to examine the multicaspase activity and cell cycle arrest. For immunomodulatory activity, the Greiss reagent and Luminex cytokine assays were used to determine the effect of the extracts on NO production and the concentration of the cytokines in the treated cells, respectively. Toxicity of selected Tulbaghia species was examined by investigating the effect of the extracts on the metabolic activity and cell membrane integrity on the treated RAW264.7 cells using the MTT and LDH assays, respectively. The zebrafish assay was used to evaluate the embryotoxicity and teratogenic effects of crude acetone and water extracts of T. violacea at 24 h intervals for 96 h post fertilisation (hpf). The percentage mortality, hatchability and heart rate were examined. Phytochemical screening of eight Tulbaghia species demonstrated the presence of flavonoids, glycosides, tannins, terpenoids, saponins and steroids. The amount of total phenol and flavonoid content varied in different plant extracts ranging from 4.50 to 11.10 milligrams gallic acid equivalent per gram (mg GAE/g) of fresh material and 3.04 to 9.65 milligrams quercetin equivalent per gram (mg QE/g) of fresh material respectively. The IC50 values based on DPPH and ABTS for T. alliacea (0.06 and 0.06 mg/mL) and T. violacea (0.08 and 0.03 mg/mL) were generally lower showing potential antioxidant activities. For antimicrobial activity, the acetone extracts of T. acutiloba, T. alliacea, T. leucantha, T. ludwigiana, T. natalensis and T. simmleri showed moderate antimicrobial activity against all test organisms while the water extracts showed moderate to no activity. One species, T. cernua, showed poor activity against all the tested microbes. The acetone and water extracts of T. violacea showed the greatest antibacterial and antifungal activity against all the tested microorganisms with minimum inhibitory concentration ranging from 0.1 mg/mL to 3.13 mg/mL. The acetone extracts of T. violacea also exhibited both bacteriostatic/fungistatic and bactericidal/fungicidal activity depending on the incubation time and concentration of the extract. The bactericidal/fungicidal activity was observed at x2 MIC. The results for anticancer activity showed that treatment of Hkesc-1 cells with acetone and water crude extracts had anti-proliferative activity with IC50 values of 0.4 mg/mL and 1.625 mg/mL, respectively while KB had 0.2 mg/mL and 1 mg/mL, respectively. Morphological changes such as blebbing, cell shrinkage and rounding were observed in the treated cells suggesting that apoptosis was taking place. AOEB staining showed that the level of apoptosis was dependent on the concentration of the extracts. The activation of multicaspase activity in both Hkesc-1 and KB treated cells was also concentration dependent leading to cell death by apoptosis and the induction of cell cycle arrest at the G2/M phase. Immunomodulatory activity results indicated that cell viability was above 80% when concentrations of 50 µg/mL or less of both acetone and water crude was used. Treatment with the acetone extract had no significant effect (p>0.05) on the LPS induced NO production in RAW264.7 cells except at 50 µg/mL where significant inhibition was observed. The water extract had no significant effect (p>0.05) on NO production at all the concentrations. Treatment of LPS–induced RAW264.7 cells with acetone extract stimulated the production of IL-1α, IL-6 and TNF-α, but had no significant effect (p > 0.05) on IL-1β. On the other hand, treatment with the water extracts stimulated the production of IL-1α, IL-6 but had no significant effect (p>0.05) on TNF-α and IL-1β. Treatment of LPS-induced RAW264.7 cells with the acetone extract had very little stimulatory effect on IL-4, IL-5 and IL-13 and no significant effect on IL-10 whereas for the water extract a significant stimulatory effect was only observed for IL-4 after 48 h of treatment. High concentrations (>10000 pg/mL) of MCP-1, MIP1-α, MIP1-β, MIP-2, GCSF, GM-CSF, RANTES and IP-10 were also observed in acetone and water extract treated RAW264.7 cells. For toxicity studies, acetone and aqueous crude leaf extracts from T. alliacea, T. simmleri, and