Humic substances (HS), the complex mixture of organic molecules produced by microbial and abiotic degradation of plant and animals residues, are the major components of organic matter in soil and natural waters. As such, they are vital to the integrity of soil, play important roles in nutrient biogeochemical cycling and determine the mobility and fate of both nutrients and contaminants. How HS enact their varied roles, however, is largely unknown due to the lack of comprehension of their molecular make-up. What is known is that they contain aromatic (e.g. phenols, condensed aromatics) and aliphatic (e.g. carbohydrates, lipids) molecules with prevailing carboxyl and hydroxyl functionalities. As for other complex chromatographically inseparable mixtures, high-resolution analytical techniques such as Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) as well as Nuclear Magnetic Resonance (NMR) spectroscopy have been applied to study HS composition at the molecular level. While FT-ICR MS can provide molecular formulae, it is only NMR that can unveil structures. Until now, however, very few unambiguous structures of individual molecules have emerged from standard NMR experiments. This is because purposely designed multidimensional NMR experiments are required to achieve ‘spectroscopic separation’ where chromatography fails. In this work, ‘spectroscopic separation’ was accomplished with the aid of chemical modification in the form of 13C methylation of COOH and OH groups. 13C containing tags allow the observation of signals only from molecules carrying these tags. In combination with purposelydesigned isotope-filtered NMR experiments, these tags act as spies reporting on their surrounding structure. This is achieved by utilising scalar and dipolar couplings to transfer the polarisation between protons and carbons of the 13CH3O groups and nuclei of the parent molecules. The necessary spectral resolution is attained using 3D/4D NMR experiments. This approach provides access to an array of correlated chemical shifts of HS molecules and represents a paradigm shift in the use of tags in investigations of complex mixtures: instead of focusing only on the tags, they are used to obtain structural information from the molecules they are attached to. The compounds at the centre of this investigation are the phenols of HS. These molecules are thought to be important in many of the key processes in organic matter rich soils, particularly peat. For example, their accumulation is thought to impair the activity of extracellular enzymes essential for the degradation of peat HS. Understanding the nature and source of phenols would provide a more fundamental framework for rationalising their role in peat, as well as other carbon rich soils. The developed methodology was initially tested on model mixtures containing 3 or 9 phenolic compounds before applying it to a operational fraction of HS, fulvic acid, extracted from a Scottish raised peat bog. For this fulvic acid, over 30 major phenolic molecules/moieties were identified, many of which can be directly traced to the flora prevalent in the vicinity of the sampling site. For the first time in 150 years of HS research, a novel analytical methodology has yielded unambiguous structural information, providing the first steps towards understanding the various roles of HS in natural systems.

Made available in DSpace on 2017-07-24T19:38:11Z (GMT). No. of bitstreams: 1 Bruna Meotti Almeida.pdf: 1294675 bytes, checksum: c27c30b3b84924bec1bb5940ff1bf17e (MD5) Previous issue date: 2013-10-05 / This dissertation aims to study the composting process of sewage sludge from a food industry with two different substrates, pruning and sawing and characterize the compounds obtained using conventional methods and spectroscopic techniques. It was created two piles, one with sawdust and sludge (SL) and another with sludge and pruning (LP) in a ratio of 1:3 by volume. It was followed for 210 days the composting process with periodic measurements of temperature, pH, moisture, elemental analysis, seed germination, metal content and spectroscopic analysis of UV / Vis and IR. THE CONCLUSION IS that the compounds obtained in LS and LP are stabilized and matured, but with different times of composting. / Este trabalho teve por objetivo estudar o processo de compostagem de lodo de esgoto de uma indústria alimentícia com dois substratos diferentes, poda de jardinagem e serragem. Para o monitoramento e caracterização dos compostos obtidos foram utilizados métodos convencionais e espectroscópicos. Montou-se duas leiras, uma com lodo e serragem (LS) e outra com lodo e poda (LP), na proporção de 1:3 em volume. Acompanhou-se durante 210 dias o processo de compostagem com medições periódicas da temperatura, pH, umidade, análise elementar, germinação de sementes, teor de metais e análises espectroscópicas de UV/Vis e IV. Como resultado das análises realizadas pode-se concluir que os compostos obtidos na LS e LP estão estabilizados e maturados, porém com tempos diferentes de compostagem.

