Orientador: Rogério Lopes Vieites / Banca: Erica Regina Daiuto / Banca: Severino Matias de Alencar / Resumo: O presente trabalho teve por objetivo determinar a atividade antioxidante, compostos fenólicos totais e cor em abacate 'Hass' submetido ao tratamento térmico, radiação gama e ultravioleta. Após a colheita, os frutos foram selecionados para tornar o lote homogêneo. Foi realizada a caracterização do fruto verde e amadurecido quanto a acidez titulável, pH, umidade, teor de lipídios, cinzas (minerais), proteína (nitrogênio bruto), açúcar total e coloração. Foram realizados 3 experimentos com frutos recém colhidos. Experimento I: imersão dos frutos em água a 45ºC por 5, 10, 15 e 20 minutos; Experimento II: radiação gama nas doses 0,2; 0,4; 0,6 e 1,0kGy e Experimento III: radiação ultravioleta (UVC) por 5, 10, 15 e 20 minutos. Após os tratamentos, os frutos foram armazenados em temperatura ambiente (21ºC±1 e 70±5% de UR) e refrigerado (10ºC±1 e 90±5% de UR). Os frutos foram analisados quanto à capacidade antioxidante pelo método DPPH e compostos fenólicos totais aos 0, 3, 9 e 12 dias. A coloração foi avaliada aos 0, 3, 6, 9, 12 e 15 dias. Os resultados foram submetidos à análise de variância e as médias comparadas pelo teste de Tukey ao nível de 1% de probabilidade. Nas condições em que os experimentos foram realizados, pode-se concluir que os frutos do tratamento testemunha e mantidos sob refrigeração apresentaram maior capacidade antioxidante e teor de compostos fenólicos totais ao longo do período de armazenamento; o tratamento térmico por 5 e 10 minutos foi efetivo na manutenção da capacidade antioxidante e dos compostos fenólicos; a dose de 0,6kGy não foi efetiva na manutenção da capacidade antioxidante; a radiação ultravioleta (UV-C) não manteve a capacidade antioxidante dos frutos, no entanto ocasionou aumento na quantidade de compostos fenólicos; o tratamento térmico e a radiação ultravioleta não afetaram a coloração da polpa / Abstract: This study aimed to determine the antioxidant activity, total phenolic compounds and color in avocado 'Hass' submitted to heat treatment, gamma and ultraviolet radiation. After harvest, fruits were selected to make the homogeneous portion. The characterization was performed of the green fruit and mature as acidity, pH, moisture, lipids, ash (minerals), protein (nitrogen gross), total sugar and coloring. 3 experiments were performed with freshly harvested fruits. Experiment I: immersion of fruits in water at 45 ° C for 5, 10, 15 and 20 minutes; Experiment II: gamma radiation doses 0.2, 0.4, 0.6 and 1.0 kGy and Experiment III: ultraviolet radiation (UV-C) for 5, 10, 15 and 20 minutes. After treatments, fruits were stored at room temperature (21±1ºC and 70±5% HR) and refrigeration (10ºC±1 and 90±5% HR). The fruits were analyzed for their antioxidant capacity by DPPH method and phenolic compounds at 0, 3, 9 and 12 days. The color was measured at 0, 3, 6, 9, 12 and 15 days. Data were subjected to analysis of variance and mean comparison using Tukey's test at 1% probability. In conditions where the experiments were performed, one can conclude that the fruits of control treatment and kept under refrigeration had higher antioxidant capacity and content of phenolic compounds during the storage period, the heat treatment for 5 and 10 minutes was effective the maintenance of antioxidant capacity and phenolic compounds, the dose of 0.6 kGy was not effective in maintenance the antioxidant capacity, ultraviolet radiation (UV-C) did not maintain the antioxidant capacity of fruits, however caused an increase in the content of phenolic compounds, heat treatment and ultraviolet radiation did not affect the pulp color / Mestre