T. violacea had a significant inhibitory (p<0.05) effect on the RAW264.7 cells after 48h treatment. Acetone extracts from T. alliacea, T. simmleri and T. violacea resulted in IC50 values of 0.48 mg/mL, 0.72 mg/mL and 0.1 mg/mL, respectively. Treatment with water extracts showed minimal toxic effect indicated by higher IC50 values of 0.95 mg/mL, 2.49 mg/mL and 0.3 mg/mL for T. alliacea, T. simmleri and T. violacea, respectively. The LDH release by macrophages after 24 h treatment with acetone extracts was observed to be concentration dependent while treatment with water extracts did not induce LDH release. The zebra fish assay showed a lethal dose (LD50) for the T. violacea acetone crude extract of 20 μg/mL whereas that for water extract was 85 μg/mL. The observed teratogenic effects included scoliosis, edema of the pericardial cavity, retarded yolk resorption, hook-like/bent tail and shorter body length. In conclusion, the results from this study indicate that the extracts from the eight Tulbaghia species examined contain phytochemicals that may have the antioxidant, antimicrobial, anticancer and immunomodulatory properties. Extracts from T. violacea were observed to be the most potent. This study thus supports the use of T. violacea in treating bacterial and fungal infections in traditional medicine. The results of this study also confirm the anticancer potential of T. violacea. The immunomodulatory activity of the acetone and water extracts from T. violacea indicated a dominantly pro-inflammatory activity. Traditional medicine prepared form T. violacea may be of benefit to individuals with weak immune systems. The toxicity of selected Tulbaghia species was observed to be concentration, extract and time dependent. Therefore, traditional medicine prepared from Tulbaghia extracts should be taken with caution preferably in small doses over a short period of time. Future studies will focus on the identification of the bioactive compound(s) responsible for the antimicrobial, anticancer and immunomodulatory activities.

PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Tulbaghia violacea Harv. has found extensive uses in traditional medicine for the treatment of numerous ailments among which are tuberculosis, oesophageal cancer, diabetes and cardiovascular diseases. Current reports show that cardiovascular diseases are now the primary cause of mortality worldwide. Thus, the potential of T. violacea plant extracts against cardiovascular diseases should be explored. The objectives of this study were, (i) to conduct qualitative and quantitative preliminary phytochemical screening of T. violacea aqueous leaf extracts, (ii) to conduct Gas chromatography–mass spectrometry (GC-MS) analysis for screening of compounds present in the plant extract, (iii) to evaluate the antioxidant activity of the T. violacea crude extracts using the DPPH:1.1-diphenyl-2-picrylhydrazyl and ABTS: 2,2-azino-bis 3-ethylebenzthiazoline-6-sulfonic acid assays, (iv) to evaluate the antimicrobial activity of the T. violacea crude extracts using disk diffusion and Minimum inhibitory concentration/Minimum bactericidal concentration (MIC/MBC), (v) to evaluate the antithrombogenic properties of T. violacea crude extracts on polystyrene, (vi) to fabricate polycaprolactone (PCL) and PCL-T. violacea incorporated scaffolds, (vii) to evaluate the antithrombogenic properties of T. violacea crude extracts on the fabricated PCL and PCL-T. violacea fabricated scaffolds and, (viii) to evaluate the growth and differentiation of adipose derived stem cells (ADSCs) on the fabricated scaffolds. The qualitative and quantitative phytochemical screening was conducted using standard procedures. Folin-Ciocalteu method was used to evaluate both total phenolic content (TPC) and total tannin content (TTC), the Aluminium chloride method was used for total flavonoid content (TFC) and GC-MS was used to screen for compounds present in the plant extract. The antioxidant activity was evaluated using DPPH and ABTS and the antimicrobial activity was evaluated using disc diffusion and MIC/MBC assays. The antithrombogenic properties of the T. violacea