This thesis describes bioanalytical methods for measuring melatonin and some anti-infective drugs in biological fluids. Solid-phase extraction (SPE) or protein precipitation was used for enrichment and purification of the analytes and Liquid Chromatography (LC) was used to analyze the samples. Developed methods were validated according to international guidelines. Melatonin is a hormone secreted by the pineal gland with a robust circadian rhythm. Bioanalytical methods for determination of melatonin in plasma and saliva have been developed which were used for monitoring melatonin levels in volunteers and patients suffering from sleep related diseases. Eflornithine (DFMO) is a chiral drug used for the treatment of human African trypanosomiasis. A bioanalytical method for determination of the DFMO enantiomers in plasma, after precolumn derivatization with o-phtalaldehyde and N-acetyl-L-cystein has been developed. The method has been used to study the L- and D-DFMO pharmacokinetics, in order to investigate the possible development of an oral treatment of DFMO. A method for simultaneous determination of three antiretroviral drugs i.e. Lamivudine (3TC), Zidovudine (AZT) and Nevirapine (NVP) in dried blood spots (DBS) was developed. The method was used for drug determination in two subjects after receiving standard antiretroviral treatment. The method seemed well suitable for the determination of 3TC and NVP and in some extent for AZT. Lumefantrine (LF) is one of the active components in a new fixed drug combination recommended by the WHO as a replacement to older drugs that has lost their effect. A method for the determination of LF in DBS was developed. The method is suitable for monitoring of drug treatment in rural settings. Tafenoquine is a new promising antimalarial drug under development. A method for the determination of Tafenoquine in plasma and in DBS is described. The method may be useful in future clinical studies in laboratory environment as well as in rural settings. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 703

Central to human intelligence, visual categorization is a skill that is both remarkably fast and accurate. Although there have been numerous studies in primates regarding how information flows in inferiortemporal (ITC) and prefrontal (PFC) cortices during online discrimination of visual categories, there has been little comparable research on the human cortex. To bridge this gap, this thesis explores how visual categories emerge in prefrontal cortex and the ventral stream, which is the human homologue of ITC. In particular, cortical spatiotemporal plasticity in visual category learning was investigated using behavioral experiments, magnetoencephalographic (MEG) imaging, and statistical machine learning methods. From a theoretical perspective, scientists from work on non-human primates have posited that PFC plays a primary role in the encoding of visual categories. Much of the extant research in the cognitive neuroscience literature, however, emphasizes the role of the ventral stream. Despite their apparent incompatibility, no study has evaluated these theories in the human cortex by examining the roles of the ventral stream and PFC in online discrimination and acquisition of visual categories. To address this question, I conducted two learning experiments using visually-similar categories as stimuli and recorded cortical response using MEG—a neuroimaging technique that offers a millisecond temporal resolution. Across both experiments, categorical information was found to be available during the period of cortical activity. Moreover, late in the learning process, this information is supplied increasingly in the ventral stream but less so in prefrontal cortex. These findings extend previous theories by suggesting that the ventral stream is crucial to long-term encoding of visual categories when categorical perception is proficient, but that PFC jointly encodes visual categories early on during learning. From a methodological perspective, MEG is limited as a technique because it can lead to false discoveries in a large number of spatiotemporal regions of interest (ROIs) and, typically, can only coarsely reconstruct the spatial locations of cortical responses. To address the first problem, I developed an excursion algorithm that identified ROIs contiguous in time and space. I then used a permutation test to measure the global statistical significance of the ROIs. To address the second problem, I developed a method that incorporates domainspecific and experimental knowledge in the modeling process. Utilizing faces as a model category, I used a predefined “face” network to constrain the estimation of cortical activities by applying differential shrinkages to regions within and outside this network. I proposed and implemented a trial-partitioning approach which uses trials in the midst of learning for model estimation. Importantly, this renders localizing trials more precise in both the initial and final phases of learning. In summary, this thesis makes two significant contributions. First, it methodologically improves the way we can characterize the spatiotemporal properties of the human cortex using MEG. Second, it provides a combined theory of visual category learning by incorporating the large time scales that encompass the course of the learning.