Estudaram-se soluções para o pós-tratamento de efluentes de reatores UASB e de lagoas de estabilização visando à utilização em irrigação de culturas agrícolas. Nesta aplicação, normalmente são desejáveis bons níveis de remoção de sólidos em suspensão e graus elevados de desinfecção, além da conservação dos principais nutrientes. No caso do efluente de reatores UASB em escala real, foram construídas unidades em escala piloto envolvendo sedimentação, filtração e radiação UV. No primeiro experimento, o efluente dos reatores UASB passou por filtro de areia pressurizado em alta taxa e por reator UV de fluxo contínuo. Nesta etapa a concentração de sólidos em suspensão (SST) no efluente dos reatores UASB teve média de 233 mg/L, devido a problemas operacionais, sendo que o filtro apresentou eficiência média de 60% de remoção de sólidos em suspensão. Porém, como o filtrado apresentou absorbância ainda elevada, média de 0,476 cm-¹, a desinfecção UV resultou ineficiente e a contagem de coliformes do efluente final incompatível com o uso agronômico pretendido. No segundo experimento, o efluente dos reatores UASB recebeu tratamento complementar passando por decantador de alta taxa e filtração em leito de manta geotêxtil, seguindo finalmente para a desinfecção UV. A concentração de SST no efluente dos reatores UASB, sanados os problemas operacionais da ETE teve valor médio de 82 mg/L. Com essa concentração afluente relativamente baixa, o decantador lamelar demonstrou-se pouco eficiente e apresentou efluente com SST médio de 67 mg/L. No entanto, foi bastante útil para conter picos de concentração de sólidos no efluente dos reatores UASB, protegendo a etapa posterior de filtração. No efluente do filtro de manta geotêxtil, o SST médio foi de apenas 7 mg/L., ABS (254nm) de 0,245 cm-1 e Turbidez de 11 UNT. A desinfecção do efluente por radiação ultravioleta com dose recebida de 2,44 W.h/m³, resultou satisfatória com média geométrica de 1,12x101 NMP/100mL de E coli. No caso do efluente de sistema de lagoas de estabilização em escala real, aplicou-se pós-tratamento em escala piloto composto de filtro de areia pressurizado de alta taxa seguido de desinfecção por hipoclorito de sódio. A concentração de SST no efluente da lagoa facultativa teve média de 117 mg/L, enquanto que a média no efluente do filtro foi de 87 mg/L. Para concentrações de cloro dosado de 2,9; 6,2 e 11,7 mg/L, as médias geométricas das densidades de E coli no efluente final resultaram 1,13x10² NMP/100mL, 1,01x101 NMP/100mL e valor não detectado pelo método, respectivamente. Concluiu-se que é possível aplicar pós-tratamentos relativamente simples aos efluentes de reatores UASB e de lagoas de estabilização, quando se deseja uso agronômico e as principais condições operacionais destas unidades puderam ser avaliadas. / It was studied the application of a simple and economical post-treatment of effluent from wastewater treatment plant for the purpose of use on irrigation of agricultural crops. The research was conducted in two different wastewater treatment plants, (1) WTP UASB reactors with two application of post-treatment: (1.1) Post treatment with a sand bed filter pressurized at high-rate followed by ultraviolet disinfection. In this step, the concentration of suspended solids (TSS) in the effluent from the UASB reactor was 233 mg/L, the filter had efficiencies of 60% removal of suspended solids in the effluent from the UASB reactor, however, was not an appropriate result for effective disinfection of the effluent in the ultraviolet reactor. Absorbance values reached an average of 0.476 cm-¹. (1.2) Post-treatment at a high rate clarifier and on geotextile bed filtration to apply the ultraviolet disinfection. The concentration of TSS in the UASB reactor effluent has an average value of 82 mg/L, the concentration of the same parameter in the effluent from the clarifier was 67 mg/L and effluent of the filter geotextile, 7 mg/L. The system removed 91% of TSS in the effluent of the UASB reactor. In this experiment there was obtained a post-treated effluent with ABS (254nm) of 0.245 cm-1 and a turbidity of 11 NTU. The disinfection of the effluent by ultraviolet radiation with a dose received of 2.44 W.h/m³, obtained satisfactory results with geometric mean of 1.12x101 NMP/100mL E coli. (2) WTP by stabilization ponds with post-treatment in a pressurized sand filter followed by highrate disinfection by sodium hypochlorite solution. A TSS concentration in the effluent from facultative pond had an averaged 117 mg/L, the concentration of the same parameter in the effluent of the filter was 87 mg/L. With application of 2.88 mgCl2/L resulted in 1.13x10² NMP/100mL E coli, for the application of 6.2 and 11.7 mg/L chlorine was obtained 1.01x101 NMP/100mL E coli and ND, respectively.

