Alpine zooplankton are exposed to a variety of stressors in their natural environment including ultraviolet radiation. Physiological coping mechanisms such as the accumulation of photoprotective compounds provide these zooplankton protection from many of these stressors. Elevated levels of carotenoid compounds such as astaxanthin have been shown to help zooplankton survive longer when exposed to ultraviolet radiation presumably due to the strong antioxidant properties of carotenoid compounds. This antioxidant capacity is important because it may ameliorate natural and anthropogenic stressor-induced oxidative stress. While previous researchers have shown carotenoid compounds impart increased resistance to ultraviolet radiation in populations of zooplankton, little work has focused on the toxicological implications of PAH induced phototoxicity on zooplankton containing high levels of carotenoid compounds. This thesis discusses research studying the role that carotenoid compounds play in reducing PAH induced phototoxicity. By sampling different lakes at elevations ranging from 9,500' to 12,700' in the front range of the Colorado Rocky Mountains, copepod populations containing different levels of carotenoid compounds were obtained. These populations were then challenged with fluoranthene and ultraviolet radiation. Results discussed include differences in survival and levels of lipid peroxidation among populations exhibiting different levels of carotenoid compounds, and the toxicological and ecological implications of these results.

"A thesis submitted in partial fulfillment of the requirements for the award of the degree of Doctor of Philosophy". / Thesis (PhD) -- Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry and Biomolecular Sciences, 2007. / Bibliography: p. 243-277. / Introduction -- A convenient synthesis of 30HKG -- Facile synthesis of the UV filter compounds 30HKyn and AHBG -- Synthesis, identification and quantification of novel human lens metabolites -- Modification of bovine lens protein with UV filters and related metabolites -- Effect of UV light on UV filter-treated lens proteins -- Conclusions and future directions. / The kynurenine-based UV filters are unstable under physiological conditions and undergo side chain deamination, resulting in α,β-unsaturated carbonyl compounds. These compounds can react with free or protein bound nucleophiles in the lens via Michael addition. The key sites of the UV filters kynurenine (Kyn) and 3-hydroxykynurenine (3OHKyn) modification in human lenses include cysteine (Cys), and to a lesser extent, lysine (Lys) and histidine (His) residues. Recent in vivo studies have revealed that 3-hydroxykynurenine-O-β-D-glucoside (3OHKG) binds to Cys residues of lens crystallins in older normal human lenses. As a result of this binding, human lens proteins become progressively modified by UV filters in an age-dependent manner, contributing to changes that occur with the development of age-related nuclear (ARN) cataract. Upon exposure to UV light, free UV filters are poor photosensitisers, however the role of protein-bound species is less clear. It has been recently demonstrated that Kyn, when bound to lens proteins, becomes more susceptible to photo-oxidation by UV light. Therefore, the investigation of 3OHKG binding to lens proteins, and the effect of UV light on proteins modified with 3OHKG and 3OHKyn, were major aims of this study. As a result of the role of these compounds as UV filters and their possible involvement in ARN cataract formation, it is crucial to understand the nature, concentration and modes of action of the UV filters and their metabolites present in the human lenses. Therefore, an additional aim was to investigate human lenses for the presence of novel kynurenine-based human lens metabolites and examine their reactivity.--As 3OHKG is not commercially available, to conduct protein binding studies, an initial aim of this study was to synthesise 3OHKG (Chapter 2). Through the expansion and optimisation of a literature procedure, 3OHKG was successfully synthesised using commercially available and inexpensive reagents, and applying green chemistry principles, where toxic and corrosive reagents were replaced with benign reagents and solvent-free and microwave chemistry was used. A detailed investigation of different reaction conditions was also conducted, resulting in either the improvement of reaction yields or reaction time compared to the literature method. Applying the same synthetic strategy, and using key precursors from the synthesis of 3OHKG, the UV filters 3OHKyn and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid-O-β-D-glucoside (AHBG), were also successfully synthesised (Chapter 3). / Chapter 4 describes the investigation of both normal and cataractous human lenses in an attempt to identify novel human lens metabolites derived from deaminated Kyn and 3OHKyn (Chapter 4, Part A). Initially, 4-(2-aminophenyl)-4-oxobutanoic acid (AHA), glutathionyl-kynurenine (GSH-Kyn), kynurenine yellow (Kyn yellow), 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid (AHB), glutathionyl-3-hydroxykynurenine (GSH-3OHKyn) and 3-hydroxykynurenine yellow (3OHKyn yellow) were synthesised and human lenses were examined for their presence. AHA and AHB were synthesised from similar precursors to those used in the synthesis of 3OHKG, while the GSH adducts and yellow