Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2015-06-18T17:20:38Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MarcadoresEstresseOxidativo.pdf: 2298812 bytes, checksum: e5d10f76398173dce7a3c6f19fd25794 (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2015-06-22T13:10:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MarcadoresEstresseOxidativo.pdf: 2298812 bytes, checksum: e5d10f76398173dce7a3c6f19fd25794 (MD5) / Made available in DSpace on 2015-06-22T13:10:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MarcadoresEstresseOxidativo.pdf: 2298812 bytes, checksum: e5d10f76398173dce7a3c6f19fd25794 (MD5) Previous issue date: 2012 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / No Brasil, o P. vivax representa a maioria dos casos de malária, totalizando quase 90% dos registros nos estados da Amazônia Legal. Vários estudos objetivaram compreender a relação do estresse oxidativo nos pacientes infectados pelo Plasmodium spp com as respostas clínica e parasitológica, pois o papel protetor ou deletério das espécies reativas de oxigênio e nitrogênio geradas por diferentes mecanismos no decurso da infecção é controverso na fisiopatogenia da malária humana. Entretanto, não há estudos que associem os danos oxidativos e as defesas antioxidantes do hospedeiro na malária vivax não complicada na Amazônia. Portanto, o objetivo deste estudo foi avaliar marcadores de estresse oxidativo no decorrer da fase aguda da malária por P. vivax, caracterizando o envolvimento das vias redox, a resposta do hospedeiro humano e a influência da quimioterapia, a fim de testar as hipóteses que os biomarcadores de estresse oxidativo não são alterados no decurso da fase aguda da malária vivax não complicada e a introdução da quimioterapia não contribui para o estresse oxidativo nestes sujeitos. Foi realizado estudo quantitativo longitudinal de casos constituídos por 38 sujeitos com malária por P. vivax adultos, de ambos os gêneros, os quais foram analisados antes, durante e após a instituição da terapia (D0, D2 e D7), comparados a grupo controle de 15 voluntários saudáveis, pareados por gênero e idade. Foram determinados por técnicas espectrofotométricas os teores de metemoglobina, a peroxidação lipídica, a capacidade antioxidante não enzimática total, a atividade da superóxido dismutase, os níveis eritrocitários de glutationa reduzida e da glutationa total plasmática. Comparado ao grupo controle, de maneira significativa, os teores de glutationa reduzida (p=0,004) foram inferiores e a peroxidação lipídica (p<0,001) foi superior, respectivamente. Entretanto, alterações significativas não foram observadas nos teores de metemoglobina, na capacidade antioxidante não enzimática total e na atividade da superóxido dismutase comparados ao grupo controle. Entretanto, com o decorrer do tratamento foram notados aumento significativo dos teores de metemoglobina (p<0,001) e da atividade da superóxido dismutase (p=0,038); porém os níveis de glutationa reduzida eritrocitária estava reduzidos (p=0,007). Além disso, não houve alterações significativas nos níveis da capacidade antioxidante não enzimática total e da glutationta total plasmática com a introdução dos antimaláricos. O nível de estresse oxidativo não foi obtido, uma vez que não foi significativa a correlação da peroxidação lipídica com a capacidade antioxidante não enzimática total durante o tratamento. Conclui-se que o aumento da peroxidação lipídica resultante do dano oxidativo foi contraposto pelo consumo de glutationa eritrocitária antes da introdução da terapia, sugerindo a participação das vias redox na malária vivax não complicada. A instituição da quimioterapia, provavelmente, interferiu no ciclo redox intraeritrocitário, o que foi caracterizado pelo continuo aumento da metemoglobina e da atividade da superóxido dismutase, bem como pela redução dos teores de glutationa reduzida nos eritrócitos. / In Brazil, P. vivax accounts for the majority of malaria cases, totaling almost 90% of the records in the Amazonian states. Several studies aimed to understand the relationship of oxidative stress in patients infected with Plasmodium spp clinical and parasitological responses because the plays role deleterious or protective of reactive oxygen species and nitrogen generated by different mechanisms during infection is controversial in the pathogenesis of human malaria . However, no studies involving