O cloridrato de raloxifeno é um fármaco utilizado para o tratamento e prevenção da osteoporose em mulheres na pós-menopausa e foi aprovado pelo FDA para prevenção de câncer de mama invasivo. Pertence à classe de moduladores seletivos de receptor estrogênico. Foi desenvolvido pela Ely Lilly Company e é comercializado como Evista® na forma de comprimidos na dosagem de 60mg. Devido à importância do fármaco e interesse do Instituto Nacional de Ciências e Tecnologia – Inovações Farmacêuticas no desenvolvimento métodos de síntese e de controle de qualidade, o presente trabalho teve como objetivo o desenvolvimento de métodos analíticos para assegurar a qualidade tanto da matéria-prima quanto do produto acabado. A matéria-prima foi analisada quanto às suas características físico-químicas através da determinação da solubilidade, do ponto de fusão e da calorimetria exploratória diferencial. A caracterização e identificação da matéria-prima foram realizadas identificando grupos característicos como o cloreto e grupo fenol e também por espectrofotometria na região do infravermelho e do ultravioleta e por cromatografia líquida de alta eficiência. A forma farmacêutica comprimidos também foi caracterizada e identificada através dos mesmos métodos que a matéria-prima e adicionalmente através de cromatografia em camada delgada e eletroforese capilar. Os métodos desenvolvidos e validados para quantificação da matéria-prima foram volumetria em meio não-aquoso e cromatografia líquida. Para os comprimidos, foram desenvolvidos métodos por espectrofotometria na região do ultravioleta, cromatografia líquida e eletroforese capilar. Os resultados de todos os métodos foram analisados estatisticamente para verificar equivalência nas determinações. / Raloxifene hydrochloride is used for the treatment and prevention of osteoporosis in post-menopausal women. It was approved by FDA for the prevention of invasive breast cancer. It was developed by Ely Lilly Company and marketed as Evista® in form of tablets of 60mg. Due to the importance of this substance and the interest of INCT-IF in its synthesis and quality control, the present work developed analytical methods to assure the quality of the raw substance and the pharmaceutical form. Qualitative and quantitative methods were developed for the analysis of raloxifene hydrochloride as raw substance and tablets. The raw substance was characterized by its physical and chemical characteristics by solubility, melting point and differencial scaning calorimetry. It was also identified by the analysis of characteristic groups such as chloride and phenol, and by infrared and ultraviolet spectrophotometry; also by high performance liquid chromatography. The tablets were also characterized and identified by the same methods as for the raw substance, but in addition by thin layer chromatography and capillary electrophoresis. The developed and validated methods for the assay of the raw substance were: non-aqueous titration and liquid chromatography. For the tablets, the assays were: ultraviolet spectrophotometry, liquid chromatography and capillary electrophoresis. The results of all methods were analyzed statistically to verify the equivalence of the determinations.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-11-29T17:27:43Z No. of bitstreams: 1 2013 - Ivaldo Jos? da Silva.pdf: 36618657 bytes, checksum: 428b35f4aa0bf23daa7adf9b14b4e7ef (MD5) / Made available in DSpace on 2018-11-29T17:27:43Z (GMT). No. of bitstreams: 1 2013 - Ivaldo Jos? da Silva.pdf: 36618657 bytes, checksum: 428b35f4aa0bf23daa7adf9b14b4e7ef (MD5) Previous issue date: 2013-03-21 / Research in Environmental Education Criticizes aims to construct and reflective critique of knowledge allowing individuals greater contextualisation-environmental relations and consequent changes, in attitudes towards collective transformation of reality. The municipality of Z? Doca, regional pole of Turi, located east of the State of Maranh?o, in the Northeast region near the border with the State of Par?, is responsibility to articulate educational actions, political and economic, social and environmental development of over 17 other municipalities in the region. It was found that municipalities the environmental sanitation and water treatment is performed unsatisfactorily and precarious, signaling the need for sustained research-driven actions on the principles of education Wake up in the population capable environmental more questioning and intervention in the local reality. This research aimed to instigate critical knowledge construction to allow greater local perception of reality, associated with insertion technique feasibility research, Community research, alternative