The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix PER/ARNT/SIM (bHLH-PAS) transcription factor that binds ligands typified by 2,3,7,8-tetracholordibenzo-p-dioxin, translocates to the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT) and associates with specific cis xenobiotic response elements to activate transcription of genes involved with xenobiotic metabolism. AHR-mediated signal transduction has been evaluated thoroughly in the C57BL/6J mouse model system. This model system, however, may not be the most accurate model for human comparisons as the AHRb-1 allele carried by C57BL/6J contains a point mutation that prematurely truncates the receptor at 805 amino acids, while the AHRb-2, rat, and human AHR all contain an additional 42-45 amino acids at their carboxy-terminus that have 70% identity. This carboxy-terminal region could be functionally significant and the analysis of AHR-mediated signal transduction in the rat, human, or other mouse strains may better represent the physiology of the AHR pathway. ARNT is another member of the bHLH-PAS family of proteins that is essential in several distinct signal transduction pathways mediated by its dimerization with a variety of bHLH-PAS proteins. Several isoforms of ARNT have been identified in mammalian and aquatic species. While ARNT and ARNT2 exhibit >90% amino acid identity in the bHLH and PAS domains, gene knock-out of either ARNT or ARNT2 results in embryonic/perinatal lethality characterized by distinct phenotypes. This suggests that neither protein can compensate fully for the loss of the other. Since overlapping tissue specific expression of ARNT and ARNT2 does exist, but neither ARNT can compensate fully for loss of the other, this suggests that the two proteins have distinct functions in the presence of various dimerization partners. Thus, the focus of these studies is to examine the discrepancies between the rat, human, or AHRb-2 possessing the extended carboxy-terminal region and that of the AHRb-1 and also to examine the role of both ARNT and ARNT2 during AHR-mediated signal transduction.

This work investigated the characteristics of polychlorinated dibenzo-p-dioxins anddibenzofurans (PCDD/F) in stack-flue gases from six stationary emission sources in five types ofincinerators: industrial waste incinerator (IWI), small-scale municipal solid waste incinerator(MSWI), medical waste incinerator (MWI), cement kilns (CK), and crematories (CR). These characteristics were further investigated using factor analysis and cluster analysis. Experimental results reveal that PCDDs dominate MSWI and CR, and PCDFs dominate IWIa, IWIb, CK and MWIs. The factor analysis results showed that CR and MSWI have similar fingerprints, as do IWIb and MWI3. The cluster analysis showed that if a vertical line is cut at a rescaled distance of four, then the PCDD/F congener profiles fall into four groups. The indicators of PCDD/Fs are OCDD, 1,2,3,4,6,7,8-HpCDF, 2,3,4,7,8-PeCDF, and 1,2,3,4,6,7,8-HpCDD. The emission factors of PCDD/Fs herein were from 0.0433

In the present investigation, microarray analysis was used to identify potential TCDD gene targets. Three microarray experiments were performed to study the effect of TCDD in an established chicken B-cell line (DT40), in a chicken macrophage cell line (HD11), and in the bursa of Fabricius from embryos exposed in ovo at 6 days of incubation. From the DT40 microarray analyses, clones with sequence similarity to the apoptotic genes caspase 8 and caspase 9, and the transcription factor NFΜB, among others, were identified. Real-time quantitative polymerase chain reaction (RT-PCR) revealed that TCDD elicits aryl hydrocarbon receptor (AhR)-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3 (see chapter III). During the course of the HD11 microarray analyses, a consistent down-regulation of the matrix metalloprotease MMP-2 was observed. This finding was the basis for the hypothesis that TCDD has an effect on the gene expression of the MMP-2 and MMP-9 in macrophages. Then, gene expression analysis and functional zymography showed that TCDD impairs the MMP-2 and MMP-9 response to LPS stimulation in HD11 chicken macrophages (see chapter V). The microarray analyses of the embryonic bursa of Fabricius provided the basis to further study of the effect of TCDD in the chicken embryo. The shifted genes were classified according to their function. The down-regulated genes included: precursor of matrix metalloprotease-inhibitor, histone acyl-transferase 1, homeobox protein CUX-2, Death Associated Protein Kinase, and UDPglucosyl transferase, among others. The up-regulated genes included: phosphoinositidespecific phospholipase, acyl Co-A oxidase, and protein effector of Cdc42, among others. Together, these microarray analyses produced a database of genes of interest that will provide sufficient hypotheses to inspire multiple investigations aimed at confirming and refining the gene expression alterations as a consequence of TCDD exposure.