oxidative damage and antioxidant defenses of the host uncomplicated vivax malaria in the Amazon. Therefore, the aim of this study was to evaluate oxidative stress markers during the acute phase of malaria by P. vivax, characterizing pathways involving redox response of the human host and the influence of chemotherapy in order to test the hypothesis that biomarkers of oxidative stress are not changed during the acute phase of vivax malaria and uncomplicated introduction of chemotherapy does not contribute for oxidative stress in these subjects. It was conducted quantitative study longitudinal case consisting of 38 subjects with malaria by P. vivax adults of both gender, which were analyzed before, during and after the introduction of therapy (D0, D7 and D2), compared to control group of 15 healthy volunteers, matched for gender and age. Were determined by spectrophotometric techniques levels of methemoglobin, lipid peroxidation, total non-enzimatic antioxidant capacity, the activity of superoxide dismutase, erythrocyte levels of reduced glutathione and total glutathione in bloodstone. Compared to the control group, significantly, the levels of reduced glutathione (p = 0.004) was lower and lipid peroxidation (p <0.001) was higher, respectively. However, no significant changes were observed in the levels of methemoglobin, in total non-enzimatic antioxidant capacity and activity of superoxide dismutase compared to the control group. However, with the course of treatment were noted significant increase in the levels of methemoglobin (p <0.001) and superoxide dismutase activity (p = 0.038), but erythrocyte levels of reduced glutathione was reduced (p = 0.007). Furthermore, no significant changes in the levels of total non-enzimatic antioxidant capacity and glutathione in bloodstone with the introduction of antimalarials. The level of oxidative stress was not obtained, since there was no significant correlation between lipid peroxidation with the total non-enzimatic antioxidant capacity during all treatment. It was concluded that increased lipid peroxidation resulting from oxidative damage is opposed by the use of glutathione before the introduction of the therapy, suggesting the involvement of redox pathways in uncomplicated vivax malaria. The institution of chemotherapy, probably, interfered in intraerythrocyte redox cycle, which was characterized by a continuous increase in methemoglobin and the activity of superoxide dismutase, as well as by the reduction of the levels of reduced glutathione in erythrocytes.

Women with pregestational diabetes have a 2-5 fold increased risk of giving birth to malformed babies compared with non-diabetic women. Diabetes-induced oxidative stress in maternal and embryonic tissues has been implicated in the teratogenic process. The malformations are likely to be induced before the seventh week of pregnancy, when the yolk sac is partly responsible for the transfer of metabolites to the embryo, and the uterine blood flow to the implantation site determines the net amount of nutrients available to the conceptus. We aimed to evaluate the effect on embryogenesis caused by a diabetes-induced disturbance in yolk sac morphology, uterine blood flow or altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state. We investigated to which extent maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes (SOD, CAT, GPX), vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis associated proteins (Bax, Bcl-2, Caspase-3) in the yolk sacs of rat embryos on gestational days 10 and 11. We found that maternal diabetes impairs, and that FA supplementation restores, yolk sac vessel morphology, and that maternal diabetes is associated with increased apoptotic rate in embryos and yolk sacs, as well as impaired SOD gene expression. We assessed uterine blood flow with a laser-Doppler-flow-meter and found increased blood flow to implantation sites of diabetic rats compared with controls. Furthermore, resorbed and malformed offspring showed increased and decreased blood flow to their implantation sites, respectively. In mice with genetically altered CuZnSOD levels, maternal diabetes increased embryonic dysmorphogenesis irrespective of CuZnSOD expression. We thus found the maternal diabetic state to be a major determinant of diabetic embryopathy and that the CuZnSOD status exerts a partial protection for the embryo in diabetic pregnancy.