aqueous leaf extracts was then evaluated using platelet activation and whole blood clotting kinetics on polystyrene discs which have been reported to induce platelet activation. The experiment was performed in the absence and presence of 100 and 1000 μg/ml T. violacea plant extracts for both the platelet activation study which used blood plasma and the whole blood clotting kinetics assay which used fresh whole blood. Platelet adhesion was evaluated using fluorescence microscopy and a scanning electron microscope (SEM) was used to evaluate their morphology. Three scaffolds designated as PCL, 10% Tvio and 15% Tvio were fabricated which consisted of a 10% PCL powder and 10% as well as 15% T. violacea aqueous plant extract with respect to the PCL powder weight. The scaffolds were then characterized using Fourier-transform infrared spectroscopy (FTIR) and Energy-dispersive x-ray spectroscopy (EDS). The scaffolds were then evaluated for their antithrombogenic properties in the presence and absence of 100 and 1000 μg/ml T. violacea plant extracts. Platelet adhesion was evaluated using a fluorescent microscope and the morphology was evaluated using SEM. For the cell study, adipose derived stem cells (ADSCs) were cultured on the designed scaffolds and evaluated for their toxicity, viability, adhesion, proliferation, morphology and differentiation into osteoblasts over a period of 3 weeks. Lactate dehydrogenase (LDH) assay was used for toxicity studies, alamar blue assay was used for viability, fluorescence microscopy was used to evaluate cellular adhesion and proliferation while the alkaline phosphate (ALP) assay was used to evaluate differentiation of the cells into osteoblasts. Cell morphology was evaluated using SEM. Phytochemical screening of the prepared T. violacea aqueous extract revealed the presence of terpenoids, flavonoids, cardiac glycosides, saponins, protein, phenols, tannins, carbohydrates and amino acids. This is the first study that has identified the presence of carbohydrates and amino acids in T. violacea aqueous leaf extracts. Different concentrations of 0.1, 1.0 and 10 mg/ml of plant extract were used to conduct the quantitative phytochemical screening assays. There was a concentration dependent increase in the amount of phenols, tannins and flavonoids as the concentration of the plant extracts increased. This was the first study that evaluated the total tannic content of T. violacea plant extracts. The amount of total phenols was higher than that of flavonoids and tannins at every concentration range studied followed by the total flavonoids and lastly total tannins. The GC-MS analysis showed the presence of 33 compounds among which were 2,4 – Dithiapentate - 2,2-dioxide, Cannabidiol, 2,4,5,7 –Tetrathiaoctane and 2,4,5,7 - Tetrathiaoctane 2-dioxide. The presence of sulphur compounds support the characteristic garlic-like smell as well as some of the biological activities of T. violacea plant extracts. The antioxidant activities based on DPPH (0.49 mg/ml) and ABTS (0.24 mg/ml) suggest that T. violacea can be used as potential antioxidant agents. For the antimicrobial activity using disc diffusion, the extracts exhibited appreciable antibacterial activities against Bacillus subtilis, Serratia marcescens, Staphylococcus aureus and S. epidermidis. The highest zone of inhibition was observed for S. epidermidis at 19.50 ± 0.87 mm. The MIC results revealed that the plant extract of T. violacea was moderately active against B. subtilis, S. aureus, S. epidermidis, E. coli, and S. marcescens with MIC value of 2.5 mg/ml. However, the antimicrobial effect of the extract on S. epidermidis was bactericidal when compared to the bacteriostatic effect on the other active microorganisms. The antithrombogenic results on the polystyrene discs showed a significant reduction in the number of platelets that adhered on the polystyrene surfaces treated with plasma mixed with 100 μg/ml of plant extract when compared to the untreated control and the 1000 μg/ml treatment. For the 1000 μg/ml treatment, there was a significant increase in the number of platelets that adhered to polystyrene surfaces. These