The dioxin receptor (DR), a transcription factor with basic-helix-loop-helix/PERARNTSIM (bHLH/PAS) homology domains, is activated by toxic xenobiotic ligands leading to severe physiological disturbances most of which are due to deregulation of receptor’s central role in normal development. Activation mechanisms of DR in the presence of exogenous or endogenous ligands are poorly understood. Elucidation of factors involved in the activation of the receptor would assist not only in development of an optimal measure for risk assessment of levels of common environmental pollutants but also in providing novel targets for therapeutic interventions. Posttranslational modifications (PTMs) play an indispensable role in all major signal transduction pathways by increasing the inventory of chemical modifications beyond those already present in the side-chains of common amino acids. Thus, by simple on/off or complex patterns generated by these PTMs, they control a myriad of different biological outcomes. Numerous studies that have suggested an important role of posttranslational modifications in DR activation has prompted a search in this direction, however, apart from phosphorylations at Ser36 and Ser68 no other PTM sites are known. Advanced mass spectrometry (MS)-based characterisation of PTMs is an established technique that can comprehensively provide an accurate cast of all PTM variants and their locations on a protein. This thesis reports the first MS-based comprehensive characterisation of all PTM sites of the purified latent DR and preliminary analysis of identified PTM sites of the activated DR in response to developmental signals (suspension-activated DR) and signals leading to toxic outcomes (ligand-activated DR). The PTM map of the latent DR revealed from this study comprises of 25 phosphorylations, 4 monomethyl-lysines, 2 dimethyl-lysines, 1 O-acetyl-serine and 2 O-sulfono-serines. Most of the phosphorylations and other PTMs were present in the conserved regions of the protein. Investigation of the activated samples of the receptor revealed loss of the above repertoire of modifications and possible presence of some rarer modifications such as O-acetyl-serines in suspension-activated instead of O-sulfonations and pyrophosphorylation at Ser716 in both suspension- as well as ligand-activated DR. A comprehensive mutagenesis study is in progress to understand the functional consequence of each of these modification sites and unravel the functional posttranslational system in DR signalling.

The dioxin receptor (DR), a transcription factor with basic-helix-loop-helix/PERARNTSIM (bHLH/PAS) homology domains, is activated by toxic xenobiotic ligands leading to severe physiological disturbances most of which are due to deregulation of receptor’s central role in normal development. Activation mechanisms of DR in the presence of exogenous or endogenous ligands are poorly understood. Elucidation of factors involved in the activation of the receptor would assist not only in development of an optimal measure for risk assessment of levels of common environmental pollutants but also in providing novel targets for therapeutic interventions. Posttranslational modifications (PTMs) play an indispensable role in all major signal transduction pathways by increasing the inventory of chemical modifications beyond those already present in the side-chains of common amino acids. Thus, by simple on/off or complex patterns generated by these PTMs, they control a myriad of different biological outcomes. Numerous studies that have suggested an important role of posttranslational modifications in DR activation has prompted a search in this direction, however, apart from phosphorylations at Ser36 and Ser68 no other PTM sites are known. Advanced mass spectrometry (MS)-based characterisation of PTMs is an established technique that can comprehensively provide an accurate cast of all PTM variants and their locations on a protein. This thesis reports the first MS-based comprehensive characterisation of all PTM sites of the purified latent DR and preliminary analysis of identified PTM sites of the activated DR in response to developmental signals (suspension-activated DR) and signals leading to toxic outcomes (ligand-activated DR). The PTM map of the latent DR revealed from this study comprises of 25 phosphorylations, 4 monomethyl-lysines, 2 dimethyl-lysines, 1 O-acetyl-serine and 2 O-sulfono-serines. Most of the phosphorylations and other PTMs were present in the conserved regions of the protein. Investigation of the activated samples of the receptor revealed loss of the above repertoire of modifications and possible presence of some rarer modifications such as O-acetyl-serines in suspension-activated instead of O-sulfonations and pyrophosphorylation at Ser716 in both suspension- as well as ligand-activated DR. A comprehensive mutagenesis study is in progress to understand the functional consequence of each of these modification sites and unravel the functional posttranslational system in DR signalling.

