Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 149-179). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (Ph.D.)--Uppsala Universitet, 2009. / This website links to the complete document in PDF format. Title from title screen (viewed on November 21, 2009). Includes bibliographical references.

"Life" may be characterized as a controlled stationary flow equilibrium, maintained by energy consuming chemical reactions. The physiological functioning of these life systems include at least 28 of the elements isolated on the periodic table thus far, most of which are metals. However, as with Paracelsus Principle: "The dose makes the poison", there exists a definite link between metal levels, essential and toxic, and the onset of neurodegenerative diseases. The economic costs of brain dysfunction are enormous, but this pales in comparison to the staggering emotional toll on the victims themselves and their families. In an attempt to improve the understanding of the causes of neurodegeneration, this study focuses on one potential aspect: the possible link between metals and neurotransmitter homeostasis utilising a variety of electronanalytical techniques. Adsorptive cathodic stripping voltammetry was employed to investigate the binding affinities and complex formation of melatonin and its precursor serotonin with calcium, potassium, sodium, lithium and aluminium. The results showed that all the metals studied formed complexes with both pineal indoleamines. However, the stability and affmity of the ligands toward the various metals varied greatly. The study suggests a further role for melatonin, that of metalloregulator and possible metal detoxifier in the brain, the in vivo studies which followed will further substantiate this notion. This research additionally focused on the cholinergic system, in particular acetylcholine complex formation studies with mercury, lead, cadmium, copper and zinc using the adsorptive cathodic stripping voltammetry method. The formation and characterisation of a solid mercury-acetylcholine complex lent further strength to the in situ electrochemical complex formation observed. The results showed the preference of acetylcholine for environmentally toxic heavy metals (such as Cd²⁺) over those divalent cations that occur naturally in the body. The possible metalloregulatory role melatonin played in the three brain regIOns: cerebellum, cortex and corpus striatum of male Wistar rats was studied as an in vivo extension of the earlier in vitro studies. Anodic stripping voltammetry was employed to detect metal levels present. The results showed that daily injections of melatonin was responsible for significantly decreasing copper(I), cadmium(II) and lead(II) levels in various regions of the rat brain of those animals that had undergone a pinealectomy in comparison to the saline injected group having undergone the same treatment. Histological and electrochemical stripping techniques were applied to investigate the implications of high A1³⁺ levels in the brain regions, particularly the hippocampus. Melatonin showed signs of promise in indirect symptom alleviation and by significantly decreasing A1³⁺ levels in rats that had been dosed with melatonin prior to A1³⁺ treatments in comparison with the control groups. Finally a preliminary study outlining a method for the production of a calcium selective microelectrode was undertaken. Further work is still needed to optimise the microelectrode production as well as its possible applications. However, whilst the overall conclusions of this entire multidisciplinary study may indeed only be in effect one piece of a very large puzzle on neurodegenerative diseases, this piece will no doubt serve as a building block for further ideas and work in this field.

Orientador: Flavio Ailton Duque Zambrone / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T12:16:07Z (GMT). No. of bitstreams: 1 Ramos_MarciaMariaReisVieira_M.pdf: 2283119 bytes, checksum: b53803c737ba4b24f8ee8fab9274456a (MD5) Previous issue date: 2007 / Resumo: A ocorrência de neurotoxicidade crônica secundária à exposição em longo prazo a níveis baixos de agrotóxicos é ainda controversa. O presente estudo teve por objetivo analisar a relação da exposição ao longo prazo aos agrotóxicos e a ocorrência de efeitos neurotóxicos crônicos. Consiste em uma revisão crítica e sistemática da literatura, de estudos epidemiológicos publicados entre 1996 e 2006, que abordaram a ocorrência de neurotoxicidade crônica em humanos, secundária à exposição a baixas doses de agrotóxicos. A revisão da literatura identificou estudos descritivos de prevalência, estudos de coorte, estudos de caso-controle e ecológicos que investigaram a relação entre neuropatologias crônicas adquiridas e a exposição aos pesticidas. Foram incluídos sessenta e três estudos nesta revisão (quarenta e seis estudos caso-controle, sete estudos seccionais e nove estudos de coorte). Somente um estudo ecológico foi identificado. Vinte e dois dos estudos caso-controle se relacionaram à doença de Parkinson ou parkinsonismo e vinte e quatro abordaram outras alterações neurológicas. Uma grande parte dos estudos documentou exposição humana a uma mistura de agrotóxicos Os