technology of water treatment based on ultraviolet radiation-UV Tube. The Methodological development used by this social research was action research procedures where awareness, dynamic cooking workshops, adaptation and installation of UV Tube, meetings and other educational practices addressed the theme of water treatment, social issues, environment and health, among other correlated in a participative manner, reflective and harmonic among respondents and researchers. The search area was chosen as the community of rural settlement good hope, located in Z? Doca, where were installed and evaluated 05 (five) UV Tube units. Data collection took place 64 (sixty-four) interviews and questionnaires applied before the dynamics of awareness and at the end of the whole educational process in order to identify improvement in the perception of the community in relation to the subject dealt with and the local reality, and consequential changes in attitudes. The treatment of data collected in the questionnaires presented variables that were tested with the non-parametric test of Chi-square and other calculated averages of answers with their respective standard deviations and their minimum and maximum values. The results indicated favourable conditions for the adaptation and insertion of the UV tube technology showing economic viability and operational compared to similar projects successfully installed in other countries, especially the acceptance of the proposal and the ease of access to the materials needed to manufacture of UV Tube. As the analysis of educational activities related to the thematic discussion addressed significant responses were expansions and modifications in order to understand the local reality criticizes pointing, including changes to posture that assisted the search for better quality of life for the community. It is concluded that the implementation of concrete action of insertion of the UV Tube use, Dialogic and critical process of construction of knowledge based on the intrinsic relationship between action and reflection associated with the permissive criticizes of knowledge built by researched and researchers was the recipe to achieve the objectives of the survey. / A pesquisa em Educa??o Ambiental Cr?tica visa ? constru??o reflexiva e cr?tica de conhecimentos, permitindo aos indiv?duos maior contextualiza??o das rela??es socioambientais e consequentes mudan?as de atitudes voltadas para transforma??o coletiva da realidade. O munic?pio de Z? Doca, p?lo regional do Alto Turi, localizado a leste do Estado do Maranh?o, pr?ximo a divisa com o Estado do Par? ? respons?vel em articular a??es educacionais, socioambientais, pol?ticas e econ?micas para o desenvolvimento de mais 17 outros munic?pios da regi?o. Verificou-se que nestes munic?pios o saneamento ambiental e, principalmente, o tratamento de ?gua, s?o realizados de forma insuficiente e prec?ria, sinalizando a necessidade de a??es orientadas por pesquisas sustentadas nos princ?pios de uma Educa??o Ambiental capaz de despertar na popula??o maior problematiza??o e interven??o na realidade local. Esta pesquisa objetivou instigar a constru??o de conhecimentos cr?ticos aptos a permitirem maior percep??o da realidade local associada ? investiga??o da viabilidade t?cnica de inser??o, junto ? comunidade pesquisada, de tecnologia alternativa de tratamento de ?gua baseado em radia??o ultravioleta ? Tubo UV. Para o desenvolvimento metodol?gico esta pesquisa social utilizou-se de procedimentos da pesquisa-a??o em que din?micas de sensibiliza??o, oficinas de confec??o, adapta??o e instala??o do Tubo UV, reuni?es e outras pr?ticas educacionais abordaram a tem?tica do tratamento de ?gua, as quest?es sociais, ambientais e de sa?de, entre outras correlacionadas de forma participativa, reflexiva e harm?nica entre pesquisados e pesquisadores. Como ?rea de pesquisa foi escolhida a comunidade do assentamento rural Boa Esperan?a - ABE, localizada em Z? Doca onde foram instalados e avaliados 05 (cinco) unidades do Tubo UV. Para coleta de dados foram realizadas 64 (sessenta e quatro) entrevistas e aplicados question?rios antes das din?micas de sensibiliza??o e ao final de todo o processo educativo, com objetivo de identificar melhoria na percep??o da comunidade em rela??o ? tem?tica abordada e a realidade local, al?m de consequentes mudan?as de atitudes. O tratamento dos dados coletados nos question?rios apresentou vari?veis que foram analisadas com o teste n?o param?trico do Qui-Quadrado e outras, calculadas as m?dias de respostas com seus respectivos desvios padr?es e seus valores m?nimos e m?ximos. Os resultados indicaram condi??es favor?veis ? adapta??o e inser??o da tecnologia do Tubo