>Magister Scientiae - MSc / Cissampelos capensis, is commonly known by the Afrikaans name ‟dawidjies” or ‟dawidjieswortel”. C. capensis is the most important and best known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western regions of South Africa. Among numerous other ailments, it is traditionally taken to treat male fertility problems. Yet, no studies have investigated the effects of this plant or its extracts on human spermatozoa. The aim of study was to investigate the effects of C. capensis rhizome extracts on sperm function.

A produção de espécies reativas de oxigênio (ROS) nos sistemas biológicos é contrabalanceada pelos sistemas antioxidantes enzimáticos e não-enzimáticos. Quando há um desequilíbrio entre a geração de ROS e esses sistemas, ocorre um aumento dessas espécies reativas causando estresse oxidativo, que pode levar a danos a macromoléculas, como lipídios, proteínas e o DNA. Os fármacos doxorrubicina (antineoplásico) e benzonidazol (antiparasitário) são conhecidos por induzir efeitos colaterais que podem estar relacionados ao aumento de ROS. Além disso, ensaios de mutagenicidade demonstram que esses fármacos apresentam atividade mutagênica por danos oxidativos. O Grupo NEQUIMED desenvolve substâncias com potencial atividade antineoplásica e antiparasitária, as quais ainda não foram avaliadas em relação às propriedades tóxicas e genotóxicas. Dessa forma, o objetivo deste trabalho foi analisar as propriedades mutagênicas e de estresse oxidativo dessas novas substâncias, comparando aos fármacos benzonidazol e doxorrubicina. Para detecção de ROS foi realizado o ensaio fluorimétrico utilizando o marcador 2,7-diacetato de diclorofluoresceína (DCFH-DA) em linhagens celulares de hepatocarcinoma humano (HepG2) e fibroblasto de camundongo (Balb/C 3T3 clone A31). O estado redox destas células foi avaliado através da quantificação da expressão gênica e do conteúdo proteico das enzimas antioxidantes através das técnicas de qRT-PCR e Western blot, respectivamente. A atividade mutagênica foi analisada com o ensaio Ames miniaturizado Salmonella/microssoma utilizando a linhagem TA102 de Salmonella typhimurium que detecta agentes mutagênicos que causam danos por oxidação. Os resultados mostraram que as substâncias estudadas pelo Grupo não induzem aumento na produção de ROS ou induzem em menores níveis do que doxorrubicina e benzonidazol, além de levar a alterações menos proeminentes que os fármacos para a expressão das proteínas antioxidantes. No ensaio mutagênico, o benzonidazol apresentou o pior perfil, doxorrubicina e Neq0438 somente foram mutagênicos com ativação enzimática, enquanto Neq0551 foi inativo. Assim, as novas substâncias (Neq0438 e Neq0551) apresentaram um perfil melhor do que os fármacos de referência, tornando-os candidatos promissores para estudos in vitro e in vivo subsequentes. / The production of reactive oxygen species (ROS) in biological systems is compensated by the enzymatic and non-enzymatic antioxidant systems. The excessive ROS production causes oxidative stress, which can damage important cellular macromolecules such as lipids, proteins and DNA. The drugs doxorubicin (antineoplastic) and benzonidazole (antiparasitic) are both known for their side effects, which can be related to the increase of ROS. Besides, mutagenicity assays show that these drugs have a mutagenic activity via oxidative damages. The research group NEQUIMED studies new substances with potential antineoplastic and antiparasitic activities, but their toxic and genotoxic properties have not been fully evaluated yet. Thus, the aim of this work is to assess the mutagenic potential and the oxidative stress generated by these substances, comparing them to benzonidazole and doxorubicin. The fluorimetric assay using the probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used for ROS detection in human hepatocarcinoma (HepG2) and mouse fibroblast (Balb/C 3T3 clone A31) cell lines. The redox state of these cells was evaluate by qRT-PCR and Western blot methods to quantifying gene expression and protein content of the antioxidant enzymes. The mutagenic potential was assessed by the miniaturized Ames text with the Salmonella/microssome mutagenicity assay, using the TA102 strain of Salmonella typhimurium, which detects oxidation damages to the DNA. The new substances did not induce an increase on ROS production, or did in lower levels when compared to doxorubicin and benzonidazole. Moreover, reference drugs also induced greater changes on the expression of the antioxidant enzymes. Benznidazole had a higher mutagenic activity, while Neq0438 and doxorubicin were mutagenic only when incubated with enzymatic activation. Neq0551 was inactive for Ames assay. Therefore, these new substances (Neq0438 and Neq0551) had a better overall profile than the reference drugs, turning out to be promising candidates for further in vitro and in vivo studies.