organofosforados formam os mais estudados, seguidos dos carbamatos. A grande maioria dos estudos utilizou métodos indiretos para a avaliação da exposição (85%). Poucos estudos levaram em consideração a história de intoxicação aguda prévia. A maioria dos estudos mostrou uma associação positiva entre a exposição aos agrotóxicos e a ocorrência de doenças neurológicas, no entanto muitas destas associações são limitadas, fracas ou não estatisticamente significantes. Diversos efeitos neurológicos eram de detecção subjetiva e de difícil confirmação diagnóstica. Esta revisão constatou que alguns efeitos neurotóxicos crônicos podem estar associados à exposição de longo prazo, aos agrotóxicos, embora nenhuma associação com substâncias específicas pode ser detectada. Muitos dos resultados dos estudos apresentaram uma relação fraca; inconsistente; ou estatisticamente insignificante. Os estudos epidemiológicos que estão por vir devem tentar sanar os vieses encontrados nos estudos desta revisão, em especial devem proceder a uma avaliação da exposição mais cuidadosa. Um programa de pesquisa é necessário para um maior esclarecimento do risco representado por exposição em longo prazo e às baixas doses destas substâncias / Abstract: The occurrence of secondary chronic neurotoxicity to the exposition in long stated period to low levels of pesticideds is still controversial. The present study it had for objective to analyze the relation of the exposition in long stated period to the pesticides and the occurrence of chronic neurotoxic effect. It consists of a critical and systematic review of literature, in epidemiologists studies published between 1996 and 2006, that they had approached the occurrence of chronic neurotoxicity in human beings, secondary to the exposition the low doses of pesticides. The review of literature identified descriptive studies of prevalence, ecological studies, coorte studies and case-control studies that had investigated the relation between acquired chronic neuropathologies and the exposition to pesticides. They had been enclosed sixty and three studies in this revision (forty and six case-control studies, seven sectional studies and nine coorte studies). Only one ecological study it was identified. Twenty and two of the case-control studies had related to the Parkinson illness or parkinsonism and twenty and four had approached other neurologic alterations. A great part of the studies registered exposition human being to a mixture of pesticides. The organophosphates ones forms the most studied, followed of the carbamates. The great majority of the studies used indirect methods for the evaluation of the exposition (85%). Few studies had taken in consideration the history of previous acute poisoning. The majority of the studies showed a positive association between the exposition to the pesticides and the occurrence of neurological illnesses, however many of these associations are limited, weak or not statistically significant. Diverse neurological effect were of subjective detection and with difficult diagnostic confirmation. This review evidenced that some chronic neurotoxic effects can be associate to the long stated period exposition to the pesticides, even so no association with specific substances can be detected. Many of the results of the studies had presented a weak relation, inconsistent, or statistically insignificant. The epidemiologists studies who are for coming must try to cure the bias found in the studies of this revision, in special must proceed to a most careful evaluation of the exposition. A research program is necessary for a bigger clarification of the risk represented for exposition in long stated period and to the low doses of these substances / Mestrado / Epidemiologia / Mestre em Saude Coletiva

Objetivo. Avaliar o campo visual em ex-trabalhadores de fábricas de lâmpadas fluorescentes com diagnóstico de mercurialismo metálico crônico ocupacional, através de testes psicofísicos de campimetria computadorizada e registros eletrofisiológicos da retina obtidos através do eletrorretinograma multifocal. Método. A avaliação psicofísica do campo visual foi realizada em 35 ex-trabalhadores (idade média = 44,2 ± 5,9 anos; 30 homens) no equipamento Humphrey Field Analyzer II (modelo 750i) em dois testes: acromático (standard automated perimetry) e azul-amarelo (short wavelength automated perimetry). O programa Visual Evoked Response Imaging System (VERISTM Science 5.0) permitiu o registro e análise dos sinais eletrofisiológicos da retina através do eletrorretinograma multifocal em 32 ex-trabalhadores (idade média = 44,6 ± 5,5 anos; 27 homens) dos 35 que realizaram os testes de campimetria computadorizada. Os resultados foram comparados com um grupo controle para o campo visual (n = 34; idade média = 43,3 ± 8,3 anos; 21 homens) e com outro grupo controle para o eletrorretinograma multifocal (n = 21; idade média = 43,5 ± 8,9 anos; 10 homens). Resultados. Os exames psicofísicos de campimetria computadorizada mostraram que há redução da sensibilidade visual em regiões centrais até 27? do campo visual. No exame acromático a diminuição da sensibilidade ocorreu, inclusive, na região foveal. O exame azul-amarelo confirmou