results were confirmed by the fluorescence and SEM results which showed a higher platelet count for the 1000 μg/ml treatment when compared to the other groups. The whole blood clotting kinetics study showed delayed blood clotting with the 100 μg/ml treatment over a period of 60 min when compared to the untreated control and the 1000 μg/ml treatment. These results correspond with the lower platelet adhesion observation and thus confirm the anticlotting properties of T. violacea aqueous leaf extracts at lower concentrations. The mean diameter of the scaffolds was recorded on the SEM as 275.60 ± 60.65 nm, 193 ± 30 nm and 537 ± 138 nm for the PCL, 10% Tvio and 15% Tvio scaffolds, respectively. The FTIR spectrum revealed the presence of amide groups as well hydroxyl O–H stretching groups which were the characteristic groups for the presence of T. violacea plant extracts in the polycaprolactone. The EDS results showed the presence of potassium, chlorine and sulphur compounds which were only present in the T. violacea scaffolds in addition to the carbon, oxygen and silicon observed in the PCL scaffold. The fabricated scaffolds were then used to evaluate platelet adhesion and activation on blood plasma in the absence and presence of 100 and 1000 μg/ml T. violacea aqueous leaf extracts. The results showed that the 10% Tvio scaffold was more effective in inhibiting platelet adhesion and activation at every treatment group especially when plasma was used in the absence of T. violacea plant extracts. A similar observation to the polystyrene study was observed were addition of 1000 μg/ml of plant extract resulted in the highest number of activated platelets. The study suggests the potential of the 10% Tvio scaffold in the prevention of platelet adhesion and aggregation. The in vitro cell adhesion, proliferation and differentiation of adipose derived stem cells (ADSCs) on the fabricated T. violacea loaded PCL nanofibers was then evaluated. The LDH assay illustrated less activity on the 10% Tvio scaffold when compared to PCL and 15% Tvio scaffolds however, none of the scaffolds were considered as toxic. The alamar blue assay was used for viability after 4 and 7 days of culture. The results showed a significant increase in cell viability for all scaffolds from day 4 to day 7 with the 10% Tvio scaffold having the highest overall cell viability for both day 4 and day 7 of cell cultures. Immunofluorescence staining was then used to count the number of cells using DAPI (4′,6-diamidino-2-phenylindole) stained images and illustrated that the T. violacea incorporated scaffolds supported better cell growth compared to the PCL scaffold. Cell morphology on the T. violacea scaffolds was denser and spread out into cellular extensions when compared to the PCL scaffold after 7 days of cell culture, supporting the higher number of adhered cells from the fluorescence results. For the long term cell study after week 1 and 3, the ALP results showed a significant difference in ALP activity between week 1 and week 3 for all scaffolds. The highest ALP activity was observed for the 15% Tvio scaffolds which is a marker for initial phase of bone matrix deposition. The designed T. violacea scaffolds supported better cell growth compared to the PCL scaffold and their morphology was more spread out and covered the entire surface of the scaffolds after week 3. Lastly, the cell count and osteocalcin differentiation was more prominent on 10% Tvio scaffold indicating higher levels of the protein marker for bone formation. Thus, supporting the use of the 10% Tvio scaffold for long-term cell studies. In conclusion, the results of this study indicated that the aqueous extract of T. violacea is rich is phytochemicals and also possess a broad range of pharmaceutically important compounds which may be attributed to the high antioxidant and antimicrobial activities identified. The results from this study suggest that T. violacea aqueous extracts have antithrombogenic properties at lower concentrations. Scaffolds fabricated with the incorporation of T. violacea plant extract also confirm the potential antiplatelet activity of the fabricated 10% Tvio scaffold. The results also suggest the potential of the fabricated 10% Tvio scaffold to enhance cell adhesion, proliferation and differentiation over long-term cell studies. It can thus be recommended that T. violacea may be useful for tissue engineering applications and bone repair with prospects of preventing cardiovascular diseases associated with bone defects. This research study has provided the foundation for clinical evaluation and outlined the potential effects of T. violacea aqueous leaf extracts as a clinical drug.