Στόχος της παρούσας μελέτης ήταν να μελετήσει αν το εκχύλισμα των στιγμάτων του Crocus sativus L. αναστέλλει την επαγόμενη από σεληνιώδες νάτριο ανάπτυξη καταρράκτη σε ένα in vivo πείραμα με νεογνά επίμυων του γένους Wistar. Μέθοδοι: Τα νεογνά των επίμυων κατατάχθηκαν τυχαία σε 3 ομάδες. Ομάδα Ι (ομάδα μαρτύρων) όπου χορηγήθηκε υποδόρια φυσιολογικός ορός τη 10η ημέρα της ζωής. Ομάδα ΙΙ (ομάδα σεληνιώδους νατρίου) στην οποία χορηγήθηκε υποδόρια σεληνιώδες νάτριο (20 µmol/kg σωματικού βάρους) τη 10η ημέρα της ζωής. Ομάδα ΙΙΙ (ομάδα σεληνιώδους νατρίου και εκχυλίσματος στιγμάτων Crocus sativus L.) στην οποία εκτός από το σεληνιώδες νάτριο τη 10η ημέρα της ζωής χορηγήθηκε και εκχύλισμα στιγμάτων του Crocus sativus L. (60 mg/kg σωματικού βάρους) την 9η και 12η ημέρα της ζωής. Την 21η ημέρα της ζωής οι επίμυες θυσιάστηκαν και οι κρυσταλλοειδείς φακοί απομονώθηκαν και εξετάστηκαν για την εμφάνιση καταρράκτη. Ακολούθησε προσδιορισμός στους κρυσταλλοειδείς φακούς της δραστικότητας των αντιοξειδωτικών ενζύμων δισμουτάση του σουπεροξειδίου (SOD), της υπεροξειδάσης της γλουταθειόνης (GPx) καθώς και της καταλάσης (CAT). Προσδιορίστηκαν επίσης τα επίπεδα της γλουταθειόνης στους φακούς. Επιπλέον, μετρήθηκαν τα επίπεδα της μηλονικής διαλδεΰδης (MDA), ως δείκτη υπεροξείδωσης των λιπιδίων, καθώς και η συγκέντρωση των ελεύθερων σουλφυδρυλομάδων, ως δείκτη οξειδωτικής βλάβης των πρωτεϊνών, στους κρυσταλλοειδείς φακούς των επίμυων. Η επίδραση των χορηγούμενων παραγόντων στο πρωτεϊνικό προφίλ των φακών εκτιμήθηκε μέσω προσδιορισμού του λόγου των υδατοδιαλυτών προς τις μη υδατοδιαλυτές πρωτεΐνες του φακού. Τέλος έγινε ανάλυση των υδατοδιαλυτών πρωτεϊνών με ηλεκτροφόρηση σε πήκτωμα πολυακρυλαμιδίου. Αποτελέσματα: Το εκχύλισμα αποξηραμένων στιγμάτων του Crocus sativus L. επέδειξε σημαντική προστασία έναντι στην επαγόμενη από σεληνιώδες νάτριο καταρρακτογένεση στο in vivo πειραματικό μοντέλο που χρησιμοποιήσαμε. Οι μέσες τιμές των δραστικοτήτων των αντιοξειδωτικών ενζύμων SOD, GPx, CAT καθώς και της συγκέντρωσης της γλουταθειόνης αυξήθηκαν σημαντικά στην ομάδα που έλαβε εκχύλισμα στιγμάτων του Crocus sativus L. σε σύγκριση με την ομάδα των επίμυων που δέχθηκε μόνο την τοξική δράση του σεληνιώδους νατρίου. Το εκχύλισμα των στιγμάτων του Crocus sativus L. απέτρεψε σε σημαντικό βαθμό την υπεροξείδωση των λιπιδίων καθώς και την οξειδωτική βλάβη στις πρωτεΐνες του φακού. Επίσης απέτρεψε την πρωτεόλυση των υδατοδιαλυτών πρωτεϊνών του φακού. Συμπεράσματα: Χορήγηση εκχυλίσματος αποξηραμένων στιγμάτων του Crocus sativus L. απέτρεψε την επαγόμενη από το σεληνιώδες νάτριο καταρρακτογένεση σε νεογνά επίμυων του γένους Wistar πιθανώς μέσω ενίσχυσης της αντιοξειδωτικής άμυνας του κρυσταλλοειδούς φακού, μέσω αναστολής του βαθμού της υπεροξείδωσης των λιπιδίων, μέσω προστασίας των σουλφυδρυλομάδων των πρωτεϊνών καθώς και μέσω αναστολής της πρωτεόλυσης των υδατοδιαλυτών πρωτεϊνών του φακού. Αυτά τα ευρήματα τονίζουν τις πιθανές αντικαταρρακτογενετικές δράσεις των αποξηραμένων στιγμάτων του Crocus sativus L. οι οποίες αποδίδονται στις αντιοξειδωτικές τους ιδιότητες. / The present study sought to investigate whether Crocus sativus L. stigmas extract prevents selenite-induced cataractogenesis in vivo and to study the possible protective mechanism. Methods: Wistar rat pups were randomized into 3 groups. Group I (control) received subcutaneous injection of normal saline on postnatal day 10. Groups II (selenite treated) and III (selenite and Crocus sativus L. treated) received subcutaneous injection of sodium selenite (20 µmol/kg body weight) on postnatal day 10. Group III received intraperitoneal injections of Crocus sativus L. stigmas extract (60 mg/kg body weight) on postnatal days 9 and 12. On postpartum day 21, rats were sacrificed and the lenses were isolated and examined for cataract formation. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and glutathione levels, as markers of antioxidant defense, were measured in the isolated lenses. Levels of the indicator of lipid peroxidation, malondialdehyde (MDA), and protein oxidation (sulfhydryl content) in lens were also determined. Effect of the different treatments on lens’ protein profile was evaluated with the estimation of soluble to insoluble protein ratio and SDS-PAGE analysis of water-soluble fraction (WSF) of lens proteins. Results: Crocus sativus L. stigmas extract demonstrated significant protection against selenite-induced cataractogenesis in vivo. The mean activities of SOD, GPx, CAT and glutathione levels were significantly increased in group III compared to the selenite-treated group. Crocus sativus L. stigmas extract significantly prevented selenite-induced lipid peroxidation, protein oxidation, as well as proteolysis and insolubilization of the lens WSF. Conclusions: Crocus sativus L. stigmas extract prevented selenite-induced cataract formation in Wistar rats possibly by reinforcement of antioxidant status, reduction of the intensity of lipid peroxidation, protection of the sulfhydryl groups, and inhibition of proteolysis of the lens WSF. These findings highlight the anti-cataractogenic potential of Crocus sativus L. stigmas by virtue of their antioxidant properties.

