The transmission of some infectious diseases requires that pathogens can survive (i.e., remain infectious) in the environment, outside the host. The viability of pathogens that are immersed in aerosols and droplets is affected by factors such as relative humidity (RH) and the chemical composition of the liquid media, but the effects of these stressors on the viability of viruses have not been extensively studied. The overall objective of this work was to investigate the effects of RH and media composition on the viability of viruses in suspended aerosols and stationary droplets. We used a custom rotating drum to study the viability of airborne 2009 pandemic influenza A(H1N1) virus across a wide range of RHs. Viruses in culture medium supplemented with material from the apical surface of differentiated primary human airway epithelial cells remained equally infectious for 1 hour at all RH levels tested. We further investigated the viability of two model viruses, MS2 and Φ6, in suspended aerosols and stationary droplets consisting of culture media. Contrary to the results for influenza virus, we observed a U-shaped viability pattern against RH, where viruses retained their viability at low and extreme high RHs, but decayed significantly at intermediate to high RHs. By characterizing the droplet evaporation kinetics, we demonstrated that RH mediated the evaporation rate of droplets, induced changes in solute concentrations, and modulated the cumulative dose of solutes to which viruses were exposed as droplets evaporated. We proposed that the decay of viruses in droplets follows disinfection kinetics. Lastly, we manipulated the chemical composition of media to explore the stability of viruses as a function of pH and salt, protein, and surfactant concentrations. Results suggested that the effects of salt and surfactant were RH and strain-dependent. Acidic and basic media effectively inactivated enveloped virus. Protein had protective effect on both non-enveloped and enveloped viruses. Results from this work has advanced the understanding of virus viability in the environment and has significant implications for understanding infectious disease transmission. / Doctor of Philosophy / Pathogenic organisms, including bacteria, viruses, fungi, protozoa, and helminths, cause infections that are responsible for substantial morbidity and/or mortality. For example, it is estimated that influenza has caused 9 million to 45 million illnesses and 12,000 to 61,000 deaths annually since 2010 in the United States. The spread of certain diseases relies on people touching the pathogenic organism on surfaces or inhaling it from the air. Successful transmission requires that the pathogen survive, or maintain its infectivity, while it is in the environment. The survival of pathogens can be affected by temperature, humidity, composition of the respiratory fluid carrying them, and other factors. However, there is limited research investigating the effects of these factors on the survival of viruses in the environment. In this work, we studied the effect of relative humidity (RH) on the survival of viruses, including influenza virus and two other types of viruses, in inhalable aerosols and larger droplets. We found that influenza viruses survive well in aerosols across a wide range of RH levels for at least 1 h. Conversely, the two model viruses survived best at both low and very high RHs, such as found indoors in the wintertime or in tropical regions, respectively, but had a pronounced decay at intermediate RHs. By measuring how fast droplets evaporated, we found that RH affected their chemistry and determined the total amount of stress that viruses were exposed to. This explained why a "U-shaped" survival pattern was observed against RH. We also investigated the survival of viruses in droplets containing different components. Results indicated that the effects of salt, surfactant, protein, and droplet pH depended on RH and the type of virus. The outcomes of this work are meaningful in predicting the survival of viruses in aerosols and droplets of various compositions in the environment and could provide insight on developing strategies to minimize the spread of infectious diseases.