Die genaue physiologische Funktion des zellulären Prion-Proteins (PrPC) ist noch immer nicht vollständig verstanden. Eine mögliche Funktion des PrPC auf das neuronale Überleben nach einem hypoxischen oder ischämischen Insult wird diskutiert. In einem Vorversuch zeigten sich nach zerebraler Ischämie deutlich größere Infarktvolumina in den Gehirnen von Prion-Knock-Out-Mäusen im Vergleich zu denen der Wild-Typ-Mäuse. Das Identifizieren der molekularen Mechanismen der PrPC-vermittelten Neuroprotektion ist daher von großem Interesse und machte die Etablierung eines Zell-Modells erforderlich. Neuere Studien konnten einen Einfluss des zellulären Prion-Proteins auf die Glykolyse nachweisen. Unter Sauerstoffmangelbedingungen kommt es zu einer vermehrten Bildung von Laktat durch das Enzym Laktat-Dehydrogenase (LDH). Neurone benötigen unter hypoxischen oder ischämischen Bedingungen dieses Laktat als Energiesubstrat. Je mehr Laktat den Neuronen zur Verfügung steht, umso höher ist das neuronale Überleben. In dieser Arbeit konnte die Beteiligung der Laktat-Dehydrogenase an der durch das zelluläre Prion-Protein vermittelten Neuroprotektion nach Hypoxie nachgewiesen werden. Das Ziel dieser Arbeit bestand darin, mögliche Unterschiede der LDH-Expression in WT-Zellen, Prnp0/0-Zellen und HEK-293-Zellen unter normalen und hypoxischen Bedingungen in vitro zu untersuchen. Die Expression der LDH war unter hypoxischen Bedingungen in den WT-Zellen im Vergleich zu den Prnp0/0-Zellen deutlich höher. Dies konnte auch in PrPC-überexprimierenden HEK-293-Zellen nach Hypoxie gezeigt werden. Ebenso konnte nachgewiesen werden, dass Hypoxie zu einem größeren Schaden des Tubulinzytoskelettes in Prnp0/0-Zellen führt als in WT-Zellen, was eine neuroprotektive Wirkung von PrPC vermuten lässt. Eine direkte oder indirekte Interaktion von LDH-A und PrPC konnte durch eine Co-Immunpräzipitation in HEK-293-Zellen nachgewiesen werden. Die genauen Mechanismen über die PrPC möglicherweise zu einer vermehrten Laktat-Produktion führt, sind noch nicht eindeutig identifiziert und müssen noch näher untersucht werden. Zusammengefasst kann gesagt werden, dass die erhobenen Daten die Vermutung verstärken, dass das Enzym LDH und sein Produkt Laktat in die durch das zelluläre Prion-Protein vermittelte Neuroprotektion nach Hypoxie involviert sind. Es ist das erste Mal, dass gezeigt wurde, durch welchen Mechanismus PrPC zur Neuroprotektion beiträgt.

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<p>Kardijalni biomarkeri u predviđanju operativnog rizika kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore Evaluacija rezultata u kardiohirurgiji podrazumeva praćenje ishoda operativnog lečenja u određenom vremenskom periodu. Najče&scaron;će je to interval od 30 dana od datuma intervencije. Najče&scaron;ći kriterijumi za praćenje su stopa mortaliteta i morbiditeta, dužina boravka u jedinici intenzivnog lečenja, ukupna dužina hospitalizacije i tro&scaron;kovi lečenja. Stratifikacija rizika podrazumeva da se bolesnici mogu podeliti u grupe u zavisnosti od broja i važnosti preoperativno utvrđenih faktora rizika, odnosno da se pre operacije može predvideti ishod hirur&scaron;ke intervencije kod svakog od njih pojedinačno. U Evropi je, u periodu između 1995. i 1999. godine, na osnovu multicentrične studije u 8 evropskih zemalja i 128 kardiohirur&scaron;kih centara u kojima je operisano 19.030 odraslih bolesnika, kreiran EvroSKOR - EuroSCORE (European System for Cardiac Operative Risk Evaluation) model za stratifikaciju rizika u kardiohirurgiji. Međutim, neminovne promene i napredak u operativnom lečenju doveli su do toga da