UV apresentando viabilidade econ?mica e operacional comparada a projetos id?nticos instalados com sucesso em outros pa?ses, destacando-se a aceita??o da proposta e a facilidade de acesso aos materiais necess?rios a confec??o do Tubo UV. Quanto a an?lise das a??es educativas relacionadas a discuss?o da tem?tica abordada registraram-se significativas respostas de amplia??es e modifica??o na forma cr?tica de perceber a realidade local apontando, inclusive, mudan?as de postura que auxiliaram a busca de melhor qualidade de vida para comunidade. Conclui-se que a realiza??o da a??o concreta da inser??o do uso do Tubo UV, em processo dial?gico e cr?tico de constru??o de conhecimento pautado na rela??o intr?nseca entre a??o e reflex?o associada ? permissiva cr?tica dos saberes constru?dos por pesquisados e pesquisadores foi a receita para alcan?ar os objetivos da pesquisa.

A via de sinalização da GTPase RhoA atua em diversos processos celulares. Para avaliar o comportamento de RhoA, após estresse causado por radiação ultravioleta, foram gerados clones mutantes que expressam RhoA em seu estado constitutivamente ativo e dominante negativo. Após exposição das linhagens à radiação ultravioleta, observou-se uma maior sensibilidade e um maior tempo de recuperação das linhagens quando a atividade de RhoA é reduzida. Estes prejuízos no reparo prejudicaram a proliferação e sobrevivência celular quando da deficiência na atividade de RhoA. Em linhagens deficientes na via de NER, percebemos que estas linhagens possuem uma capacidade ainda mais reduzida de reparo quando a atividade de RhoA é inibida. / The RhoA GTPase signaling pathway acts on many cellular processes. To evaluate this possible RhoA function after stress caused by ultraviolet radiation, mutant clones expressing RhoA in its constitutively active or dominant negative forms were generated. After exposure of the cells to ultraviolet radiation, cell lines showed a higher sensitivity and a delayed recovery capacity when the RhoA activity is reduced. The impaired repair reduced the cells proliferation and survival under RhoA deficiency. In cell lines deficient in NER pathway, we notice that these cell lines, have a further reduced ability to repair damaged DNA under RhoA inhibition.

Este trabalho teve por objetivo estudar a desinfecção seqüencial cloro-radiação ultravioleta e ozônio-radiação ultravioleta a fim de avaliar a resistência de microrganismos indicadores de poluição fecal - E. coli, coliformes totais e Clostridium perfringens - e compará-la à dos protozoários patogênicos: Giardia spp. e Cryptosporidium spp.. Houve, primeiramente, monitoramento do reator anaeróbio UASB cujo efluente foi utilizado neste estudo. Foi, então, verificado o comportamento das espécies residuais de cloro no esgoto e conduzido um estudo de parâmetros indicadores (pH, potencial de oxi-redução e condutividade) para o breakpoint. Na desinfecção, foram empregados o cloro, ozônio e radiação ultravioleta; separados, e seqüencialmente. Os ensaios realizados com ozônio promoveram remoção de DQO, sólidos e dos valores de absorbância em 254 nm, diferentemente do cloro. A ordem crescente de resistência à desinfecção foi: E. coli < coliformes totais < C. perfringens < protozoários. Houve correlação em alguns ensaios entre a bactéria esporulada C. perfringens e Giardia spp.. O efeito sinérgico, promovido pela desinfecção seqüencial, foi evidenciado em alguns experimentos para C. perfringens e Giardia spp. / The present dissertation reports on the study of the sequential disinfection with chlorine-ultraviolet radiation and ozone-ultraviolet radiation to evaluate the resistance of microorganisms that indicate fecal pollution - E. coli, total coliforms and Clostridium perfringens - and compare it to the resistance of pathogenic protozoa: Giardia spp. and Cryptosporidium spp.. Firstly, the UASB reactor, whose effluent was utilized in this study, was monitored. Then the behavior of the residual species of chlorine in the sanitary sewage was verified and a study of the indicating parameters (pH, oxi-reduction potential and conductivity) for the breakpoint was conducted. Chlorine, ozone and ultraviolet radiation were used separately and sequentially in the disinfection. Tests performed with ozone promoted removal of COD, solids and absorbance values of 254 nm, differently from chlorine. The order of resistance to disinfection was: E. coli < total coliforms < Clostridium perfringens < protozoa. There was a correlation in some tests between spore-forming bacterium Clostridium perfringens and Giardia spp.. The synergic effect caused by the sequential disinfection was observed in some experiments for C. perfringens and Giardia spp.