Orientador: Fernando Braz Tangerino Hernandez / Resumo: A ocorrência de geadas nas pastagens causa perdas de alimento para os animais, reduzindo a produção de leite e carne. Existe uma diversidade de espécies forrageiras com grande potencial produtivo e ampla adaptação térmica, que podem ser cultivadas em áreas de risco desse fenômeno. No entanto é necessário caracterizar a tolerância dessas espécies a baixas temperaturas e as regiões com condições climáticas adequadas. Os métodos de avaliação de danos por baixas temperaturas são predominantemente qualitativos, baseados em critérios visuais que têm o viés da subjetividade. Esta tese teve como objetivos determinar as temperaturas mínimas de início de danos para sete espécies forrageiras e efetuar o zoneamento de risco de geadas para o estado do Paraná. Foram incluídas no estudo as forrageiras: Alfafa (Medicago sativa), Sorgo (Sorghum bicolor), Aveia Preta (Avena strigosa), Brachiaria brizantha cv. Marandu, Milheto (Pennisetum glaucum), capim Mombaça (Panicum maximum) e Tifton 85 (Cynodon spp). As plantas foram conduzidas em vasos em casa de vegetação até 60 dias e submetidas a baixas temperaturas no interior de uma câmara de crescimento com condições de luminosidade e temperatura controladas, atingindo valores mínimos de 0,2 -0,9, -1,8, -2,7, -4,1, -4,6 e -6,2oC, durante uma hora. Foram realizadas avaliações quantitativas pós testes de fluorescência da clorofila, condutividade elétrica de solução embebida com discos de folhas e atividade das enzimas ascorbato peroxidase (APX), cata... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

>Magister Scientiae - MSc / There is an influx in heavy metals into soils and ground water due to activities such as increased mineral mining, improper watering and the use of heavy metal contaminated fertilizers. These heavy metals are able to increase the ROS species within plants which may result in plant metabolism deterioration and tissue damage. Heavy metals may also directly damage plants by rendering important enzymes non-functional through binding in metal binding sites of enzymes. The heavy metal focused on in this study was vanadium due to South Africa being one of the primary produces of this metal. Two related Brassica napus L cultivars namely Agamax and Garnet which are economically and environmentally important to South Africa were exposed to vanadium. Physiological experiments such as cell death, chlorophyll and biomass determination were conducted to understand how these cultivars were affected by vanadium toxicity. A low cost, sensitive and robust vanadium assay was developed to estimate the amount of vanadium in samples such as water, soils and plant material. The oxidative state as well as the antioxidant profile of the two cultivars were also observed under vanadium stress. A chlorophyll assay which was conducted on the two cultivars exposed to vanadium showed a marked decrease in chlorophyll A in the suspected sensitive cultivar which was Garnet. However, the suspected tolerant cultivar Agamax fared better and the decrease in chlorophyll A was much less. A similar trend was observed for the two cultivars when the cell death assay was conducted. The vanadium assay showed that Garnet had higher concentrations of vanadium within its leaves and lower concentrations in its roots when compared to Agamax. This observation displayed that Agamax had inherent mechanisms which it used to localize vanadium in its roots and which assisted in its tolerance to the vanadium stress. The oxidative state was determined by doing assays for the specific reactive oxygen species namely hydrogen peroxide and superoxide. It was observed that vanadium treated Garnet leaves had higher reactive oxygen species (ROS) production when compared to the Agamax treated leaves. In-gel native PAGE activity gels were conducted to determine the antioxidant profile for the two cultivars which were exposed to vanadium. The