Submitted by TATIANA PAGAN LOEIRO DA CUNHA null (tatiana.pagan@hotmail.com) on 2016-11-04T20:36:05Z No. of bitstreams: 1 Tese - Tatiana Pagan Loeiro da Cunha.pdf: 1659409 bytes, checksum: a80eea0627fa170f0797cedff8944fe6 (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-11-10T15:46:00Z (GMT) No. of bitstreams: 1 cunha_tpl_dr_jabo.pdf: 1659409 bytes, checksum: a80eea0627fa170f0797cedff8944fe6 (MD5) / Made available in DSpace on 2016-11-10T15:46:00Z (GMT). No. of bitstreams: 1 cunha_tpl_dr_jabo.pdf: 1659409 bytes, checksum: a80eea0627fa170f0797cedff8944fe6 (MD5) Previous issue date: 2016-09-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A crescente demanda da sociedade por alimentos saudáveis e produzidos de forma mais sustentável, impulsionou a agricultura começar a produzir alimentos de forma consorciada, onde em uma mesma área pode-se cultivar duas ou mais espécies. Porém, fatores como as interações fisiológicas, taxas de crescimento e a viabilidade agronômica e econômica das culturas devem ser avaliadas, pelo fato da alface sofrer com o sombreamento causado pelo tomateiro, além da condutividade elétrica utilizada, quando cultivado em substrato. Assim, o objetivo deste trabalho foi avaliar a viabilidade agronômica e econômica, além dos parâmetros fisiológicos de trocas gasosas, pigmentos fotossintéticos e crescimento das plantas em cultivo consorciado em relação ao monocultivo, em função das épocas de estabelecimento e da condutividade elétrica da solução nutritiva. Para isso, foram conduzidos dois experimentos, onde no primeiro, conduzido no município de Jaboticabal, SP, avaliou-se a viabilidade agronômica e os parâmetros fisiológicos do consórcio entre alface e tomateiro, em função das épocas de estabelecimentos do consórcio. No segundo, conduzido no município de Almería, Espanha, foi avaliada a viabilidade agronômica e econômica do cultivo consorciado em função da condutividade elétrica da solução nutritiva, ambos conduzidos em casa de vegetação usando como substrato a fibra de casca de coco. No primeiro estudo foi confirmada a viabilidade agronômica e as respostas fisiológicas positivas, quanto às trocas gasosas, teores de pigmentos clorofilianos e taxa de crescimento nas cultivares de alface em consórcio com o tomateiro, quando a alface é transplantada no mesmo dia ou até sete dias após o transplantio do tomateiro. Com os resultados obtidos no primeiro experimento, no segundo, também foi observada viabilidade agronômica quando a alface é transplantada no mesmo dia que o tomateiro. As maiores produtividades para ambas as culturas foram alcançadas quando cultivadas na condutividade elétrica de 2,5 dS m-1, condição em que também se obteve o maior rendimento econômico, evidenciando a viabilidade econômica do consórcio. / The increasing demand of society for healthy food produced in a more sustainable way, boosted the agriculture to start producing food using the intercropping way, which means that it is possible cultivate two or more species in the same field. However, factors such as physiological interactions, growth rates, and the agronomic and economic viability of crops should be assessed, since the lettuce suffers with the shading caused by tomato plants, as well as the electrical conductivity used when grown on the substrate. So, the objective of this study was to evaluate the agronomic and economic viability, in addition to the physiological parameters of gas exchange, photosynthetic pigments and biomass accumulation of intercropping in relation to monocultures, depending on the seasons of establishment and the electrical conductivity of the nutrient solution. For this, two experiments were conducted. The first was conducted in Jaboticabal, SP and it was evaluated the agronomic viability and physiological parameters of the lettuce intercropped with tomato, according to the seasons of the establishments of the intercropping. The second experiment was conducted in the city of Almeria, Spain and it was evaluated the agronomic and economic viability of intercropping due to the electrical conductivity of the nutrient solution. Both experiments were conducted in a greenhouse using as substrate the coconut husk fiber. The first study confirmed the agronomic viability and positive physiological responses for the gas exchange, chlorophyll pigment content, and growth rate in the lettuce cultivars intercropping with tomato when the lettuce was transplanted in the same day or up to seven days of the tomato transplanting. As the results obtained in the first experiment, it was also observed in the second experiment, the agronomic viability when lettuce was transplanted in the same day of the tomato. Higher yields for both crops were achieved when grown in the electrical conductivity of 2.5 dS m-1 , a condition that also had the highest economic performance, demonstrating the economic viability of the intercropping.