Nesse projeto, foi desenvolvido um sistema biológico que denominamos Dosímetro de DNA, com o objetivo de avaliar a ação genotóxica da radiação UV solar a partir da produção de lesões na molécula de DNA. Para determinar os diferentes tipos de danos, utilizamos enzimas de reparo de DNA e anticorpos específicos. Complementando estas análises, foram ainda realizados ensaios de frequência de mutação e da taxa de inativação biológica de DNA. Através da utilização dessa tecnologia foi possível confirmar a produção de lesões CPDs e 6-4PPs após a irradiação em lâmpadas de UVB e UVA. Outro fator importante foi a maior indução de danos oxidativos que a banda de UVA apresentou em relação à de UVB. Exposições ambientais demonstram claramente que a luz solar possui diferentes perfís de indução de lesões de DNA, que variam de acordo com a localidade da irradiação, e que fenômenos biológicos como inativação de DNA e mutagênese são diretamente dependentes da presença de fotoprodutos na molécula de DNA e não estão associados aos danos oxidativos. Portanto, o sistema Dosímetro de DNA é capaz de fornecer uma ampla compreensão da ação genotóxica da luz solar e informações inovadoras que poderão ser utilizadas no desenvolvimento de substâncias fotoprotetoras mais eficientes. / To better understand the impact of solar UV radiation upon DNA molecule, we developed a biological system based on the exposure of plasmid DNA to artificial and natural UV sources. The quantification of DNA lesions was performed through the use of specific DNA repair enzymes and antibodies. The biological effects of sunlight, as well as artificial UV-radiation, were evaluated through the determination of the DNA inactivation rate and mutagenesis frequency. Through the application of this technology, we could detect the induction of CPDs and 6-4PPs after exposures to UVB and UVA lamps. Interestingly, the induction of oxidative damages was significantly higher after UVA than UVB radiation. Surprisingly, the profiles of induction of DNA damages observed after sunlight exposures have presented variations especially according to the latitude. In addition, our data clearly indicate that the induction of these biological effects is directly related to the presence of photoproducts in UV-exposed DNA samples, and suggest that oxidative damages are not related to these biological processes. Therefore, a very suitable system was developed capable of providing a wide comprehension of the biological effects of solar-UV radiation upon the DNA molecule.