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A produção de resíduos orgânicos tem aumentado em anos recentes e uma alternativa para o seu aproveitamento é o processo de compostagem, no qual se desenvolvem reações bioquímicas para estabilização do material que estão diretamente associadas com a humificação da matéria orgânica. Considerando a complexidade das transformações no processo de humificação e a importância de se conhecer com maior precisão estas reações e as características dos materiais gerados, foi proposto neste trabalho um estudo detalhado do processo de compostagem. A combinação de métodos convencionais e técnicas espectroscópicas permitiu o monitoramento contínuo da compostagem, de diferentes resíduos orgânicos. Para execução dos experimentos foram montadas 6 leiras (L) de 3,6 m3 com os seguintes resíduos: poda de árvores, esterco bovino fresco, bagaço de laranja, torta de filtro e solução de ácido pirolenhoso (como possível catalisador do processo). O monitorado foi realizado por 7 meses com medidas diárias de temperatura, controle semanal do teor de umidade e coletas mensais das amostras para extração de ácidos húmicos e análises químicas. Foram observadas as fases típicas da temperatura em todas as leiras, exceto na L1 (constituída apenas de poda de árvores). Na fase termofílica a temperatura atingiu os 60 o C e esta fase permaneceu por, aproximadamente, 90 dias. Este também foi o tempo necessário para a estabilização da relação da capacidade de troca catiônica pelo teor de carbono do composto (CTC/C), que alcançou valores acima de 2,2 mmolc g-1 . As análises de macro e micronutrientes evidenciaram o potencial fertilizante dos compostos produzidos, cujos valores, para alguns elementos, estão acima dos limites exigidos pela Instrução Normativa no 23 do Ministério de Agricultura, Pecuária e Abastecimento. Já nas análises espectroscópicas foram encontrados índices de estabilização a partir dos espectros de fluorescência de luz UV - Vis. Estes apresentaram boa correlação com a razão C/N e os radicais livres orgânicos do tipo semiquinona, detectados por Ressonância Paramagnética Eletrônica. As correlações dos índices com a razão C/N mostraram que este último parâmetro tem limitação para monitorar a compostagem, pois a partir de 60 dias a relação C/N estabiliza, ainda na fase termofílica do processo, enquanto que os índices espectroscópicos continuam variando, indicando que o processo não se estabilizou completamente. Dentre as informações obtidas por fluorescência, foi possível identificar reações de transformação de moléculas orgânicas mais simples para estruturas mais complexas, detectadas através da diminuição de intensidade das bandas de emissão em comprimentos de onda menores (em torno de 300 nm) com simultâneo aumento da intensidade em regiões de maiores comprimentos de onda (próximos de 400 nm). Este comportamento também foi observado nos espectros de absorção UV - Vis. A partir dos dados de Ressonância Magnética Nuclear (RMN de 13 C) foi possível evidenciar a decomposição de estruturas como carboidratos, polissacarídeos e grupos alquil, assim como um aumento das estruturas aromáticas, durante o processo de humificação. Assim através da combinação das análises por métodos convencionais e espectroscópicos foram propostos indicadores analíticos mais sensíveis para o monitoramento e a caracterização da compostagem de resíduos orgânicos. / The production of organic residues has increased in recent years and an alternative to their recycling is the composting process, where various biochemical reactions are developed for the material stabilization. These reactions are directly associated with organic matter humification. Considering the complexity of the transformations occurred during the humification process and the importance of knowing these reactions and the characteristics of the produced materials more precisely, this work has proposed a detailed study of the composting process. The combination of conventional methods and spectroscopic techniques allowed the continuous composting assessment and monitoring from different organic residues. Six piles (P) of 3.6 m3 were assembled using the following residues: garden trimming, fresh cattle manure, orange pomace, filter cake and pyroligneous acid solution (used as possible catalyst of the process). The process was monitored for 7 months with daily measurements of temperature, weekly moisture control and monthly collection of samples for humic acid (HA) extraction and chemical analyses. Typical phases of temperature in all piles were observed, except for P1 (composed only garden trimming). In the thermophilic phase the temperature reached 60 o C and this phase remained for about 90 days. This was also the time necessary for stabilization of cation exchange capacity and total carbon ratio (CEC/C), which reached values above of 2.2 mmolc g-1 . The macro- and micronutrient analyses showed the fertilizer potential of the produced composts. Concentration values for same elements are above those required by Normative Instruction number 23 of the Brazilian Ministry of Agriculture Livestock and Food Supply. As a result of the spectroscopic analyses, stabilization indices were obtained from fluorescence spectra of UV-Visible light. These indices showed good correlation with C/N ratio and with the semiquinone-type free radical, detected by Electron Paramagnetic Resonance (EPR). The correlation of the indices with C/N ratio showed that the latter parameter has limitation to monitor the composting, because after 60 days the C/N ratio stabilizes and this occurs during the thermophilic phase, while the spectroscopic indices continue to change, which is an indicative that the process was not completely stabilized. Among several observations by fluorescence of UV-Visible light it was possible to identify transformation reactions of simpler organic molecules to more complex structures, which were detected by decrease in emission band intensity in shorter wavelengths (near 300 nm) with simultaneous increase in band intensity in longer wavelengths (near 400 nm). This behavior was also observed in the UV - Visible absorption spectra. It was possible to show the decomposition of carbohydrate type structures, polysaccharides and alkyl groups and the increase in aromatic structures, by 13 C Nuclear Magnetic Resonance (13C NMR), during humification process of the organic residues. Therefore, more sensible analytic indicators were proposed for monitoring and characterization of the composting of organic residues through the combination of conventional and spectroscopic methods.