compounds were synthesised from Kyn and 3OHKyn via base induced deamination. Following isolation and structural elucidation, AHA, AHB and GSH-Kyn were confirmed as novel human lens metabolites. They were quantified in low pmol/mg lens (dry mass) levels in normal and cataractous lenses of all ages, while GSH-3OHKyn, Kyn yellow and 3OHKyn yellow were not detected. In contrast to AHA, the lens metabolites AHB, GSH-Kyn and GSH-3OHKyn were found to be unstable at physiological pH. The spectral properties of these compounds suggest that they may act as UV filters. --Chapter 4 (Part B) also describes the identification and characterisation of a novel human lens UV filter, cysteinyl-3-hydroxykynurenine -O-β-D-glucoside (Cys-3OHKG). An authentic standard was synthesised via Michael addition of cysteine to deaminated 3OHKG. Cys-3OHKG was detected in low pmol/mg lens (dry mass) levels in normal lenses only after the 5th decade of life and was absent in cataractous lenses. Cys-3OHKG showed rapid decomposition at physiological pH. / Chapter 5 describes the identification and quantification of amino acids involved in covalent binding of 3OHKG to lens proteins. Model studies with bovine lens proteins and 3OHKG at pH 7.2 and 9.5 were undertaken. The amino acid adducts were identified via total synthesis and spectral analysis, and subsequently quantified upon acid hydrolysis of the modified lens proteins. Under both pH conditions, 3OHKG was found to react with lens proteins predominantly via Cys residues with low levels of binding also detected at Lys residues. Comparative studies with Kyn (pH 9.5) and 3OHKyn (pH 7.2 and 9.5) resulted in modified lens proteins at Cys residues, with only minor modification at Lys residues at pH 9.5. The extent of modification was found to be significantly higher at pH 9.5 in all cases. His adducts were not identified. 3OHKG-, Kyn- and 3OHKyn-modified lens proteins were found to be coloured and fluorescent, resembling those of aged and ARN cataractous lenses. In contrast, AHB and AHA, which can not form α,β-unsaturated carbonyl compounds, resulted in non-covalent modification of lens proteins. AHB may contribute to lens colouration and fluorescence as further reactions of this material yielded species that have similar characteristics to those identified from 3OHKyn modification. These species are postulated to arise via auto-oxidation of the o-aminophenol moiety present in both 3OHKyn and AHB.--In Chapter 6, the potential roles of 3OHKG and 3OHKyn, and the related species AHA and AHB, in generating reactive oxygen species and protein damage following illumination with UV light was examined. The UV filter compounds were examined in both their free and protein-bound forms. Kyn-modified proteins were used as a positive control. Exposure of these compounds to UV light (λ 305-385 nm) has been shown to generate H2O2 and protein-bound peroxides in a time-dependent manner, with shorter wavelengths generating more peroxides. The yields of peroxides were observed to be highly dependent on the nature of the UV filter compound and whether these species were free or protein bound, with much higher levels being detected with the bound species. Thus, protein-bound 3OHKyn yielded higher levels of peroxide than 3OHKG, with these levels, in turn, higher than for the free UV filter compounds. AHB-treated lens proteins resulted in formation of low but statistically significant levels of peroxides, while AHA-treated lens proteins resulted in insignificant peroxide formation. The consequences of these photochemical reactions have been examined by quantifying protein-bound tyrosine oxidation products (3,4-dihydroxyphenylalanine [DOPA], di-tyrosine [di-Tyr]) and protein cross-linking. 3OHKG-modified proteins gave elevated levels of di-Tyr, but not DOPA, whereas 3OHKyn-modified protein gave the inverse. DOPA formation was observed to be independent of illumination and most likely arose via o-aminophenol auto-oxidation. AHB- and AHA-treated lens proteins resulted in statistically insignificant di-Tyr formation, while a light independent increase in DOPA was observed for both samples. Both reducible (disulfide) and non-reducible cross-links were detected in modified proteins following illumination. These linkages were present at lower levels in modified, but non-illuminated proteins, and absent from unmodified protein samples. / This work has provided an optimised synthetic procedure for 3OHKG and other lens metabolites (Chapters 2 and 3). Four novel lens metabolites have been identified and quantified in normal and cataractous human lenses (Chapter 4). Subsequent experiments, described in Chapter 5, identified the major covalent binding sites of 3OHKG to lens proteins, while AHA and AHB showed non-covalent binding. Further work described in Chapter 6 showed that protein-bound 3OHKG, Kyn and 3OHKyn were better photosensitisers of oxidative damage than in their unbound state. Together, this research has provided strong evidence that post-translational modifications of lens proteins by kynurenine-based metabolites and their interaction with UV light appear, at least in part, responsible for the age-dependent colouration of human lenses and an elevated level of oxidative stress in older lenses. These processes may contribute to the progression of ARN cataract. / Mode of access: World Wide Web. / xxxix, 308 p. ill. (some col.)