Plant form is a compromise between resource gathering, reproduction and the tolerance to physical demands of the abiotic and biotic environment. In an agricultural field in addition to the natural factors causing stress, humans also introduce physical and mechanical stresses, and chemical pesticides into the environment. Many of these factors are hazardous, since they represent stresses to which plants are unable to develop defense mechanisms. However, weeds have persisted in the agricultural environment despite the efforts to eradicate them. They have adapted to environmental changes such as crop rotation and have developed tolerance to stressors like pesticides in very short periods of times (less than 10 years), much less time than normally expected for evolutionary responses to occur. Perhaps a key to why weeds persist in stress-dominated habitats is the way they compromise between yield and survival. The mechanisms that explain which process is relevant in the control of seed production or seedling growth relate to the ecophysiology of the individual plants. However, trade-offs between plants physiological functions will have implications at both population and community levels. Climate change, air pollution and water scarcity are examples of environmental stresses that particularly affect agriculture. Herbicides are a major technological tool for agriculture and are responsible, at least in part, for significant increases in crop production during the last quarter of the century. The research presented in this dissertation was developed to understand the extent that individual responses to multiple environmental stresses can be extrapolated to population-level responses in an annual weed species. The specific objectives were to assess (1) the impact of three anthropogenic stresses (herbicide, UVB light and ozone) and their interactions on individual Italian ryegrass ontogeny and reproduction and (2) the potential evolutionary effect of these stresses and combinations on changes in population size and structure over time. Plants were capable of growth and reproductive compensation under the studied stresses. Stress factors with similar biochemical mechanisms had different effects at the individual plant and population levels of organization. Compensation occurred at all levels of organization: as individuals modifying growth and allocation to different organs and as populations modifying birth, and death rates and density dependent responses. The ability to compensate sometimes decreased with the number of stress factors (e.g. herbicide and UVB). In other cases, compensation ability increased with the number of stress factors (e.g. ozone and herbicide). / Graduation date: 2004

Ph.D. / Molecular and Medical Genetics / The objective of this study is to examine, in light of the expression of multiple p53 family member isoforms, the specific role of p73 in malignant conversion, cellular response to DNA damage, and direct or indirect cooperation with other p53 family members in a clonal model of epidermal carcinogenesis. We first focused on the role of p73 in malignant conversion. Whether sporadic or siRNA induced, loss of p73 in initiated p53+/+ keratinocytes lead to conversion to squamous cell carcinoma (SCC) in vivo which was reversible upon reconstitution of TAp73α but not ΔNp73α. Second, we investigated the cellular response to ionizing radiation (IR) in the presence and absence of p73, showing that loss of p73 at malignant conversion was associated with resistance to IR in vitro. The loss of radiation sensitivity and malignant conversion was characterized by reduced steady state DNA binding levels of transcriptionally active p63 isoforms to the p21 promoter, failure to induce specific p53 family transcriptional targets, and failure to arrest in G1. Reconstitution of TAp73α, but not ΔNp73α, increased steady state DNA binding capabilities of TAp63β, TAp63γ, and ΔNp63γ, and steady state levels of p53 family target mRNA, but did not restore cellular sensitivity to IR. We thus uncovered a functional cooperation between TA isoforms of p73 and p63 and showed that p73-mediated DNA damage response was uncoupled from its tumor suppressive role. We observed preferential DNA binding of the inhibitory ΔNp63α isoform both in vitro and invivo in SCC suggesting that in the absence of TAp73α a balance is tipped toward DNA binding of the inhibitory isoforms. Third, we studied the role of the p53 family inkeratinocyte response to UVB. Tumorigenic cells lacking p73 that were resistant to IR remained sensitive to UVB, accompanied by DNA binding of the TAp63γ isoform, suggesting that keratinocyte response to UVB is not dependent upon p73 and suggesting a hierarchy of p53 family member responses to DNA damage. Finally, we examined TAp73α interaction with the p53 family inhibitor Mdm2. Mdm2 was in complex with DNA-bound p53 family members in malignant cells, but reconstitution of cells withTAp73α correlated with removal of Mdm2 from the complex, making them more like primary keratinocytes or initiated cells. Like the initiated cells, cells expressing TAp73α were refractory to treatment with the Mdm2-p53 inhibitor Nutlin-3 while cells lacking p73 expression or expressing ΔNp73α were sensitive. Thus, we suggest that p73 may be acting as a molecular shield to keep p53 family member inhibitors, such as ΔNp63α andMdm2, at bay. Further understanding of p53 family interplay in tumor development and DNA damage response could lead to new therapies or optimization of current therapeutic strategies in solid tumors of epithelium, particularly where deregulation or loss of p63 and p73 expression is associated with increased tumor invasiveness, treatment resistance, and poor patient prognosis.