antioxidant enzymes which were under investigation were ascorbate peroxide (APX), superoxide dismutase (SOD) and glutathione-dependent peroxidases (GPX-like) as these enzymes are known to be responsible for controlling the ROS produced in the plants. The GPX-like profile consisted of three isoforms. No isoforms were inhibited by vanadium treatments but one isoform had increased activity in both the Garnet and Agamax treated samples. The SOD profile for Garnet consisted of six isoforms and Agamax had seven isoforms. One isoform which was visualized in both Agamax as well as Garnet was inhibited by vanadium treatments. Agamax also had two isoforms which were up-regulated however the corresponding isoforms in Garnet showed no change. The Ascorbate peroxidase profile consisted of seven isoforms for both Garnet and Agamax. No isoforms were inhibited by vanadium treatment. Three isoforms were up-regulated in Garnet and Agamax under vanadium treatments. Here, it is illustrated that Garnet lacked certain mechanisms found in Agamax (and thus experienced more cell death, yield and chlorophyll loss) and performed worst under high vanadium concentrations. Although Garnet increased the activity of some of its antioxidant isoforms in response to increasing ROS levels it was not adequate to maintain a normal oxidative homeostasis. This disruption in oxidative homeostasis lead to plant damage. Agamax was observed to produce less ROS than Garnet and was able to control the ROS produced more effectively than Garnet and thus less damage was observed in Agamax.

Orientadora: Profa. Dra. Giselle Cerchiaro / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, Santo André, 2018. / Em doencas neurodegenerativas amiloidais o estresse oxidativo tem um papel importante juntamente com a proteina ¿À-amiloide (A¿À), associada a formacao de placas senis na Doenca de Alzheimer. Tais condicoes demonstraram desbalanco de metais, como cobre e zinco, tanto na concentracao celular e quanto nos processos antioxidantes. A Cu,Zn-Superoxido Dismutase (SOD1), em condicoes neurodegenerativas, pode demonstrar alteracoes estruturais e funcionais, tendo menor afinidade pelo cobre e pelo zinco. Diante destas condicoes, o objetivo principal desta tese foi em condicoes oxidativa (H2O2) e neurodegenerativa (A¿À1-42) avaliar os danos a biomoleculas, concentracao metais e a influencia da enzima SOD1 em linhagens de celulas neuronais (NSC-34 e mHippoE2). Diferentes respostas quanto a sensibilidade das linhagens neuronais foi observada durante as condicoes oxidativas e neurodegenerativa. Quanto os danos ao DNA a linhagem NSC-34 demonstrou maior sensibilidade a condicao oxidativa, com aumento de danos ao DNA, lesoes oxidativas em bases nitrogenadas que indicaram a presenca de lesoes tipo 8-oxo-G, corroborando com anormalidades nucleares e inibicao do processo de divisao celular. Nesta mesma linhagem quantidades aumentadas de Cu foram observadas, juntamente com a presenca da enzima SOD1 a nivel citoplasmatico e nuclear na condicao oxidativa (H2O2), alem de resultados significantes para danos permanentes ao DNA (anormalidades nucleares e quebras cromossomicas). A linhagem mHippoE2 apresentou aumentos significativos mediante a condicao oxidativa e neurodegenerativa, como oxidacao de proteinas e lipidios, demonstrando tambem alteracoes morfologicas citoplasmaticas. O tratamento com A¿À1-42 demonstrou aumento de danos ao DNA, lesoes oxidativas 8-oxo-G e tambem em bases purinicas. Podemos observar nesta mesma linhagem a forte influencia do Zn na condicao neurodegenerativa, atividade da SOD1 em ambas condicoes e tambem danos permanentes ao DNA mediante condicao neurodegenerativa. Dentre os resultados obtidos salientamos a relevancia dos achados na condicao neurodegenerativa ocasionada pelo peptideo A¿À1-42 nos ensaios para avaliacao genotoxica e mutagenica. Tal condicao demonstrou a presenca de danos importantes a bases nitrogenadas, tanto purinicas quando pirimidinicas, apontando tambem para possiveis efeitos mutagenicos detectados pelos eventos de quebras cromossomicas associados as anormalidades nucleares, bem como a presenca da enzima SOD1 no nucleo das celulas. / In neurodegenerative diseases, oxidative stress plays an important role associated with â-amyloid protein (Aâ), associated with the formation of amyloid plaques in Alzheimer's Disease (AD). In AD condition it has been demonstrated an imbalance of essential metals, such as copper and zinc, their cellular concentration and antioxidant processes alterations. The antioxidant enzyme Cu, Zn-Superoxide