Comissão Nacional de Energia Nuclear (CNEN) / O ser humano está exposto diariamente a várias fontes de radiação natural sendo que a principal delas é o gás nobre 222Rn, pertencente à cadeia radioativa do 238U. A grande importância do estudo do 222Rn se deve ao fato do mesmo ser responsável, juntamente com seus produtos de decaimento, por cerca da metade da dose efetiva proveniente das fontes de radiações ionizantes naturais que é recebida pela população mundial. Além disso, o gás 222Rn ao ser inalado produz isótopos que passam por oito decaimentos radioativos (metade por emissão de partículas alfa e metade por emissão de partículas beta) para chegar ao isótopo estável de chumbo. Essa radiação interage com as células dos tecidos biológicos apresentando alta probabilidade de induzir o desenvolvimento de tumores pulmonares. O objetivo desta dissertação é avaliar os valores de concentração de 222Rn em ambientes de trabalho da região de Curitiba – Paraná, assim como, mensurar os níveis de 222Rn provenientes do solo e de materiais de construção que permeiam os postos de trabalho avaliados. Para o estudo dos níveis de 222Rn indoor foram utilizados detectores de estado sólido CR-39, submetidos à revelação química e leitura manual em microscópio óptico. Os cálculos das concentrações de 222Rn nos postos de trabalho foram obtidos a partir de metodologia de calibração desenvolvida pelo Centro de Desenvolvimento da Tecnologia Nuclear (CDTN) em conjunto com o Laboratório de Radiações Ionizantes da Universidade Tecnológica Federal do Paraná (UTFPR). O detector ativo AlphaGUARD (SaphymoGmbH) foi utilizado para análise das concentrações médias de 222Rn do solo e dos materiais de construção que também foram submetidos ao método de espectrometria gama para avaliação qualitativa e quantitativa dos radionuclídeos presentes nas amostras de areia, argamassa, brita azul, brita vermelha, concreto e tijolo vermelho. A metodologia utilizada para as medidas de solo permitiu encontrar as concentrações médias de 222Rn (radônio) e 220Rn (torônio) presentes no solo. Os resultados das concentrações médias de 222Rn obtidos nas medidas indoor dos postos de trabalho encontram-se entre 36 ± 49 Bq/m³ e 164 ± 51 Bq/m³. Valores esses considerados dentro do limite de referência de 200 Bq/m³ estabelecido por agências internacionais como United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) e o International Commission on Radiological Protection (ICRP), mas um pouco acima do limite de 148 Bq/m³ preconizado pela United States Environmental Protection Agency (EPA). Em relação aos materiais de construção, os valores de concentração de 222Rn encontrados estão em uma faixa de 427 ± 310 Bq/m³ a 2053 ± 700 Bq/m³. As concentrações de 222Rn no solo variaram de 31 ± 2 kBq/m³ a 35 ± 4 kBq/m³ e os valores médios encontrados de 220Rn estão em um intervalo de 41 ± 6 kBq/m³ e 25 ± 11 kBq/m³, os quais constatam que as concentrações do gás radônio do solo estão abaixo do critério sueco que determina que valores inferiores a 50 kBq/m³ não caracterizam uma situação de alto risco. / In every day life people are exposed to various types of radiation arising from different artificial and natural sources and among all of them the main role belongs to the isotope of noble gas 222Rn that makes part of the 238U radioactive chain. The isotope 222Rn is responsible for approximately half of the effective annual dose received by the world population. Being inhaled, the radon isotopes have to undergo 8 radioactive decay events (one half by emitting alpha particles and another half by emitting betas) to get to a stable isotope of lead. This radiation interacting with the cells of biological tissue have very high probability to induce the lung cancer. The goal of present research is to evaluate the activity concentration of 222Rn in the air of workplaces at Curitiba – Paraná State. Simultaneously there were performed the measurements of 222Rn emanation from soil and building materials occurred at evaluated workplaces. Indoor measurements of 222Rn activity were performed using Polycarbonate track etched detectors CR-39 that after the exposition in air were submitted to chemical etching and manual reading using the optical microscope.The calculations of the activity concentration of 222Rn in the air of workplaces were completed using the results of calibration performed by the Center of Nuclear Technology Development (CDTN) in cooperation with