Um dos maiores interesses atuais no âmbito mundial, está focalizado no aumento da radiação ultravioleta (UV) e sua influência no equilíbrio dos sistemas aquáticos. O Rio Negro-AM, um dos principais afluentes da Bacia Amazônica, constitui-se em um lugar ideal para o estudo do impacto da radiação UV na biogeoquímica da matéria orgânica dissolvida (MOD), pelas elevadas concentrações de carbono orgânico (CO), altas taxas de radiação solar registradas e variabilidade sazonal do sistema. O presente trabalho teve por finalidade avaliar a dinâmica e funcionalidade das substâncias húmicas aquáticas (SHA) do Rio Negro frente a fatores ambientais como índices fluvio e pluviométricos e a radiação UV-visível. Com este objetivo as SHA foram classificadas em relação às estações do ano (verão, outono, inverno e primavera) e caracterizadas, através da composição elementar, capacidade complexante (CC) com Cu+2 e das espectroscopias de ressonância paramagnética eletrônica (RPE), ressonância magnética nuclear de carbono 13 (RMN 13C), infravermelho com transformada de Fourier (FTIR), absorbância e fluorescência de luz UV-visível. O resultado revelou o efeito das mudanças sazonais do rio na estrutura da MOD, sendo que as SHA do inverno (época de cheia) evidenciaram o maior carácter alifático por RMN 13C, diretamente relacionado com altos níveis fluviométricos que favoreceriam o ingresso da MOD fresca de origem terrestre, hidrofílica e em vias de estabilização. Por outro lado, as SHA de verão e outono (época de seca), mostraram o maior carácter aromático com altos teores de radicais livres do tipo semiquinona, os maiores índices de humificação por fluorescência e as maiores absorbâncias a comprimentos de onda mais longos. Em uma segunda parte, avaliaram-se os efeitos da radiação UV-visível nas propriedades ópticas e estruturais das SHA de verão e inverno, para isso empregou-se uma lâmpada de Hg-Xe (290 - 475 nm). Em geral observou-se uma diminuição progressiva da absorbância e da cor, uma mineralização com redução aproximada de 75% de CO e a formação de estruturas de menor complexidade, evidenciada através do deslocamento para menores comprimentos de onda dos máximos de fluorescência; uma acidificação do meio (6 - 4,5) e uma perda total da CC de Cu+2, nos primeiros 300 minutos da irradiação. Desse modo, a variabilidade estrutural sazonal também foi observada no comportamento na fotodegradação das SHA de verão e inverno. Assim, as de verão (com maior aromaticidade) apresentaram uma cinética lenta (k=10,33 x 10-4), embora mais efetiva com a perda total de absorbância do 87%, atribuída a uma maior ressonância dos grupos cromóforos presentes em maior quantidade. Em contrapartida, as SHA de inverno (com a maior alifaticidade) degradaram se mais rapidamente (k=12,33 x 10-4) atingindo um estado limite estacionário, até uma perda de absorbância de 79%, devida à acelerada decomposição dos grupos alifáticos e/ou a produção de estruturas absorventes que são recalcitrantes e inertes aos comprimentos de onda da lâmpada. Contudo, estes resultados contribuem para um melhor entendimento das tendências dos fluxos de CO nos rios e a subseqüente quantificação de C das fontes terrestres e aquáticas e sua dependência com diversos fatores ambientais. / Nowadays, one of the major world concerning is focused in the increasing of the damaging ultraviolet (UV) radiation and its impact on the aquatic ecosystems. In addition, the Rio Negro, one of the principal tributaries of the Amazon Basin, is an appropriate site for studying the influence of the UV radiation in the dissolved organic matter (DOM) biogeochemistry, since its high level of organic carbon (OC) production, the high rates of solar exposition and the seasonal variation of the system. The present research had the intent to evaluate the dynamics and functionality of the Rio Negro aquatic humic substances (AHS) influenced by environmental factors such as pluviometric and fluviometric levels and the UV-vis irradiation. In order to reach these objective, the AHS from one year were classified in seasonal groups (summer, autumn, winter and spring) and characterized through the elemental analysis, copper (Cu+2) complexation capacity (CC) and through the spectroscopic techniques as electronic paramagnetic resonance (EPR), nuclear magnetic resonance of 13C (NMR 13C), Fourier transformed infrared spectroscopy and UV-visible absorbance and fluorescence. The results revealed the river seasonal changes effects on the DOM structure, being that, the winter AHS (flooding season), evidenced the highest aliphatic character from the NMR 13C analysis, that can be related with the high fluviometric levels that would increasing the presence of fresh hydrophilic and not totally stabilized (MOD) from terrestrial sources. In contrast, the summer and autumn AHS (dry season) showed the greatest aromatic presence, high concentration of semiquinone free radicals, the greatest humification degrees from the fluorescence and the elevated long wavelength absorbance. Complementarily, the AHS optical and structural properties were assessed as a function of its exposure to UV-visible irradiation using an Hg-Xe