The subject of this diploma thesis is the influence of selected chemicals on cell lines viability. The theoretical part contains options of cancer treatment by using chemotherapeutics including their mechanism of action and side effects. Additionally, there are described alternative DNA structures with focus on G-quadruplexes and ligands that interact with G-quadruplexes. These compounds are promising drugs in cancer treatment due to their high specificity to G-quadruplexes, which are found in telomeres of chromosomes. G-quadruplex interacting ligands by stabilization of G-quadruplexes can inhibit the enzyme telomerase, which is necessary for telomere lengthening of rapidly dividing cancer cells. Additionally, the possibilities of viability assays are summarized in the theoretical part. The aim of the experimental part was comparing cytotoxic activity between commercially available chemotherapeutics and selected G-quadruplex interacting ligands. Another task was the study of apoptosis and necrosis after the treatment of selected chemicals on cell lines and after the localization of ligands interacting with G-quadruplexes in the cells of the breast cancer cell line. In the experimental part, G-quadruplex interacting ligands have been shown to exhibit similar cytotoxic activity to commercially available chemotherapeutic agents.

Objectives: To investigate the effects of serotonin and melatonin (MLT) on the regulation of malignant growth and the activity of serotonin receptors (5HTR1a/-1b) in prostate cancer (PCa) cell lines. Materials and Methods: In four PCa cell lines (LNCaP, 22RV1, PC3, DU145) and two reference cell lines 5HTR1a and -1b, relative mRNA expression levels were assessed. Different serotonin and MLT receptor agonists and antagonists were used in stimulation and inhibition experiments. Results: mRNA expression of 5HTR1b was higher than that of 5HTR1a in all PCa cell lines. Serotonin showed a significant growth stimulatory effect in all PCa lines. The 5HTR1a and -1b agonists/antagonists did not significantly affect viability. MLT inhibited viability only in PC3 cells. Similarly, the 5HTR1a antagonist induced apoptotic changes in PC3 cells only at 10–4M, while the 5HTR1b antagonist induced necrosis at 10–4M in all cell lines. Cell cycle alterations were seen in PC3 and DU145 cells under the influence of the 5HTR1a antagonist. Conclusions: Serotonin receptor antagonists and agonists as well as MLT influence viability and apoptosis of PCa cell lines at supraphysiologic concentrations. In contrast to other reports, our results do not support a regulatory role of serotonin or MLT receptor activation or inhibition in PCa growth. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Metals environment aquatic contamination has been a growing problem with serious consequences to life of different species over time, even after interrupted their emission into the environment. Among relevance metals, manganese (Mn) has shown importance to be related to several activities such as oil exploration, coal deposits extraction, fertilizer use in agriculture, among others. In living organisms, Mn is an essential trace element for a number of vital functions, and involves energy regulation by blood clotting. On the other hand, high concentrations of Mn can cause irreversible damage to living organisms primarily affecting central nervous system (CNS). Thus, waterborne Mn toxicity can switch from aquatic species, while metal bioaccumulation in marine fish or freshwater has been found around 0.2 to 19.0 mg/kg dry weight. In this study, silver catfish (Rhamdia quelen) were exposed to different Mn concentrations (4.2; 8.4 e 16.2 mg/L), under two different dissolved oxygen levels, normoxia (7.48 ± 0.28) and hypoxia (3.88 ± 0.41) for 15 days. At the end of protocol, Mn bioaccumulation as well parameters of oxidative stress and mitochondrial viability were evaluated in different tissues. In hypoxic conditions, the highest Mn concentration (16.2 mg/L) showed the lowest silver catfish kidney and brain lipoperoxidation (LPO) levels, while brain reduced glutathione (GSH) levels were increased in lower Mn concentration (4.2 mg/L) and