Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-05T15:42:35Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Luís final (2).pdf: 2354969 bytes, checksum: 99edddc3a89f16f62d7e6fc6517c4a93 (MD5) / Made available in DSpace on 2016-04-05T15:42:35Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Luís final (2).pdf: 2354969 bytes, checksum: 99edddc3a89f16f62d7e6fc6517c4a93 (MD5) Previous issue date: 2013-09-09 / FACEPE / As lectinas são proteínas conhecidas por sua capacidade de ligar específica e reversivelmente a carboidratos resultando em uma variedade de propriedades biológicas. Este trabalho tem por objetivo avaliar os efeitos imunomodulador e citoprotetor das lectinas Cramoll 1,4 (pCramoll) e rCramoll 1 (rCramoll). Na determinação da ação imunomoduladora macrófagos peritoneais de camundongos Balb/c foram tratados com diferentes concentrações das lectinas (0,625-10 μM) e foram analisados os efeitos na produção óxido nítrico (NO), viabilidade celular, produção de ânion superóxido, alterações no potencial de membrana mitocondrial (ΔΨm) e na fagocitose de Staphylococcus aureus. A produção de citocinas pró-inflamatorias (IL-1β, IL-6, INF-γ e TNF-α) foi avaliada em macrófagos infectados e não infectados com S. aureus. Ambas lectinas induziram significantemente a produção de NO e de citocinas. As proteínas foram consideradas não-citotóxicas pelo ensaio com MTT, entretanto a análise por Citometria de Fluxo revelaram um aumento de células mortas. A produção de superóxido foi estimulada pelas lectinas o que foi confirmado pelas mudanças induzidas no ΔΨm. A ação fagocítica dos macrófagos foi aumentada em 27,1% e 22,47% após o tratamento destes com pCramoll e rCramoll . Por fim, as lectinas inibiram a expressão de TNF-α e IL-6 e estimularam a produção de IL-1β e INF-γ por macrófagos infectados por S. aureus. Paralelamente, o potencial protetor das lectinas contra a morte celular induzida pelo estresse oxidativo foi avaliada. Para tanto, células Vero (fibroblastos renais de macaco) foram pré-tratadas com crescentes concentrações das lectinas (0,625-10 μM) por 30 minutos e em seguida foram expostas ao H2O2 (1 mM). Após 24 horas, o efeito citoprotetor foi avaliado. Os mecanismos celulares envolvidos foram determinados por citometria de fluxo envolvendo: proteção contra morte celular, danos nos lisossomos e DNA, produção de ânion superóxido (MitoSOX), alterações no potencial de membrana mitocondrial (ΔΨm) e proliferação celular. pCramoll e rCramoll atenuaram a citotoxicidade induzida por H2O2 de forma dose-dependente, os efeitos máximos foram 96.85 ± 15.59% (rCramoll) e 59.48 ± 23.44% (pCramoll). A análise com Live/Dead mostrou redução da morte celular de 65.04 ± 3.29% (H2O2) para 39.77 ± 2.93% (pCramoll) e 13.90 ± 9.01% (rCramoll). Os efeitos deletérios de H2O2 na proliferação celular foram reduzidos em 10.83% (pCramoll) e 24.17% (rCramoll). As lectinas atenuaram a produção excessiva de superóxido, o collapso do ΔΨm e os danos lisossomais e ao DNA das células Vero expostas à H2O2. Em conclusão nossos estudos demonstram que pCramoll e rCramoll possuem elevado potencial imunomodulador e citoprotetor. / This study aims to evaluate the immunomodulatory effects on macrophages and cytoprotector action against oxidative stress of Cramoll 1.4 (pCramoll) and rCramoll 1 (rCramoll). In the determination of the immunomodulory action, peritoneal macrophages from Balb/c mice were treated with different concentrations of lectins (0.625 to 10 mM) and we analyzed the effect on NO production (Griess Reagent), cell viability (MTT Reagent), induction of apoptosis (Kit Live/Dead and Acridine orange), superoxide anion production (MitoSOX), changes in mitochondrial membrane potential (ΔΨm) and phagocytosis of Staphylococcus aureus. The production of proinflammatory cytokines (IL-1β, IL-6, INF-γ e TNF-α) was analyzed in S. aureus infected and non-infected macrophages. Both lectins significantly enhanced Macrophages NO production and cytokines. The lectins were not cytotoxic as observed by MTT assay. However Live/Dead analysis revealed an increase of apoptosis in treated cells, the reduction of lysosomal