je neophodno ažurirati postojeći sistem stratifikacije. Tako je 2012. godine u rutinsku upotrebu uveden novi sistem Euroscore II. Na Klinici za kardiohirurgiju Instituta za kardiovaskularne bolesti Vojvodine (IKVBV), EuroSCORE model uveden je u rutinsku upotrebu od početka 2001. godine. Analizom rezultata, posle dvogodi&scaron;nje primene, pokazalo se da je model bio precizan, odnosno da nije postojala značajna razlika između očekivanog (3,7%) i stvarnog mortaliteta (3,47%). U poslednjih nekoliko godina, kod bolesnika kojima sledi kardiohirur&scaron;ka intervencija, u smislu razmatranja njihove prediktivne vrednosti, sve vi&scaron;e pažnje se poklanja kardijalnim biomarkerima. Najznačajniji biomarkeri u kardiovaskularnoj medicini su: Troponin, Kreatin kinaza MB izoenzim (CKMB), N-terminalni pro B-tip natriuretski peptid (NT-proBNP), C-reaktivni protein (CRP), Laktat dehidrogenaza (LDH), Mokraćna kiselina (Acidum uricum). Ciljevi ovog rada su bili da se kreira model za predviđanje preoperativnog rizika kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore na osnovu preoperativnih vrednosti određenih biomarkera i da se kreira novi model sa kombinacijom prethodnog modela i već postojećeg modela EuroSCORE II. Ispitana su 704 bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcione frakcije manje ili jednake 50%. Bolesnici su operisani na Institutu za kardiovaskularne bolesti Vojvodine, od 20. januara 2014. do 20. aprila 2016. Kod bolesnika su urađene tri vrste operacija: revaskularizacija miokarda-koronarna hirurgija, hirurgija stečenih srčanih mana - valvularna hirurgija i kombinovane operacije. Od biohemijskih analiza, 24 sata pre operacije, urađene su sledeće analize: troponin I, kreatin kinaza, kreatin kinaza MB izoenzim, masena kreatin kinaza, laktat dehidrogenaza, C-reaktivni protein, NT-proBNP i mokraćna kiselina. Praćen je postoperativni mortalitet, postoperativni infarkt miokarda i postoperativni cerebrovaskularni incident i njihova povezanost sa preoperativnim vrednostima nabrojanih biomarkera. U studiju su bili uključeni svi bolesnici sa stečenim bolestima srca, stariji od 18 godina, kod kojih je ejekciona frakcija leve komore bila manja ili jednaka 50% i kod kojih su izvr&scaron;ene sledeće vrste operacija: revaskularizacija miokarda - koronarna hirurgija, hirurgija stečenih srčanih mana - valvularna hirurgija i kombinovane operacije - koronarna i valvularna hirurgija. Rezultati su pokazali da je postoperativni mortalitet bio 3,13%, da je postoperativni infarkt miokarda imalo 7,95% a postoperativni cerebrovaskularni incident 9,23% od ukupnog broja ispitanika. 1. Povezanost vrednosti biomarkera sa postoperativnim infarktom miokarda kod bolesnika sa oslabljenom ejekcionom frakcijom leve komore: povi&scaron;ene preoperativne vrednosti troponina I su bile povezane sa postoperativnim infarktom miokarda. Povezanost preoperativnih vrednosti biomarkera sa postoperativnim cerebrovaskularnim incidentom kod bolesnika sa oslabljenom ejekcionom frakcijom leve komore: povi&scaron;ene preoperativne vrednosti troponina I i CRP-a su bile povezane sa postoperativnim cerebrovaskularnim incidentom. 2. Analiziran je uticaj preoperativnog nivoa svih biomarkera, pojedinačno, na značajne neželjene kardijalne i cerebrovaskularne događaje - Major Adverse Cardiac and Cerebrovascular Events (MACCE) kao ishod posle operacije na srcu, kod bolesnika sa oslabljenom ejekcionom frakcijom leve komore. Dobijeni su sledeći rezultati: Preoperativna vrednost nivoa troponina I veća od 0,01&mu;g/L i MACCE bili su povezani. Povećane preoperativne vrednosti nivoa C-reaktivnog proteina (CRP) i postoperativni MACCE bili su povezani. Povećane preoperativne vrednosti nivoa laktat dehidrogenaze (LDH) i MACCE bili su povezani. Zaključci ove teze su: 1. Nezavisni prediktor postoperativnog infarkta miokarda i značajnih neželjenih kardijalnih i cerebrovaskularnih događaja, kod kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcionom frakcijom manjom ili jednakom 50%, jeste povi&scaron;ena preoperativna vrednost troponina I. 2.Vrednost preoperativnog troponina I je slab marker za predviđanje postoperativnog infarkta miokarda i značajnih neželjenih kardijalnih i cerebrovaskularnih događaja, kod kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcionom frakcijom manjom ili jednakom 50%. 