a redução encontrada no exame acromático para regiões paracentrais até 27° de excentricidade. O eletrorretinograma multifocal apresentou redução nas amplitudes das respostas retinianas em regiões centrais até 25°, sem alteração no tempo implícito das respostas. As regiões paracentrais mostraram redução significativa para os valores de amplitude do primeiro componente negativo (N1) e do primeiro componente positivo (P1). Discussão. A redução na sensibilidade visual em diferentes regiões do campo visual, confirma que há prejuízos no sistema visual decorrentes da exposição crônica ao vapor de mercúrio. Nesse caso, não se pode especificar as regiões afetadas, porque a metodologia utilizada não permite isolar estruturas da via visual e, consequentemente, não permite localizar as regiões específicas que o mercúrio estaria prejudicando preferencialmente. Os prejuízos causados pela intoxicação ao vapor de mercúrio na retina parecem ser difusos, considerando que a redução de amplitude das respostas de N1 e P1 pode indicar prejuízos em diferentes grupos celulares da retina. Os resultados mostram que parte dos prejuízos de campo visual causados pelo vapor de mercúrio estão relacionados com alterações retinianas. Os resultados estão de acordo com trabalhos preliminares que monstraram alterações visuais que permanecem mesmo anos após o afastamento da fonte de exposição, sugerindo que a intoxicação por vapor de mercúrio pode não ser totalmente reversível. Conclusão. Os sujeitos expostos cronicamente ao vapor de mercúrio durante um período de 10 anos (em média) apresentam redução da sensibilidade visual em diferentes regiões do campo visual, mesmo após 7 anos (em média) de afastamento da fonte expositora. Pode haver prejuízos em diferentes regiões da via visual envolvidos nas alterações de campo visual, mas parte desses prejuízos causados pela exposição crônica ocupacional ao vapor de mercúrio possui origem retiniana. / Purpose. To analyse visual field sensitivity in a group of workers retired from thefluorescent lamp industry diagnosed with chronic occupational metallic mercurialism using psychophysical tests such as automated perimetry and measuring the retina cells\' electrical responses with the multifocal electroretinogram. Methods. The psychophysical evaluation of the visual field was performed in 35 retired workers (mean age = 44.2 ± 5.9 years; 30 males) using Humphrey Field Analyzer II (model 750i) device in two different tests: SAP (standard automated perimetry) and SWAP (short wavelength automated perimetry). The Visual Evoked Response Imaging System (VERISTM Science 5.0) provided us the electrophysiological recordings and analysis of the retina based on measurement data from the multifocal electroretinogram in 32 retired workers (mean age = 44.6 ± 5.5 years; 27 males) that were included in the 35 automated perimetry test subjects. The results were compared with an age-matched control group using the visual field tests (n = 34; mean age = 43.3 ± 8.3 years; 21 males) and to another age-matched control group at the multifocal electroretinogram (n = 21; mean age = 43.5 ± 8.9 years; 10 males). Results. The automated perimetry tests have shown visual sensitivity reductions in the central areas around 27° of eccentricity. In the SAP test sensitivity decrease was found even in the foveal region. The SWAP test results are in agreement with the reduction found around 27° in the SAP test at mid-peripheral areas. The multifocal electroretinogram has shown decreases in amplitude in the retina recordings in the central areas around 25° of eccentricity, but there were no implicit time reductions. The mid-peripheral areas have shown significant reductions in the amplitude values in the first negative component (N1) and in the first positive component (P1) as well. Discussion. The visual sensitivity reductions in the different visual field areas confirm the visual damages in patients with long-term mercury vapor exposure. In this case the affected visual pathway sections could not be determined since the applied psychophysical method does not allow us to indicate the specific visual structure principally damaged by the mercury vapor. The damages found in the retina due to mercury vapor intoxication can be considered broadly dispersed, since the reductions in N1 and P1 amplitudes might be the indications of damages in multiple retina cell groups. Our results show that some visual field losses are related to various retinal alterations caused by the mercury vapor. The results are in agreement with preliminary works that showed visual dysfunctions after several years away from the mercury vapor source suggesting that mercury vapor intoxication may not be completely reversible. Conclusion. The long-term (10 years in average) mercury vapor exposed workers have shown visual sensitivity reductions in different visual field areas after 7 years (in average) away from the mercury vapor source. In our present study we would like to indicate that visual field reductions cannot only be related to damages in the various sections of the visual pathway, but some of these visual field losses can occur due to retinal alterations caused by cronic mercury vapor exposure.