the Laboratory of Applied Nuclear Physics of the Federal University of Technology - Paraná (UTFPR).The instant radon detector AlphaGUARD (Saphymo GmbH) was used in the measurements of the average concentrations of 222Rn in soil gas and building materials. Building materials were also submitted to gamma spectrometry analysis for qualitative and quantitative evaluation of the radionuclides present in samples of sand, mortar, blue crushed stone, red crushed stone, concrete and red bricks. The method used for the measurements of radon activity in soil gas allowed to find the average concentrations of two isotopes 222Rn and 220Rn. The average concentration of indoor 222Rn obtained in the measurements in air of workplaces vary between 36 ± 49 Bq/m³ and 164 ± 51 Bq/m³. These values are considered within the reference limit of 200 Bq/m³ established by international agencies such as the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) and the International Commission on Radiological Protection (ICRP), but slightly above the limit of 148 Bq/m³ established by the United States Environmental Protection Agency (USEPA). The measurements involving building materials presented the concentration values of 222Rn in a range from 427 ± 310 Bq/m³ to 2053 ± 700 Bq/m³. The 222Rn concentrations in soil ranged from 31 ± 2 kBq/m³ to 35 ± 4 kBq/m³ and the average values of 220Rn are found in a range of 41 ± 6 kBq/m³ and 25 ± 11 kBq/m³, thus the concentrations of radon gas soil are below the Swedish criterion of 50 kBq/m³ that represent the minimum value for high-risk situation.

Par leur capacité de moduler la réponse immune, les rayons ultraviolets (UV) ont trouvé des applications dans le traitement de diverses maladies immunes. Leurs mécanismes d’action sont encore incomplètement définis. L’un d’entre eux comporte l’induction de cytokines immunosuppressives et antiinflammatoires. Ce processus peut être provoqué par la phagocytose de corps apoptotiques, l’apoptose constituant une des lésions cellulaires élémentaires provoquée par les UV. Le but de notre travail a été de préciser les cytokines impliquées dans la réponse aux UV, de définir certains mécanismes de leur production et de la potentialiser par des agents pharmacologiques.<p>Notre étude a comporté deux parties: (1) l’une in vivo chez des malades souffrant de GVH chronique résistante aux traitements conventionnels et traités par photochémothérapie extracorporelle, procédure dans laquelle les leucocytes du malades, prélevés par leucaphérèse puis traités par un psoralène et par UVA lui sont finalement réinjectés; (2) l’autre in vitro où des PBMC de volontaires sains ont été irradiés avec 10J/m2 de rayons UVC qui ne nécessitent pas de photosensibilisation par psoralène. Deux cytokines, l’IL-10 et l’IL-1Ra ont été évaluées par RT-PCR dans un système de coculture autologue entre PBMC et PBL rendus apoptotiques par irradiation. L’évolution du processus apoptotique déclenché par les UV a été mesurée par cytomètrie de flux. Celui-ci concernait essentiellement les lymphocytes, les monocytes/macrophages révélant une résistance relative à l’apoptose, il était progressif, culminant entre la 24ème et la 48ème heures. Lors des cocultures entre PBMC et PBL irradiés, un accroissement très significatif du nombre de copies d’ARNm, concernant les deux cytokines étudiées, l’IL-10 et l’IL-1Ra était observé. L’induction d’IL-1Ra était dépendante de l’IL-10. Une préactivation par du LPS était nécessaire pour la révélation du phénomène. <p>Ensuite, nous avons évalué l’implication sur la synthèse de cytokines du processus de phagocytose de lymphocytes rendus apoptotiques par irradiation UV et divers moyens pharmacologiques pour la potentialiser. La préincubation du matériel irradié pendant une nuit (16h) à 37° dans le but d’accroître la proportion de cellules en voie d’apoptose avant mise en contact avec les PBMC a permis d’obtenir un accroissement très marqué sans nécessiter de LPS, portant essentiellement sur la production d’IL-1Ra tant sur l’ARNm que la protéine secrétée; l’induction d’IL-10 était cette fois négligeable. L’implication de la phagocytose dans le processus a été démontrée par deux agents bloquants (a) l’anticorps monoclonal anti-CD36 (corécepteur avec l’intégrine &61537;V&61538;3 de la thrombospondine) activant la production d’IL-1Ra et mimant par ce fait le processus phagocytaire et (b) la cytochalasine E la bloquant.