activity was also reduced. The superoxide production was stimulated by both lectins, which was confirmed with the reduction on ΔΨm. S. aureus phagocytic activity of macrophages were enhanced in 27.1% and 22.47% by pCramoll and rCramoll, respectively. Finally, pCramoll and rCramoll downregulated the production of IL-6 and TNF-α and upregulated the expression of IL-1β, INF-γ during S. aureus infection of macrophages. In addtion, the protective effects of lectins against cell death induced by oxidative stress were evaluated. For this purpose, Vero cells (monkey kidney fibroblasts) were pretreated with increasing concentrations of lectins (0.625 to 10 μM) for 30 minutes and then were exposed to H2O2 (1mM). After 24 hours, the cytoprotective effect was evaluated and the cellular mechanisms involved were determined by flow cytometry involving: protection against cell death (Live/Dead kit), damage to lysosomes (Acridine Orange) and DNA (TUNEL), superoxide anion production (MitoSOX), changes in mitochondrial membrane potential (ΔΨm) (Rhodamine 123) and cell proliferation. pCramoll and rCramoll significantly attenuated the H2O2-induced cytotoxicity in a dose-dependent way, the maximum protective effects were 96.85 ± 15.59% (rCramoll) and 59.48 ± 23.44% (pCramoll). The Live/Dead analysis showed a reduction in apoptotic cells from 65.04 ± 3.29% to 39.77 ± 2.93% (pCramoll) and 13.90 ± 9.01% (rCramoll). The deleterious effects of H2O2 on cell proliferation were reduced 10.83% (pCramoll) and 24.17% (rCramoll). The lectins attenuated the excessive superoxide production, the collapse of ΔΨm, lysosomal and DNA damage that occurred in Vero cells exposed to H2O2. In conclusion, our results show that pCramoll and rCramoll have high immunomodulatory potential on macrophages and cytoprotective effects against H2O2.

The unique floral biodiversity of Southern Africa would be reflected in the phenolic acid and flavonoid composition as well as the antioxidant activity of honeys from this region. In this exploratory investigation the total polyphenolic (TPC) and flavonoid (TFC) content, antioxidant activity as well as the cellular protective effects of a selection of honeys collected in this region was evaluated. Thirteen honey samples representative of the Western Cape (WCa, WCb and WCc), Eastern Cape (ECa, ECb and ECc), South East Mozambique (SEMa, SEMb and SEMc) and Agricultural: A-E (Eucalyptus) (A-E1 and A-E2), A-L (Litchi) and A-O (Orange) were collected. These samples were subjected to physicochemical analysis, the antioxidant content (TPC and TFC) and both enzymatic (catalase activity) and non-enzymatic activity, using the 2,2-diphenyl-2-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays was determined. From the DPPH, TEAC and ORAC data the Relative Antioxidant Capacity Index (RACI) was calculated. To determine whether high antioxidant activity translates into significant cellular protection, biological and cellular assays were undertaken. Using the pBR322 plasmid assay and the erythrocyte haemolysis assay the ability of honeys to protect against 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH) oxidative damage was evaluated. Further evaluation was undertaken in the SC-1 fibroblast cell line and the physiologically more relevant Caco-2 cell line. Toxicity and antioxidant effects were evaluated in the SC-1 cell line while antioxidant effects were only evaluated in the Caco-2 cell line. The long-term mitogenic and toxic effects were determined in the SC-1 cell line using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Neutral Red (NR) and Crystal Violet (CV) assays. Short term, total- and intracellular antioxidant effects were determined in both cell lines using the dichlorofluorescein diacetate assay (DCFH-DA) assay. For all cellular experiments honey at concentrations of 0.01% and 1% were used. The physiochemical properties of the honeys evaluated fulfilled the regulatory standards compiled in the Codex Alimentarius (CODEX STAN12-1981 revision 2001). The results were as follows: SEMb had the highest TPC (167.96 mg GAE/100g) and TFC (51.60 mg CE/100g) while A-E2 had the highest