No description available.

Orientadores: Sérgio Marangoni, Luís Alberto Ponce Soto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T10:47:27Z (GMT). No. of bitstreams: 1 Carregari_VictorCorasolla_M.pdf: 2297387 bytes, checksum: fc95267d0ed3c753978294fe999d0cf7 (MD5) Previous issue date: 2011 / Resumo: O presente trabalho apresenta a purificação e caracterização bioquímica de uma nova PLA2 neurotóxica nomeada BbilTX-I isolada do veneno da serpente Bothriopsis bilineta. Esta proteína foi purificada através de dois passos cromatográficos, em Sephadex G75 e HPLC de fase reversa C18 (?Bondapak Watters). A BbilTX-I é uma PLA2D49, com elevada atividade catalítica, massa molecular de 14185,48 Da, e alta similaridade na sua sequência N-terminal com outras PAL2 de veneno de serpentes. A temperatura ótima para a atividade enzimática da BbilTX-I está entre 25Cº e 37 ºC. A fração BbilTX-I é cálcio dependente e tem a atividade enzimática bastante diminuída quando testadas com os íons divalentes. Da mesma forma como outras PLA2 descritas na literatura, a BbilTX-I mostra o pH ótimo em torno de 8. A fração F3 isoladas de crotapotinas crotálicas e a DA2-II extraída do soro de Didelphis albiventris apresentaram um efeito inibitório de mais de 50% sobre a atividade enzimática da toxina BbilTX-I. O efeito da concentração do substrato 4-nitro-3-(octanoiloxi) ácido benzóico sobre a atividade enzimática da BbilTX-I demonstrou que a toxina é uma enzima monomérica com tendência alostérica sugerindo provavelmente um mecanismo de cooperatividade positiva mnemônical. A PLA2 BbilTX-I inserida em preparação isolada de biventer cervicis de pintainho apresentou um efeito bloqueador da transmissão nervosa na junção neuromuscular, em doses de 10, 20, 30 e 50?g/ml e causou 50% de bloqueio em 20 minutos com 10 ?g/ml, 30 minutos com 20 ?g/ml, 22 minutos com 30 ?g/ml e 20 minutos com 50 ?g/ml. Como não houve alteração significativa na resposta ao KCl (usado para detectar danos na membrana celular) e à Acetilcolina (usada para identificar atividade sobre os receptores colinérgicos nicotinicos) podemos dizer que a PAL2 BbilTX-I é uma neurotoxina pré-sináptica não miotoxica, que atua no terminal nervoso da sinapse neuromuscular interferindo sabre a liberação da ACh. A crotapotina F3 isolada do veneno da serpente Crotalus durissus cascavella e o fator anti-homorrágico DA2-II isolado do soro de Dildelphis albiventris inibiram em 64% e 57% respectivamente a atividade edematizante da fração BbilTX-I demonstrando que a ação inflamatória desta proteína é dependente de sua ação catalítica / Abstract: In this present work we described the purification and functional characterization of a new neurotoxic PLA2, named BbilTX-I from Bothriopsis bilineata snake venom. This protein was by the combination of two chromatographic steps, Molecular Exclusion on a Sephadex lunm and Reverse Phase HPLC (RP-HPLC) on a C18 ?-Bondapack (Waters) column. BbilTX-I is a monomeric PLA2-D49 showing high purity degree in SDS-PAGE in both reducing and non-reducing conditions. The purity of the toxin was confirmed by MALDI-TOF mass spectrometry and shows a molecular mass of 14185.48 Da. BbilTX-I posses a high catalytic activity upon 4-nitro-3-(octanoiloxi) benzóic acid as a substrate compare with other bothropic PLA2. The optimal temperature and pH for BbilTX-I activity was determinate between 25-37 °C and around 8, respectively. This PLA2 is Ca+2 dependent but others divalent ions significatively reduce its catalytic activity, when pre­incubated with the protein. Also were tested the effects of fraction F3, crotapotin from Crotalus durrisous cascavela, and anti-hemorrhagic factor DA2-Il from Didelphis albiventris opossum sera under optimal conditions; both fraction showed an inhibitory effect over the BbilTX-I catalytic activity. At different concentrations of the substrate this monomeric