Masters of Science / Bacterial endophytes are able to improve the growth of their hosts through a number of different mechanisms such as nutrient uptake regulation, plant hormone production and regulation, siderophore production and phosphate solubilisation. They have also been shown to be able to provide protection to plants against various abiotic stressors, through various means such as oxidative stress protection. The purpose of this study was therefore to isolate endophytic bacteria from the stems of different weeds, to characterize their ability to use some of the most important growth promoting mechanisms including the ability to produce IAA, siderophores and ACC deaminase, what effect they had on the nutrient uptake in their hosts and to determine to what extent they could promote growth in the roots, stems and leaves of Brassica napus plants. In addition to this the endophytes were tested to see to what extent they could protect Brassica napus from the negative effects of vanadium stress and how this affected the plant physiologically in terms of morphology, overall biomass, the plants nutrient profile, lipid peroxidation and levels of cell death. The effect of vanadium stress on the oxidative state of Brassica napus was also monitored by determining the levels of stress induced reactive oxygen species (ROS) and the corresponding antioxidants that are responsible for regulating these reactive oxygen species. Six different endophytes (P1, P2, P3, P4, P5, P6) were isolated from different weed samples. Each endophyte was found to be able to significantly improve germination and growth in their host plant. Each isolate was able to improve the uptake of certain macronutrients and micronutrients in their respective hosts, while all of the isolates were shown to be capable of producing siderophores and ACC deaminase. One isolate had high levels of IAA production, with the remaining isolates producing small amounts of IAA. All isolates were also unable to solubilize phosphate. The five best performing endophytes (P1, P2, P3, P5, P6) in the preliminary growth trials were used in the follow up vanadium stressed growth trials, with endophyte P4 being left out of the remaining experiments. All of the endophytes showed improvements in growth promotion in comparison to the control, with endophyte treated plants showing both increased growth and biomass in both the non-stressed and vanadium stressed treatments of the vanadium stressed growth trial; however, the leaves of the vanadium stressed plants were significantly smaller than their non-stressed counterparts. When looking at the oxidative state it was found that vanadium stress caused a significant increase in the development of O2 -, H2O2 and •OH in the control and in addition to this it was shown that treatment with endophytes was able to cause a significant decrease in the levels of stress induced H2O2 and •OH in all of the treatments and O2 - for plants treated with endophyte P5. The noted change in the oxidative state of endophyte treated plants was attributed to an increase in the antioxidant activity of these plants, as it was found that endophyte treated plants showed a combination of increased activity for Superoxide dismutase, catalase and ascorbate peroxidase. This study has shown that endophytic bacteria from plant stems can be used to improve crop growth and yield, while simultaneously producing more nutrient dense crops from the same amount of land. It has also determined that endophytes P1, P2, P3, P5 and P6 are able to successfully provide protection to crop plants from the harmful effects of exposure to vanadium stress. This has great potential for improving food security locally and around the world, by allowing those who cannot gain access to large amounts of food to take in more nutrients from the same amount of food. Furthermore, it also presents the opportunity to use endophyte treatments to grow crops on land that has been previously contaminated with certain heavy metals. / 2023-12-01

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18899号 / 医博第4010号 / 新制||医||1009(附属図書館) / 31850 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Previous studies have established that ozone-oxygen mixtures can be used to inactivate a variety of microorganisms including bacteria, fungi and viruses. Ozone is a potent reactive oxygen species (ROS) that rapidly decays into a variety of additional short half-life ROS which have been shown to cause oxidative damage to biological molecules. I hypothesize that controlled ozone exposure and the subsequent generation of additional ROS would reduce viral infectivity by lipid and/or protein peroxidation. A proprietary ozone-oxygen delivery system was used to inactivate a series of enveloped [herpes simplex virus type-1 strain McIntyre (HSV-1), vaccinia strain Elstree (VAC), vesicular stomatitis virus strain Indiana (VSV), and influenza A strain (H1N1) A/WS/33] and non-enveloped [human adenovirus type2 (Ad2)] viruses. Plaque reduction and suspension-infection viral antigen assays were used to determine inactivation kinetics. After ozonation, HSV-1 and VSV lost up to 6 log10 infectious particles in 15 min, while VAC and influenza A lost up to 5 log10 in 40 min and 30 min, respectively. In comparison, the non-enveloped Ad2 lost up to 5 log10 in 60 min. Increasing amounts of serum supplementation in the ozone treated virus suspensions slowed the rate of inactivation in both enveloped and non-enveloped viruses, suggesting the protective effect of serum against ozone. Lipid peroxidation was determined through a chromogenic assay for malondialdehyde (MDA), a byproduct of peroxidation events. MDA concentrations were inversely correlated with virus infectivity, as MDA concentrations elevated with virus exposure time to ozone. Transmission electron microscopy images of Ad2, HSV-1, VAC and VSV confirmed the drastic morphological changes that resulted from ozone treatment. The ROS-mediated attack compromised the integrity of the lipid envelopes and protein shells of the viruses. These data suggest that a wide range of viruses can be inactivated through use of an innovative ozone delivery system, thus validating my hypothesis.