Dismutase (SOD1), under neurodegenerative conditions has structural and functional changes, such as lower affinity for copper and zinc. According to these conditions, the main objective of this thesis was to investigate how the oxidative (H2O2) and neurodegenerative (Aâ1-42) conditions cause biomolecules damage, metal alteration and SOD1 location in neuronal cell lines (NSC-34 and mHippoE2). Different responses in neuronal cell lines were observed during the conditions evaluated. For DNA damage, the NSC-34 cells demonstrated greater sensitivity to the oxidative condition, with increased DNA damage, oxidative lesions on nitrogenized bases indicating the presence of 8-oxo-G type lesions. In this same cell line we observed an increase of Cu amount, together with the presence of the SOD1 enzyme at the cytoplasmic and nuclear level in the oxidative condition (H2O2). The mHippoE2 cell line presented increased protein oxidation through the oxidative and neurodegenerative condition. Treatment with Aâ1-42 demonstrated increased DNA damage in this cell, 8-oxo-G oxidative lesions and also purine bases. We observed, in this same cell line, the strong influence of Zn on the neurodegenerative condition, SOD1 activity in both conditions and it was observed permanent damages to DNA in the neurodegenerative condition. Among the results, we highlight the relevance of the findings in the neurodegenerative condition caused by the Aâ1-42 peptide in the genotoxic and mutagenic evaluation trials. This condition demonstrated the presence of important damages to nitrogenated bases, both purine and pyrimidine, also pointing to possible mutagenic effects detected by the events of chromosomal breaks associated with nuclear abnormalities, as well as the translocation of the SOD1 enzyme to nuclei.

3.2. Abstract Soil contamination by heavy metals represents rather serious environmental problem for both human health and an environment itself. One of the perspective technologies dealing with this threat that only recently has been intensely developed is phytoremediation by means of short rotation coppice plantations. As plants used in this technology (mostly poplars and willows) host two major groups of mycorrhizal fungi substantially influencing plant physiology it is important to study plant-mycobiontheavy metals interactions rather than just plant-heavy metals interactions. The present thesis aimed to contribute to the growing knowledge of the field by search for suitable mycobionts of poplar or willow tolerant to heavy metals, by evaluating an activity of the key antioxidative enzyme in selected mycobionts and by looking at physiological responses of plant hosts to their mycobionts in a soil polluted by heavy metals. The first experiment in vitro focused on screening of morphometric criteria of fungi growing on solid growth media amended with mixture of heavy metals. Based on the results, several tolerant ectomycorrhizal strains were chosen for the next inoculation of fast growing trees serving phytoextraction and phytostabilisation strategies. The second, re-synthetic experiment was conducted in...

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (>170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis.

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About ten percent of cases are inherited, of which twenty percent are due to mutations in the superoxide dismutase 1 (SOD1) gene. Since the only FDA approved ALS drug prolongs survival by just a few months, new therapies for this disease are needed. Experiments in transgenic ALS mouse models have shown that decreasing levels of mutant SOD1 protein alters and in some cases entirely prevents disease progression. We explored this potential therapeutic approach by using a single stranded AAV9 vector encoding an artificial microRNA against human SOD1 injected bilaterally into the cerebral lateral ventricles of neonatal SOD1G93A mice. This therapy extended median survival from 135 to 206 days (a 50% increase) and delayed hind limb paralysis. Animals remained ambulatory until endpoint, as defined by a sharp drop in body weight. Treated animals had a reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons. As compared to untreated SOD1G93A mice, the AAV9 treated mice also had significant improvements in multiple parameters including the number of motor neurons, diameter of ventral root axons, and degree of neuroinflammation in the spinal cord. These studies clearly show that an AAV9-delivered artificial microRNA is a translatable therapeutic approach for ALS.