PLA2 show an allosteric behavior suggesting a positive cooperative mnemonical enzyme mechanism in the catalysis. BbilTX-I showed an inhibitor effect on neuromuscular nerve transmissions on the isolated muscle biventer cervicis. At doses at 10, 20, 30 and 50 g/ml it elicited a blockade of 50% of 20, 30, 22 and 20 minutes, respectively. The effect displayed by BbilTX-I did not with the responses evoked by the addition of KCl (cell membrane damage) and (Ach functionality of cholinergic receptors), thus, we can argue that BbliTX-I is a pre-synaptic neurotoxin impairing the Ach liberation. BbilTX-I shows itself as an edematogenic PLA2 when injected in the mouse right footpad, it elicited a 70% higher than of edema when compare with the control. The edemetogenic activity of this PAL2 is related to its catalytic activity since both are inhibited by F3 crotapotin and DA2-II, 64 and 57% respectively. The reproducibility of biological activity, through the pharmacologic effects, only is possible by the use of chemically homogeneous fractions that hold biological function integrity. This fraction was obtain with high efficiency methodologies like HPLC, mass spectrometry, this result can be associated with the biological activity, disregarding the subjectivity caused by the venom or by impure fractions. This kind of methodologies can be applied to biochemical, structure and function, logical and pharmacological studies, which can reveal unknown mechanism in the function-structure relationship of snake venom PLA2 / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

This thesis will address the mechanisms underlying amphetamine addiction, as well as the psychosis that may develop with amphetamine use.

Indiana University-Purdue University Indianapolis (IUPUI) / Lead (Pb) is one of the most harmful and most abundant neurotoxins in the environment. Despite the extensive movement made to eradicate toxic levels of Pb in the environment, children, predominately in lower socioeconomic areas, are still exposed to varying levels of Pb. Human studies suggest that Pb exposure leads to altered drug consumption in adults by altering underlying neural mechanisms, specifically dopamine (DA) activity. However, there is limited research on this at blood Pb levels below 10 μg/dL, levels often seen in children growing up in neighborhoods located in old industrial and urban areas. To model how early-life low-level Pb exposure effects DA-dependent behaviors associated with addiction in adulthood, we used C57BL/6J mice. Litters were weaned at PND 21 and assigned to either a three-week history of 30 parts per million (ppm) Lead (IV) Acetate exposure or a control condition of 0 ppm Pb in DI drinking water. After the Pb exposure period, mice were switched to regular tap water until they reached adulthood. Afterward, separate animals were tested in one of three experiments: two-bottle choice alcohol preference drinking, alcohol-induced behavioral sensitization (EBS), and cocaine-induced behavioral sensitization (CBS). In experiment 1, our hypothesis was met, and both male and female mice with a prior Pb exposure displayed significantly higher alcohol intake and preference scores over the three-week period than control mice. In experiment 2, there were no differences in EBS and no evidence of EBS in any of the groups. However, there was an increased acute response to 2.0 g/kg EtOH in the Pb-exposed chronic group as compared to the control animals. Lastly, in experiment 3, Pb-exposed animals in the chronic cocaine group were more sensitive to the effects of cocaine (10 mg/kg) across days than the controls, both the acute cocaine groups and both saline control groups. Thus, with these experiments, we concluded that low levels of developmental Pb exposure might be targeting DA in the reward pathway, which is essential for alcohol intake and drug sensitization.