3. Na pojavu postoperativnog cerebrovaskularnog incidenta, kod kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcionom frakcijom manjom ili jednakom 50%, ne utiče nijedna od ispitivanih varijabli. 4. Nezavisni prediktori postoperativnog mortaliteta kod kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcionom frakcijom manjom ili jednakom 50%, na osnovu kojih je moguće kreirati prediktivni Model su godine starosti i povi&scaron;ene preoperativne vrednosti NT-proBNP. 5. Kreirani Model je dobar marker za predikciju ishoda posle operacije na srcu, kod kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcionom frakcijom manjom ili jednakom 50%. 6. Povi&scaron;ena preoperativna vrednost NT- proBNP može da bude dobar marker u predikciji smrtnog ishoda posle operacije na srcu kod bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcionom frakcijom manjom ili jednakom 50%. 7. Model EuroSCORE II se pokazao kao slabiji marker za predikciju ishoda posle operacije na srcu kod kardiohirur&scaron;kih bolesnika sa oslabljenom sistolnom funkcijom leve komore, ejekcionom frakcijom manjom ili jednakom 50%. 8. Testiranjem kreiranog modela, podelom na manje rizične i vi&scaron;e rizične bolesnike, u odnosu na visinu ejekcione frakcije leve komore, pokazalo se da je model dobar marker za predviđanje smrtnog ishoda posle operacije na srcu, u obe grupe.</p> / <p>Cardiac surgery operative risk assessment in patients with imapired systolic left ventricular function using cardial biomarkers Evaluation of results in cardiac surgery involves monitoring the outcomes of operative treatment in a given time period. Typically, this interval includes 30 days from the date of operation. The most common criteria used for monitoring are the rate of mortality and morbidity, length of stay in the intensive care unit, the total length of hospitalization and medical costs. Risk stratification means that patients can be divided into groups depending on the number and importance of preoperatively identified risk factors, and that the outcome of surgery for each of the patients can be predicted preoperatively. In Europe, in the period of 1995-1999 on the basis of a multi-center study in 8 European countries and 128 cardiac centers in which 19,030 adult patients were operated on, EuroSCORE (European System for Cardiac Operative Risk Evaluation) model for risk stratification in cardiac surgery was developed. However, the inevitable changes and progress in the surgical treatment rendered the EuroSCORE model obsolete warranting updated system. It was in 2012 when a new system EuroSCORE II was introduced into practice At the Clinic for Cardiac Surgery of the Institute of Cardiovascular Diseases, EuroSCORE model was introduced in routine clinical use since the beginning of 2001. By analyzing the results, two years after application, it was shown that the model was accurate, and that there was no significant difference between the expected (3.7%) and the actual mortality (3.47%) In recent years, in patients who are candidates for cardiac surgery, more attention is paid to cardiac biomarkers in terms of evaluating their predictive power. The most significant biomarkers in cardiovascular medicine are: Troponin, creatine kinase MB isoenzyme (CKMB), N-terminal pro B-type natriuretic peptide (NT-proBNP), C-reactive protein (CRP), Lactate dehydrogenase (LDH), and uric acid (Uric uricum). The objectives of this study were to create a model to predict preoperative risk for cardiac surgery patients with impaired systolic left ventricular function on the basis of preoperative levels of certain biomarkers and to create a new model with a combination of the previous model and already existing EuroSCORE II model. The study included 704 patients with impaired systolic left ventricular function, ejection fraction less than or equal to 50%. All patients underwent cardiac surgery at the Institute of Cardiovascular Diseases, from January 20th 2014 until 20th April 2016. Patients were