lamp (290-475 nm). In general there were observed a progressive decreasing of absorbance and color, a mineralization with a 75% carbon loss and the formation of the less complex structures inferred from the change of the maximum fluorescence to shorter wavelengths. In addition there were registered the system acidification (6- 4,5) and a total loss of the Cu+2 CC during the first 300 minutes of the radiation exposure. As expected, the structure seasonal dependence influenced in the photodegradation behavior of the winter and summer AHS. The summer AHS sample (with the highest aromaticity) showed slower kinetics (k=10,33 x 10-4), but higher susceptibility to the UV irradiation with a 87% loss of absorbance, explained by the highest concentration of aromatic structures more resonant with the irradiation applied. On the counterpart, the winter AHS (with the highest aliphaticity) presented faster kinetics (k=12,33 x 10-4), reaching rapidly a stationary state with a total absorbance loss of 79%, as a result of an accelerated decomposition of its aliphatic fractions and/or the production of absorbing light recalcitrant compounds, unreactive to the wavelengths applied in this study. Finally, these results contribute for a better understanding of the OC river fluxes and the subsequent quantification of the terrestrial and aquatic C sources and its dependence with diverse environmental factors.

Investigate the interaction of nanomaterials with biological systems, known as nano-bio interaction is of great interest for the assessment of the concern arising from nanomaterials progressive use. Such interaction determines nanomaterials potential effect on human and environment becomes more and more important to understand how they interact with living organisms and the environment. The novel physicochemical characteristics of nanomaterials, such as their small size, large surface area to volume ratio and surface energy, may initiate new toxicological effects due to nanomaterials ability to enter into the biological systems through adsorption and dissolution and modify the structure of various macromolecules An example of these interactions is the adsorption of proteins on nanoparticles surface forming what is known as the 'protein corona'. Therefore, being able to understand how these molecules and other biologically important species are adsorbed and interact, should help us to reduce any adverse impacts of nanoparticles on human health and the environment. Due to the importance of surface composition and surface functionality in nanotoxicology, analytical tools that can probe the change in the structure and composition of the nanoparticles in aqueous media are crucial but remain limited. Therefore in this work, in situ characterization of the liquid–solid interface to probe surface adsorption of environmentally and biologically relevant media on nanoparticle surfaces has been conducted. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy provides the molecular information that allows for the determination of the adsorption mode such as conformational and structural changes of the coordinating ligand. Surface adsorption of titanium dioxide (TiO2) nanoparticles have been investigated in different biological media typically used for toxicity studies and show that the surface composition of TiO2 nanoparticles depends to a large extent on the composition of the medium due to surface adsorption. Moreover, hydrodynamic diameter and surface charge of TiO2 NPs were evaluated using dynamic light scattering DLS. The results indicated that TiO2 NPs undergo different trends in aggregation upon the adsorption of biological media on its surface and zeta potential measurements showed surface charge alterations which are consistent with the aggregation study. In order to understand the dynamic transformations of nanomaterials in biological environments, the effect of dissolution has been predicted. Copper oxide CuO and zinc oxide ZnO nanoparticles were used to study dissolution due to their instability in biological media. Once these particles exposed to solutions they release their ions and tend to aggregate. Therefore, the dissolution of these materials was conducted at size ca. 24 nm and nanoparticles coated with proteins and humic acid employing simulated lung fluids as models to develop a better understanding of how these properties effect the solubility and stability in biological systems. From this study, it was found that both copper oxide and zinc oxide NPs showed different trends in dissolution. Cu and Zn ions once coated with proteins and HA highly dissolved in ALF at low pH 4.5 compared with other fluids (Gamble’s solution and water) at extracellular pH which shows only slightly enhanced in the basal condition. The acidity of ALF may explain the higher solubility of metals that are phagocytized versus those that remain extracellular. Some general conclusions can be drawn from these investigations. It seems that analytical tools to characterize the interfacial region between nanopaerticles and these complex systems provide a reasonably good qualitative and quantitative description of the interactions.