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京都大学 / 新制・論文博士 / 博士(医学) / 乙第13490号 / 論医博第2258号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 稲垣 暢也, 教授 長船 健二 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Saccharomyces cerevisiae teve seu seqüenciamento genômico finalizado em 1996, sendo composto de cerca de 6600 ORF\'s ( \"open reading frames\", quadros abertos de leitura) das quais aproximadamente 20% estão anotadas como não caracterizadas e de função molecular desconhecida. Com o objetivo de caracterizar algumas dessas ORF\'s relacionadas aos processos antioxidantes, escolhemos a proteína Yjl077cp. Através de nossos experimentos foi possível relacionar a proteína Yjl077cp com a resposta antioxidante, pois a linhagem mutante para o gene YJL077C apresentou letalidade quando exposta por 24 horas a uma concentração de 3mM de peróxido de hidrogênio (H2O2) . Testes de tolerância ao cobre, demonstraram que esse metal na concentração de 10mM, adicionado diretamente ao meio de cultura YPD, após um período de 4 horas de incubação afeta a cinética de crescimento, e após 24 horas de incubação, essa concentração é letal para a linhagem mutante. Observamos que a adição de CuSO4, causa modificação de pH do meio de cultura YPD, foram realizados experimentos para que verificássemos o efeito da acidificação do meio de cultura na viabilidade de Δics3, através de experimentos de tolerância em meio YPD ácido (pH 4.0) e com pH corrigido com adição de CuSO4. De acordo com os resultados obtidos, o efeito da letalidade de 10 mM de CuSO4 observado sem correção de pH, não ocorre quando em meio de cultura com o pH corrigido (pH6.0), demonstrando que, a letalidade da linhagem Δics3 é causada pelo acréscimo do metal e a mudança de pH (ácido, pH4.0). Testes de ICP-AES detectaram uma maior absorção intracelular de cobre na linhagem mutante Δics3, em condições de pH 4.0, quando comparada a absorção desse mesmo metal em condições de pH 6.0, demonstrando que grandes quantidades de cobre, estão sendo incorporadas para o meio intracelular, devido a acidificação do meio, sugerindo o envolvimento de Δics3, às ações das VATP-ases. Em conjunto esses resultados demonstram que o gene ICS3 é importante para a manutenção da viabilidade celular de S. cerevisiae crescida em meio de cultura com pH ácido e em condições de altas concentrações de CuSO4. O objetivo do presente trabalho foi entender a relação de ICS3 na resposta antioxidante e sensibilidade ao cobre através de experimentos de tolerância e em meio YPD ácido (pH 4.5) e com pH corrigido após adição de CuSO4. / Saccharomyces cerevisiae had its genome sequencing completed in 1996, consisting of about 6600 ORF\'s (\"open reading frames\" open reading frames) of which approximately 20% are annotated as uncharacterized and unknown molecular function. In order to characterize some of these ORF\'s related to antioxidants processes, we chose Yjl077cp protein . Through our experiments it was possible to relate the Yjl077cp protein with the antioxidant response, because the mutant strain to YJL077C gene showed lethality when exposed for 24 hours at a concentration of 3 mM hydrogen peroxide (H2O2). Copper tolerance tests demonstrated that this metal at concentration 10mM added directly to the medium YPD culture after a 4-hour period of incubation affects the growth kinetics, and after 24 hours of incubation, these concentration is lethal to mutant strain. We found that the addition of CuSO4, causes pH modification in the YPD culture medium, experiments were conducted to we check the effect of acidification of the culture medium on the viability of Δics3 through experiments of acid tolerance in YPD medium (pH 4.0) and pH adjusted with addition of CuSO4. According to the results, the effect of mortality of 10 mM CuSO4 observed without pH correction, does not occur when the culture medium with the Ph adjusted (pH6.0), demonstrating that the lethality of the strain Δics3 is caused by adding the metal and the change of pH (acid, pH4.0). ICP-OES tests detected greater intracellular uptake of copper in the mutant strain l1ics3 under conditions of pH 4.0 when compared to absorption of the same metal under conditions of pH 6.0, demonstrating that large amounts of copper, are being incorporated into the intracellular medium due to acidification of the medium, suggesting the involvement of Δics3 to the actions of the VATP-ases. Together, these results demonstrate that ICS3 gene is important for maintaining cell viability of S. cerevisiae grown in culture medium at acidic pH, and under conditions of high concentrations of CuSO4. The objective of this study was to understand the relationship ICS3 in antioxidant response and sensitivity to copper through experiments tolerance and YPD medium acid (pH 4.5) and fixed pH after addition of CuSO4.