catalase (38.48 µmol H2O2/g) activity. RACI revealed that WCb had the highest antioxidant activity.SEMc showed the highest protection of plasmid DNA against oxidative-induced strand breaks while SEMa showed the highest protection of erythrocytes against AAPH-induced haemolysis. Although correlations were found between antioxidant content and antioxidant activity assays, no correlation was found these parameters and the biological assays. For the long-term cytotoxicity assay, AAPH showed significant cytoxicity at 0.78mM, 1.56mM and 0.28mM when measured using the MTT, NR and CV assays, respectively. Some honeys 4/13 and 3/13 showed a mitogenic effect at a concentration of 0.01% and 1% respectively. Toxic effects, were observed for 1/13 and 8/13 at 0.01% and 1% honey respectively. Toxicity after 72 h exposure varied from 10-30% (CV assay). The same concetrations of honey was used to determine the short-term, 2h, antioxidant effects in both the SC-1 and Caco-2 cell lines. No oxidative effect was found for all honeys at these concentrations. For the DCFH-DA assay using the SC-1 cell line at 1%, 12/13 and 7/13 honeys showed total and intracellular protection respectively. The highest extracellular protection was for SEMa (% Protection (%P) = 95) and SEMb (%P = 93). Intracellular protection was the highest for SEMc (%P = 21) and A-L (%P = 20). At 0.01%, 7/13 and 8/13 honeys exhibited total and intracellular protection, respectively. For both the highest protection was found for SEMc (%P = 43, total and %P = 30, intracellular). For the Caco-2 cell line at 1%, 11/13 and 4/13 showed total and intracellular protection, respectively. Of these the highest extracellular protection was for SEMb (% Protection (%P) = 90). Intracellular protection was the highest for ECa (%P = 28) and WCc (%P = 26). At 0.01%, 4/13 and 8/13 honeys showed total and intracellular protection respectively. The highest extracellular protection was found for SEMc (%P = 62) and intracellular protection was ECc (%P = 28). The SC-1 cell line was found to be the most sensitive to the antioxidant effects of honey compared to the Caco-2 cell line. The honeys SEMa, SEMb and SEMc showed protection against oxidative damage in both cell lines. In conclusion, the antioxidant activity of honeys from Southern Africa is of a high quality. The WC, SEM and EC honeys showed the highest antioxidant effects and could provide health benefits against diseases associated with oxidative stress as indicated by these results. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Anatomy / unrestricted

The subject of the thesis is study of possible applications of isoform of glutamic acid polymer (-PGA). The theoretical part is focused on the properties of this biopolymer and potential applications in various areas. Producers and mechanisms of biosynthesis are also mentioned. In the experimental part, the polymer was firstly characterised by following methods: FT-IR spectroscopy, TGA, DSC and SEC-MALS. Its isoelectric point, antimicrobial activity and solubility in various solvents were also determined. The biopolymer was also precipitated by divalent cations and its interaction with oppositely charged CTAB surfactant was studied. The main experimental study was researching the effect of -PGA on viability of Saccharomyces cerevisiae and Lactobacillus rhamnosus under stress conditions by flow cytometry. The performed stresses included ethanol exposure, high temperature and freezing stress, in which its effects were compared to conventional cryoprotectants. The cells of the mentioned microorganisms were also stressed osmotically and exposed to model gastrointestinal juices - gastric, pancreatic and bile. The protective effects of -PGA on the cells were recorded in ethanol stress on Lactobacillus rhamnosus. Its excellent cryoprotection properties were confirmed and its protective effect of gastric juice exposure on Saccharomyces cerevisiae cells was also observed. At the end of the experimental part, -PGA/alginate beads suitable for encapsulation of probiotic bacteria and -PGA/chitosan nanoparticles for encapsulation of biologically active substances.