Doctor of Philosophy (PhD) / This thesis presents the development of new techniques for measuring the mean redshifted 21 cm line of neutral hydrogen during reionization. This is called the 21 cm cosmological reionization monopole. Successful observations could identify the nature of the first stars and test theories of galaxy and large-scale structure formation. The goal was to specify, construct and calibrate a portable radio telescope to measure the 21 cm monopole in the frequency range 114 MHz to 228 MHz, which corresponds to the redshift range 11.5 > z > 5.2. The chosen approach combined a frequency independent antenna with a digital correlation spectrometer to form a correlation radiometer. The system was calibrated against injected noise and against a modelled galactic foreground. Components were specified for calibration of the sky spectrum to 1 mK/MHz relative accuracy. Comparing simulated and measured spectra showed that bandpass calibration is limited to 11 K, that is 1% of the foreground emission, due to larger than expected frequency dependence of the antenna pattern. Overall calibration, including additive contributions from the system and the radio foreground, is limited to 60 K. This is 160 times larger than the maximum possible monopole amplitude at redshift eight. Future work will refine and extend the system known as the Cosmological Reionization Experiment Mark I (CoRE Mk I).

This thesis discusses developing new tools to probe DNA damage resulting from photoinduced chemical processes and probing the initial excited-state structural dynamics of nucleic acids and sunscreen agents. The results of this thesis show that UV resonance Raman spectroscopy (UVRRS) is an information-rich probe of chemical compositions in in situ analysis of sunscreen formulations. The results indicate that 244-nm excited UV resonance Raman spectra can provide a limit of detection of 0.23% w/w of sunscreen active ingredients (AIs), far below typical active ingredient (AI) concentrations in sunscreen formulations. UVRRS was used to probe the photostability of sunscreens, by determining the initial excited-state structural dynamics of Benzophenone-3 (BZ3), a common sunscreen active ingredient. Only minor excited-state structural distortions were obtained, based on the low displacement values, suggesting an inherently stable molecule. The largest structural change occurs along the carbonyl stretch, suggesting a strong interaction with the methanol solvent and a dissipative decay path for the molecule. Similarly, to gain an insight into how the structure of nucleic acids determine their photochemistry, UVRRS was used to probe the initial excited-state structural dynamics of 9-methyladenine (9-MeA). As for BZ3, the initial excited-state structural dynamics obtained for 9-MeA, are low, and much lower than for pyrimidine bases, providing a strong evidence for the photochemical stability of this purine base analog, compared to pyrimidines. Since DNA does not exist in isolation in cells, the role of a UV-absorbing amino acid, tryptophan, in UV-induced DNA photodamage was explored using molecular beacons (MBs) to detect the damage. The results indicate that tryptophan protective effects on DNA far outweighs their photodamage potential. However, MBs are expensive and they have some technical limitations, despite their exquisite sensitivity. Therefore, a new DNA damage detection method was developed based on smart probes (SPs). These probes are shown to be sensitive and sequence specific for detecting DNA photodamage.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.

In the absence of telomerase activity, a subset of cancerous and immortalized cells maintain telomere length by means of a poorly understood mechanism, termed alternative lengthening of telomeres (ALT). Many details of telomere maintenance in ALT positive cells remain unclear, but significant evidence implicates a homologous recombination mechanism. ALT specific nuclear structures, known as ALT-associated promyelocytic leukemia nuclear bodies (APBs), are thought to serve as the site of telomere extension. Using electron spectroscopic imaging we have demonstrated that APBs contain substantial amounts of nucleic acid sequestered within the bodies. In contrast, promyelocytic leukemia nuclear bodies in non-ALT cell lines contain no significant nucleic acid. We show that the nucleic acid found in APBs is not RNA and provide evidence that it is in fact telomeric repeat DNA. This evidence supports a role for APBs to sequester extrachromosomal telomeric DNA in order to suppress the activation of DNA repair.