Made available in DSpace on 2016-06-02T20:36:38Z (GMT). No. of bitstreams: 1 4321.pdf: 5011742 bytes, checksum: 61d67530b35683f34c3ef4b4e30917aa (MD5) Previous issue date: 2012-03-30 / Financiadora de Estudos e Projetos / Sugarcane bagasse ash is a byproduct of sugar and alcohol industry with potential for field application. However, the presence of organic contaminants such as dioxins turns necessary the development of strategies for removal of those pollutants, such as bioremediation. Because of the high toxicity of dioxins, it is wise to develop effective and low risk methods for initial evaluation of microbial degradation capacity of those compounds by using less dangerous alternatives such as screening tools to select possible microorganisms as candidates for bioremediation. In such context, this work aimed to apply statistical models to evaluate microbial growth capacity under influence of components of a compost windrow already used for other sugarcane industry residues, and also development of qualitative and quantitative methods to evaluate microbial degradation of the dye RBBR and 1,2,3,4-TCDD, molecules selected as model compounds. The Scott-Knott method grouping multiple means showed good results, indicating microbial growth inhibition of all fungi in culture medium with compost windrow, and microbial growth promotion for Aspergillus strains in sugarcane ash doped medium. Furthermore, quantification of RBBR dye in liquid broth was developed, validated and applied through a dilute-and-shoot technique followed by LC-MS/MS, where it was possible to detect up to 4,8pM of dye, being observed a degradation from 40-99% of dye over 30 days. In a similar way, it was developed and validated a GC-MS method to quantify 1,2,3,4-TCDD in liquid broth after extraction, with detection limits of 10ppb, observing a removal of up to 20% of dioxin after 30 days. Another point evaluated was the identification of RBBR degradation products, where it was observed the presence of four dye metabolites during the course of the experiment, which allowed us to propose a degradation pathway of RBBR dye by Aspergillus flavus. / A cinza do bagaço da cana-de-açúcar é um subproduto da indústria sucroalcooleira que apresenta potencial para aplicação no campo. Porém, a presença de contaminantes orgânicos como dioxinas torna necessário o desenvolvimento de estratégias para a remoção destes poluentes, como a biorremediação. Por serem as dioxinas extremamente tóxicas, é sensato o desenvolvimento de métodos eficazes e de baixo risco para avaliação inicial da capacidade microbiana de degradação destes compostos usando alternativas menos perigosas como ferramentas de triagem e seleção de micro-organismos candidatos à biorremediação. Neste contexto, este trabalho teve como objetivo o uso de modelos estatísticos para avaliação do crescimento microbiano em componentes de uma composteira já utilizada para tratamentos de outros resíduos sucroalcooleiros, bem como o desenvolvimento de métodos analíticos qualitativos e quantitativos de avalição da capacidade microbiana de degradação do corante RBBR e da 1,2,3,4-TCDD, moléculas selecionadas como modelos. O agrupamento de médias por Scott-Knott apresentou bons resultados na avaliação do crescimento microbiano, apontando inibição do crescimento de todos os fungos pela composteira, e promoção de crescimento das cepas de Aspergillus no meio com cinza. Ademais, foi desenvolvido e validado um método para quantificação do corante RBBR direto do meio de cultura líquido usando a técnica dilute-and-shoot seguido por LC-MS/MS, onde foi possível detectar até 4,8pM de corante, sendo observada uma degradação entre 40-99% do corante em 30 dias. Da maneira similar, foi desenvolvido e validado um método por GC-MS para quantificação da 1,2,3,4-TCDD em meio líquido após extração, com limites de detecção de 10ppb, onde foi observada uma remoção de até 20% da dioxina em 30 dias de cultivo. Outro ponto avaliado foi a identificação dos produtos de degradação do RBBR durante o experimento, sendo observada a presença de quatro metabólitos do corante nas amostras, o que nos permitiu propor a rota de degradação do corante por Aspergillus flavus.

As Tim O'Brien advises in The Things They Carried, "You can tell a true war story by the way it never seems to end" (76). If the war story never seems to end, then how does it manifest in future generations? In my case, as the first-born son of a Vietnam veteran, the war story has played out physically, within my body, in the form of an Agent Orange-related disability. How has my response to disability affected both the fine details and the overall texture of my life? My father also suffered from Post-Traumatic Stress Disorder (PTSD) for several years after his return, a timeframe that happens to coincide with the first and most impressionable years of my life. How has this affected my relationships to my disability and to the world at large? Lastly, what can a chronicle of Agent Orange in Vietnam tell me about my own story?

For centuries chemists have sought to improve humankind’s quality of life and address many of society’s most pressing needs through the development of chemical processes and synthesis of new compounds, often with phenomenal results. Unfortunately, there also are many examples where these chemicals have had unintended, detrimental consequences that are not apparent until years or decades after their initial use. There are numerous halogenated molecules in this category that are globally dispersed, resistant to natural degradation processes, bioaccumulative, and toxic to living organisms. Chemicals such as these are classified as persistent organic pollutants (POPs), and due to their negative environmental and health effects, they require safe, effective, and inexpensive means of remediation. This research focuses on the development and optimization of a reaction matrix capable of reductively dehalogenating several POPs. Initial experiments determined that powdered magnesium and 1% V/V acetic acid in absolute ethanol was the most effective system for degrading polychlorinated biphenyl (PCB), an extraordinarily recalcitrant environmental contaminant. Further studies showed that this matrix also was capable of degrading polychlorinated dibenzo-p-dioxins (PCDDs), polybrominated diphenyl ethers (PBDEs), and four organochlorine pesticides (OCPs); dieldrin, heptachlor, heptachlor epoxide, and chlordane. During this phase of testing, field samples contaminated with chlordane were washed with ethanol and this ethanol/chlordane solution was degraded using the same reaction matrix, thereby demonstrating this technology’s potential for “real-world” remediation projects. Finally, a set of experiments designed to provide some insight into the mechanism of dechlorination seems to indicate that two distinct processes are necessary for degradation to occur. First, the passivated iv outer layer of the magnesium must be removed in order to expose the zero-valent magnesium core. Next, an electron is transferred from the magnesium to the target molecule, causing the cleavage of the halide bond and the subsequent abstraction of either a hydrogen or proton from a solvent molecule. It is anticipated that an understanding of these fundamental chemical processes will allow this system to be tailored to a wide range of complex environmental media

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