It is now generally acknowledged that particulate air pollution can cause respiratory symptoms and that indoor dust particles may be associated with mucous membrane irritation and odour annoyance. One reason for this may be that dust particles adsorb large quantities of gases and other volatile compounds. It is therefore important to be able to determine the chemical compounds adsorbed onto indoor dust particles. In this thesis, a new technique was developed that can analyse chemical compounds in indoor dust particles in a simple yet accurate way. In its basic configuration, it comprises a one stage thermal desorption oven, a gas flow cell with a miniaturized GC column, and a nitrogen-flushed photo diode array (PDA) detector for fast UV spectra recording. The dust sample is thermally desorbed in the oven and the released compounds are flushed onto the GC column by means of a carrier gas stream; the separated compounds are then registered by the PDA detector and identified by their characteristic gas-phase UV spectra. Using this set-up, a number of volatile organic as well as inorganic compounds were identified in indoor dust particles, e.g. nitric oxide, ammonia, hydrogen sulphide, pyridine, 2-furaldehyde, 2-methylfuran, and isoprene. Moreover, acrylate monomers were identified in dust samples from a secondary school with problems due to powdering floor polish. An instrumental set-up with higher performance was achieved by interfacing the gas flow cell to a capillary GC column. When airborne indoor dust samples were analysed by this system and by GC-MS under similar conditions of thermal desorption (150 °C) and GC separation, the two analytical systems were found to be complementary. GC-UV together with GC-MS was thus demonstrated to be considerably more powerful than GC-MS alone for the analysis of volatile organic compounds (VOC) in indoor dust. When airborne dust samples from damp (n=9) and control (n=9) residences were analysed for VOC and microorganisms, identifications made by culture and microscopy of the major moulds found, i.e. Aspergillus, Cladosporium and Penicillum, coincided with the identification of VOC known to be produced by these species. A number of additional VOC were also found, some of which may be irritating to the skin, eyes or respiratory tract if present at higher concentrations. Quantitative GC-UV analysis of indoor dust from 389 residences in Sweden showed that the VOC found at the highest concentrations were saturated aldehydes (C5-C10), furfuryl alcohol, 2,6-di-tert-butyl-4-methylphenol, 2-furaldehyde, and benzaldehyde. Alkenals were also found, notably 2-butenal (crotonaldehyde), 2-methyl-propenal (methacrolein), hexenal, heptenal, octenal, and nonenal. GC-UV was also applied (together with GC-MS) to determine VOC in dust from residences of 198 children with symptoms of asthma and/or allergy (cases) and from residences of 202 children without symptoms (controls). The mean concentration of nicotine was found to be significantly higher in dust from case residences, while the mean concentrations of hexane, nonanal, octane, 2-pentylfuran and tridecanol were significantly higher in dust from control residences. In a stepwise logistic regression model, nicotine, hexanal, furfuryl alcohol, nonane, butanol, and octenal showed increased relative risks, expressed as odds ratios comparing cases with controls. By contrast, benzaldehyde, nonanal, butenal, hexane, tridecanol, and pentylfuran showed decreased relative risks. These findings point to the possibility that not only environmental tobacco smoke but also other emissions in the indoor environment may be linked to the increased prevalence of asthma and/or allergy in children. It is concluded that GC-UV may be used as an alternative or complement to GC-MS for measuring chemicals in indoor dust, thus improving the survey and control of human exposure to particle-bound toxicants and other chemicals. / Copyright Agreement: Figure 3 included in the PDF file abowe is the exclusive property of SAGE Publications (http://www.sagepublications.com/), or its licensors and is protected by copyright and other intellectual property laws. The download of the file(s) is intended for the User's personal and noncommercial use. Any other use of the download of the Work is strictly prohibited. User may not modify, publish, transmit, participate in the transfer or sale of, reproduce, create derivative works (including coursepacks) from, distribute, perform, display, or in any way exploit any of the content of the file(s) in whole or in part. Permission may be sought for further use from Sage Publications Ltd, Rights and Permissions Department, 1 Oliver's Yard, 55 City Road, London EC1Y 1SP Fax: +44 (020) 7324-8600. By downloading the file(s), the User acknowledges and agrees to these terms.