kidney catalase activity was reduced in the same metal concentration, in relation to normoxia. Lowest Mn concentration (4.2 mg/L) in hypoxia showed higher gills mitochondrial viability, compared to normoxia. Hematocrit of silver catfish exposed to two highest Mn concentration (8.4 and 16.2 mg/L) was reduced in normoxia conditions while under hypoxia, these values were similar to control. Plasma except, Mn bioaccumulation in liver, kidney and gills were higher in normoxia than hypoxia. From these results is possible to suggest that hypoxia stimulates the development of adaptive mechanisms and/or hormesis in silver catfish exposed to Mn, mainly because the lower metal bioaccumulation occurred in this oxygen level. Contributing with this, under hypoxia, oxidative damage indicators were lower than those observed in normoxia, which were accompanied by changes in antioxidant system represented by GSH and catalase. In conclusion, our results show in the first time that silver catfish exposed to Mn contamination is able to show a better survival under hypoxia. These findings indicate need for continuing studies in search of molecular mechanisms involved in the adaptation and or hormesis processes, which were suggested here. / A contaminação do meio aquático por metais tem sido um problema crescente para à vida de diferentes espécies, mesmo depois de interrompida sua emissão no ambiente. Entre os metais de relevância, o manganês (Mn) apresenta importância por estar relacionado a uma série de atividades tais como exploração de petróleo, extrativismo em jazidas de carvão mineral, utilização de fertilizantes na agricultura, entre outros. Nos organismos vivos, o Mn constitui um oligoelemento essencial para uma série de funções vitais, e envolve desde a regulação da energia até coagulação sangüínea. Por outro lado, quando em concentrações elevadas, o Mn pode causar danos irreversíveis aos organismos afetando principalmente o sistema nervoso central (SNC). A toxicidade do Mn dissolvido na água pode variar entre as espécies aquáticas, a bioacumulação do metal em peixes marinhos ou de água doce tem sido encontrada em torno de 0,2 até 19,0 mg/kg de peso seco. No presente estudo, jundiás (Rhamdia quelen) foram expostos a diferentes concentrações de Mn (4,2; 8,4 e 16,2 mg/L), sob dois diferentes níveis de oxigênio dissolvido: normóxia (7,48±0,28 mg/L) e hipóxia (3,88±0,41 mg/L), durante 15 dias. Ao final do protocolo, a bioacumulação de Mn bem como, parâmetros de estresse oxidativo e a viabilidade mitocondrial foram avaliados em diferentes tecidos vitais. Em condições de hipóxia, a maior concentração de Mn (16,2 mg/L) mostrou os menores níveis de lipoperoxidação (LPO) em rim e encéfalo dos jundiás, enquanto os níveis de glutationa reduzida (GSH) encefálico foram aumentados na menor concentração de Mn (4,2 mg/L) e a atividade da catalase renal foi reduzida nesta mesma concentração de metal, em relação à normoxia. A menor concentração de Mn (4,2 m/L) em hipóxia proporcionou maior viabilidade mitocondrial das brânquias, em relação à normóxia. O hematócrito dos jundiás expostos às duas maiores concentrações de Mn (8,4 e 16,2 mg/L) foi reduzido em condições de normóxia, enquanto sob hipóxia, estes valores foram semelhantes ao grupo sem a presença do metal. Sob normóxia, a bioacumulação de Mn em fígado, rim e brânquias foi maior que sob hipóxia, excetuando o plasma. A partir desses resultados é possível sugerir que a hipóxia pode ter estimulado o desenvolvimento de mecanismos adaptativos ou de hormesis nos jundiás, em presença de Mn na água, principalmente porque neste nível de oxigênio, a bioacumulação do metal ocorreu em menor grau. Contribuindo com este resultado, sob hipóxia, os indicadores de danos oxidativos foram mais baixos que os observados sob normóxia, os quais foram acompanhados de alterações do sistema antioxidante representado pela GSH e catalase. Neste sentido, nossos resultados apontam para uma melhor sobrevivência da espécie em estudo, sob hipóxia, quando exposta ao Mn. Estes achados indicam a necessidade da continuidade dos estudos, em busca dos mecanismos moleculares envolvidos no processo de adaptação aqui sugeridos.