<p>Nous avons testé diverses substances pharmacologiques dont l’action activatrice de l’IL-1Ra est connue, en l’occurrence les immunoglobulines G à usage IV (IgIV) et le GM-CSF. L’adjonction d’IgIV (1mg/ml) ou GM-CSF (10 ng/ml) une heure après le début de la coculture exerce sur la sécrétion d’IL-1Ra un effet additif avec les UV. Selon la concentration utilisée, les IgIV peuvent agir par deux mécanismes. Outre l’effet d’activation macrophagique lié au récepteur Fc, nous avons démontré à haute concentration un mécanisme nouveau, du à la présence dans les IgIV d’anticorps naturels antiFas induisant l’apoptose des lymphocytes. Une incubation de 16h des lymphocytes avec 25 mg/ml d’IgIV avant mise en culture provoque outre une apoptose importante une augmentation significative de l’IL-1Ra. Dans ce cas, le processus est indépendant du fragment Fc, la fraction F(ab’)2 gardant la capacité d’induire l’apoptose et de provoquer la production d’IL-1Ra. <p><p>En conclusion, nous avons mis en évidence un mécanisme nouveau d’induction d’IL-1Ra, non décrit auparavant et défini diverses modalités qui pourraient accroître sa production: <p>- L’incubation de 16h du matériel irradié permet d’orienter le système en accroissant la production de l’IL-1Ra sans que la production de l’IL-10 soit modifiée et sans nécessiter de LPS. Nous attribuons cet effet à l’accroissement du processus apoptotique qui en résulte.<p>- Nous avons potentialisé la production d’IL-1Ra par deux agents pharmacologiques, le GM-CSF et les IgIV. Les mécanismes d’action des IgIV dépendent de la concentration utilisée.<p>1. Aux concentrations de l’ordre de 1mg/ml, les IgIV exercent, avec les UV un effet additif sur l’induction d’IL-1Ra par une action dépendant du fragment Fc. <p>2. Aux concentrations élevées de 25mg/ml, un effet apoptotique attribuable à l’action d’anticorps anti-Fas agonistes est observé. Une préincubation de 16h de lymphocytes avec cette concentration d’ IgIV avant mise en culture avec les PBMC autologues provoque outre l’apoptose importante des lymphocytes un accroissement significatif de la production d’IL-1Ra. Le processus est indépendant du fragment Fc, la fraction F(ab’)2 gardant la capacité d’induire l’apoptose et la production d’IL-1Ra. \ / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

This study set out to examine the levels of galactic cosmic radiation exposure to Australian aircrew during routine flight operations, with particular attention to the high southern latitude flights between Australia and South Africa. Latitudes as high as 65?? South were flown to gain the data and are typical of the normal flight routes flown between Sydney and Johannesburg on a daily basis. In achieving this objective it became evident that suitable commercially available radiation monitoring equipment was not readily available and scientific radiation monitors were sourced from overseas research facilities to compliment my own FH4lB and Liulin monitors provided by UNSW. At the same time it became apparent that several predictive codes had been developed to attempt to model the radiation doses received by aircrew based on flight route, latitudes and altitudes. Further, it became apparent that these codes had not been subjected to verification at high southern latitudes and that they had not been validated for the effects of solar particle events. Initially measurements were required at the high latitudes followed by mid-latitude data to further balance the PCAIRE code to ensure reasonableness of results for both equatorial and high latitudes. Whilst undertaking this study new scientific monitors became available which provided an opportunity to observe comparative data and results. The Liulin, QDOS and a number of smaller personal dosimeters were subsequently obtained and evaluated. This appears to be the first time that such an extensive cross comparison of these monitors has been conducted over such a wide range of latitudes and altitudes. During the course of this study a fortuitous encounter with GLE 66 enabled several aspects of code validation to be examined, namely the inability of predictive codes to estimate the increased dose associated with a GLE or the effects of a Forbush decrease on the code results. Finally I review the known biological effects as discussed by numerous authors based on current epidemiological studies, with a view to high-lighting were the advent of future technology in aviation may project aircrew dose levels.