submitted to three types of operations: revascularization - coronary surgery, surgery of acquired heart defects - valvular surgery and combined operations. Following biochemical analyses were performed 24 hours prior to surgery: troponin I, creatine kinase, creatine kinase MB isoenzyme, mass creatine kinase, lactate dehydrogenase, C-reactive protein, NT-proBNP and uric acid. Postoperative mortality, postoperative onset of myocardial infarction and occurence of cerebrovascular accident and their correlation with preoperative values of listed biomarkers were registered. The study included all patients with acquired heart disease, older than 18 years, with the left ventricular ejection fraction less than or equal to 50% who were submitted to the following types of operations: revascularization - coronary surgery, surgery of acquired heart diseases - valvular surgery and combined operations - coronary and valvular surgery. The results showed that the postoperative mortality was 3.13%, new onset of postoperative myocardial infarction was detected in 7.95% of the patients and postoperative cerebrovascular accident developed in 9.23% of patients. Correlation of preoperative biomarkers values with postoperative myocardial infarction in patients with impaired left ventricular ejection fraction - elevated preoperative troponin I were associated with postoperative myocardial infarction. Correlation of preoperative biomarkers values with postoperative cerebrovascular incident occurence in patients with impaired left ventricular ejection fraction - elevated preoperative troponin I and CRP were associated with postoperative cerebrovascular incident. The influence of preoperative levels of all biomarkers, separetly, on the rate of significant adverse cardiac and cerebrovascular events - Major Adverse Cardiac and Cerebrovascular Events (MACCE) as the heart surgery outcome, in patients with impaired left ventricular ejection fraction. The following results were obtained: Increased preoperative levels of C-reactive protein (CRP) and postoperative MACCE were related. Increased preoperative levels of lactate dehydrogenase (LDH) and MACCE were related. The conclusions of this thesis are: 1. Independent predictor of postoperative myocardial infarction onset and significant adverse cardiac and cerebrovascular events in cardiac surgery patients with impaired systolic left ventricular function (ejection fraction less than or equal to 50%) is elevated preoperative value of troponin I. 2. Preoperative Troponin I value was poor marker for predicting postoperative myocardial infarction and significant adverse cardiac and cerebrovascular events in cardiac surgery patients with impaired systolic left ventricular function (ejection fraction less than or equal to 50%). 3. None of the studied variables showed influence on the postoperative cerebrovascular accident occurence, in cardiac surgery patients with impaired systolic left ventricular function (ejection fraction less than or equal to 50%). 4. Independent predictors of postoperative mortality in cardiac surgery patients with impaired systolic left ventricular function (ejection fraction less than or equal to 50%), that could be used to create a predictive model are: age and elevated preoperative value of NT-proBNP. 5. Developed model showed satisfactory results for predicting outcome after heart surgery in cardiac surgery patients with impaired systolic left ventricular function (ejection fraction less than or equal to 50%). 6. Elevated preoperative value of NT-proBNP may be a good marker for mortality prediction after the cardiac surgery in patients with impaired systolic left ventricular function (ejection fraction less than or equal to 50%). 7. EuroSCORE II model showed poor performance when predicting outcomes after cardiac surgery in patients with impaired systolic left ventricular function (ejection fraction less than or equal to 50%). 8. Validation of the newly-created model, considering low and medium risk patients, based on the value of left ventricular ejection fraction, showed that the model is a good marker for the mortality prediction in both groups.</p>