Objectives: To investigate the effects of serotonin and melatonin (MLT) on the regulation of malignant growth and the activity of serotonin receptors (5HTR1a/-1b) in prostate cancer (PCa) cell lines. Materials and Methods: In four PCa cell lines (LNCaP, 22RV1, PC3, DU145) and two reference cell lines 5HTR1a and -1b, relative mRNA expression levels were assessed. Different serotonin and MLT receptor agonists and antagonists were used in stimulation and inhibition experiments. Results: mRNA expression of 5HTR1b was higher than that of 5HTR1a in all PCa cell lines. Serotonin showed a significant growth stimulatory effect in all PCa lines. The 5HTR1a and -1b agonists/antagonists did not significantly affect viability. MLT inhibited viability only in PC3 cells. Similarly, the 5HTR1a antagonist induced apoptotic changes in PC3 cells only at 10–4M, while the 5HTR1b antagonist induced necrosis at 10–4M in all cell lines. Cell cycle alterations were seen in PC3 and DU145 cells under the influence of the 5HTR1a antagonist. Conclusions: Serotonin receptor antagonists and agonists as well as MLT influence viability and apoptosis of PCa cell lines at supraphysiologic concentrations. In contrast to other reports, our results do not support a regulatory role of serotonin or MLT receptor activation or inhibition in PCa growth. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Michael R. Kanost / Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms, which are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence identity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We purified recombinant Tribolium castaneum (red flour beetle) MCORP. As expected, no oxidase activity was detected. We analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP. They are constitutively expressed at a low level in many tissues, including ovaries. TcMCORP larval RNAi led to 100% mortality before adult stage. These deaths occurred during the larval to pupal and pupal to adult molts. Pharate pupal RNAi resulted in 20% mortality during the pupal to adult molt, and 100% mortality by one month after adult eclosion. In addition, knockdown of TcMCORP in females prevented oocyte maturation, thus greatly decreasing the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests. A multicopper oxidase-1 (MCO1) ortholog has been identified in all insect species examined so far; thus, MCO1 probably has a conserved physiological function in insects. Most of the well-studied MCOs are laccases, ferroxidases, or ascorbate oxidases. Previously we found Drosophila melanogaster MCO1 has ferroxidase activity and we identified three putative iron binding residues in DmMCO1. Our kinetic analysis of recombinant MCO1 from Drosophila melanogaster, Anopheles gambiae, Tribolium castaneum and Manduca sexta showed that MCO1 orthologs are much better at oxidizing ascorbate than laccase substrates or ferrous iron, suggesting that MCO1 orthologs function as ascorbate oxidases. The putative iron binding residues are required for ascorbate oxidase activity but not ferroxidase and laccase activities. Ascorbate oxidases have been identified only in plants. This is the first identification of ascorbate oxidase in insects. Further studies are needed to understand their physiological function in insects.

Call number: LD2668 .T4 1985 M62 / Master of Science

Call number: LD2668 .T4 1985 M63 / Master of Science

The ecological importance of drifting will depend on the abundance of drifting algae and whether it is reproductively viable. However, the ability of adult plants to successfully disperse long-distances by drifting is largely unknown, particularly for fucoids. The abundance, species composition, and reproductive status of drifting algae was examined by transect surveys around Kaikoura and Banks Peninsula. Abundance and species composition varied between sites, but all drifting algae that were in reproductive season, and had reproductive structures intact, were reproductively active. The reproductive longevity and viability of drifting and beach-cast Hormosira banksii, Durvillaea antarctica and Cytophora torulosa was compared with attached populations. Drifting algae remained reproductively viable, and fecundity did not differ from that of attached algae. Viable propagules were released from drifting algae for the duration of the experiments (H. banksii 57 days, D. antarctica 62 days, and C. torulosa 43 days). In contrast, beach-cast algae ceased to release propagules after 14 days. Dispersal by drifting relies on offshore transport after detachment. To determine the influence of wind and tidal currents on the nearshore transport of drifting algae, tagged H. banksii, D. antarctica, C. torulosa and GPS-tracked drifters were released from shore. Drifters generally moved in the direction of the prevailing wind, but some influence of tidal direction and bathymetry was detected. Offshore winds and outgoing tides were favourable for the offshore transport of surface drifting algae. Following dispersal and arrival at new locations, the distance between gametes may be important in determining the fertilisation success of dioecious species. Experiments testing the fertilisation success of H. banksii and D. antarctica, over increasing distances, showed that fertilisation success decreased with increasing distance between male and female gametes. Despite this, eggs were fertilised when male and female gametes were up to 2m apart, and sperm remained viable for 2 hours.