Orientador: Prof. Dr. Daniel Carneiro Carrettiero / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / A estreptozotocina (STZ) e uma toxina derivada da bacteria Streptomyces acromogenes. A administracao cerebral de STZ promove alteracoes metabolicas que mimetizam as encontradas em pacientes com doenca de Alzheimer (DA) esporadica. Por este motivo, a STZ vem, sendo utilizada intracerebroventricularmente na criacao de modelos animais para o estudo de DA. A DA possui duas importantes caracteristicas histopatologicas, o acumulo de peptideo ¿À amiloide, levando a formacao das placas senis, e o aumento da fosforilacao da proteina tau, que culmina com a formacao dos emaranhados neurofibrilares, que possuem papel eminente no processo neurodegenerativo da DA. Alguns fatores estao relacionados com o surgimento dessas caracteristicas no paciente, como por exemplo, a geracao de estresse oxidativo, ativacao de Caspase e morte neuronal. A co chaperona BAG2 e fundamental para degradacao da proteina Tau fosforilada inibindo sua ubiquitinacao e favorecendo sua degradacao pela via proteossomica independente de ubiquitina. O objetivo do estudo foi avaliar os efeitos da STZ nos parametros relacionadas a citotoxicidade (MTT, Senescencia, Caspasee potencial mitocondrial), na geracao de estresse oxidativo (Diclorofluorisceina e citometria), dosagem de oxido nitrico (Griess), bem como estudar sua acao nos niveis das proteinas oxido nitrico sintase (nNOS e iNOS), Tau fosforilada e BAG2. Para o estudo, foram utilizadas cultura primaria de celulas do hipocampo. As celulas foram tratadas com STZ (0,05; 0,5 e 5 mM) pelos periodos de 6, 12 e 24 horas. A STZ apresentou citotoxicidade na concentracao de 5 mM por 24 horas, no ensaio de MTT e pelo teste de Senescencia celular nas concentracoes de 0,05 e 5 mM em 12 e 24 horas, promovendo aumento na morte celular e niveis de caspase total ativa em celulas tratadas 0,05; 0,5 e 5 mM por 12 e 24 horas. Apesar disso, nao houve alteracao no potencial mitocondrial em nenhuma das concentracoes e tratamentos com STZ. O tratamento promoveu aumento tanto na quantidade de especies reativas de oxigenio avaliado pela Diclorofluorisceina e estresse oxidativo por citometria de fluxo em celulas submetidas as concentracoes de 0,5 e 5 mM por 12 e 24 horas. Os niveis de Nitrito apresentaram uma diminuicao nas concentracoes de 0,5 e 5 mM em 12 horas de tratamento e um aumento em celulas tratadas com 5 mM por 24 horas. Em relacao aos niveis de nNOS houve uma reducao em seus niveis em celulas tratadas na concentracao de 5 mM por 24 horas. Ja a iNOS nao teve seus niveis alterados em nenhum dos tratamentos. Apos o tratamento com STZ foi observado a diminuicao nas formas fosforiladas de proteina Tau, nas concentracoes de 0,05; 0,5 e 5 mM por 12 e 24 horas. O tratamento com STZ 14 promoveu uma reducao nos niveis de Tau total nas concentracoes de 0,5 e 5 mM em 12 horas e 0,05; 0,5 e 5 mM por 24 horas. A razao entre a proteina Tau fosforilada e total nao apresentou alteracao significativa. A proteina BAG2 apresentou diminuicao em seus niveis em celulas tratadas com 0,5 e 5 mM por 12 horas de STZ e 5 mM por 24 horas. Desse modo, conclui-se que a STZ, em celulas de cultura primaria de hipocampo, mostrou ser uma ferramenta interessante de estresse oxidativo, caracteristica da DA, alem de apresentar morte celular tambem um fator envolvido com a doenca. / Cancer is the leading cause of death worldwide and it is considered a public health problem. Tumor cells exhibit a variety of features that enable tumor growth and dissemination, like resistance to cell death mechanisms. Phenothiazines is a group of drugs that have been used in the treatment of psychiatric disorders for a long time. Literature data demonstrate that these compounds exhibit relevant biological effects including antitumor activity. Publications from our group have evidenced extremely important effects of phenotiazines and analogues on mitochondria related to the increase of calcium cytosolic concentration promoted by this drug. In our earlier work with isolated mitochondria, it was demonstrated that these drugs promote the mitochondrial membrane permeability due to the opening of permeability transition pore complex with consequent dissipation of mitochondrial transmembrane potential, calcium efflux and cytochrome c release. Additionally, in our latest publication, we demonstrated the cytotoxicity of phenothiazines in hepatoma cells, accompanied by cellular morphological alterations, plasma membrane permeability and immediate dissipation of mitochondrial transmembrane potential. Among the studied phenothiazines, the most potent was thioridazine, which was chosen for the present work. The goal of this work was to underlie the molecular mechanisms of thioridazine-­induced cell death in leukemic cells, evaluating the role of BCL-­2 family proteins, as well as changes in signaling pathways associated to cell death, including endoplasmic reticulum (ER) stress. This compound was able to induce apoptosis in a concentration and time-­dependent manner, and also inhibit the cell cycle progression in K562 cells. Furthermore, tioridazine-­induced cell death was accompanied by dissipation of mitochondrial transmembrane potential, alterations in BCL-­2 proteins expression as well as activation of the kinases JNK, ERK1/2 and p38. Our results also demonstrated that thioridazine was able to activate the pro ­apoptotic protein BAX and the release of the mitochondrial protein Omi, resulting in mitochondrial outer membrane permeabilization. In addition, we observed that thioridazine was able to induce a severe ER stress, promoting an increase in the expression of the major sensor proteins in this signaling pathway, leading to cell death. We can conclude that both mitochondrial and ER stress contributes to thioridazine-­induced cell death in K562 cells. Besides the importance of our results to the elucidation of phenothiazines-­induced tumor cells death, it also confirms the promising therapeutic potential of this class of drugs as antitumor agents.

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.