Common bean (Phaseolus vulgaris L.) is an important legume crop in Kenya and is a cheap source of proteins. The small scale farmers in Kenya produce common bean under low agricultural input systems and this predisposes the crop to pests and diseases. Among the diseases, angular leaf spot (ALS) is a major constraint to common bean production and contributes to yield losses as high as 80%. The causative pathogen Pseudocercospora griseola (Sacc.) Crous & Braun is highly variable and several races have been reported. There are few common bean genotypes with resistance to this disease. Therefore breeding for resistance to ALS is important for the country. This study was carried out to; i) evaluate the common bean production systems, constraints and farmer varietal preferences in Kenya, ii) evaluate local landraces and selected introductions of common bean for yield performance and reaction to ALS, iii) study the genetics of resistance to ALS in common bean and iv) develop a breeding method for durable resistance to ALS in common bean. To determine the common bean production systems, farmers’ preferred traits and their knowledge on common bean constraints including ALS, a survey was conducted in Kiambu county using a semi-structured questionnaire, interviews, and focus group discussions. The study revealed that farmers cultivate common beans during the short and long rain seasons. However, they experience better yields in the short rains due to reduced disease incidence. The majority of the farmers (71%) intercrop common bean and this ensures maximum utilisation of space. A high percentage (70%) of the farmers utilise their retained seed for production. The farmers identified ALS as one of the most important constraints to production. The only preventative measure they undertake to control the disease is weeding. The farmers reported that they would prefer improved varieties that were resistant to ALS. Farmers have a preference for particular common bean traits that include high yield (80%), resistance to insect-pests and diseases (72%), type I growth habit (52%), early maturity (68%), seed size and colour (21%) and cooking time (20%). These should be incorporated in breeding programmes. Two hundred common bean landraces and market class varieties were evaluated for ALS resistance in a nethouse at University of Nairobi, Kabete Field Station and for ALS resistance and yield in the field in KARI-Tigoni. The results showed that disease severity scores for the genotypes were similar in the two locations, with the top three resistant genotypes being Minoire, GBK 028123 and Murangazi with disease severity scores of 2.9, 2.9 and 3.2 in Kabete and 2.6, 2.8, and 2.9 in Thika respectively. These resistant genotypes can be used as sources of resistance in a breeding programme or they can be used as resistant varieties. All the market class varieties were susceptible to ALS (disease severity score 6.7-8.0). There was a non-significant correlation between disease and yield most likely because most of the resistant genotypes were exotic and hence not adapted to the local conditions. There was also a non-significant correlation between disease and seed size. The two hundred common bean genotypes were evaluated for yield at University of Nairobi, Kabete Field Station and KARI-Thika. The results indicated that the 2011 and 2012 seasons had similar mean yields and that yields at Kabete were higher than at KARI-Thika. The highest yielding genotypes across the two locations were; GLP 2 (766 kg ha-1), Nyirakanyobure (660 kg ha-1), GBK 028110 (654 kg ha-1), GLP 585 (630 kg ha-1) and Mukwararaye (630 kg ha-1). There was a significant genotype x environment interaction and hence it is important for breeders to carry out stability analysis, so as to recommend varieties for a wide range of environments. To study the genetics of ALS resistance in common bean, three inter-gene pool crosses: Super-rosecoco x Mexico 54, Wairimu x G10909 and Wairimu x Mexico 54 were made. The resistant genotypes were Mexico 54 and G10909, while Super-rosecoco and Wairimu were susceptible. The generations F1, F2, BC1P1 and BC1P2 for each of the crosses were developed. The parents P1, P2 and the five generations of each cross were evaluated for resistance to ALS in Kabete Field Station. Results showed that both dominance and additive gene action were important in the expression of resistance to ALS. However, additive gene action was predominant over dominance gene action. There was a moderately high narrow sense heritability estimate (52.9-71.7%). The minimum number of genes controlling resistance to ALS was between 2 and 3. The predominance of additive gene effects and the moderately high narrow sense heritability estimates recorded imply that progress in resistance to ALS could be made through selection in the early segregating generations. A double cross followed by selection against resistant genotypes was used to develop a method to breed for durable resistance to ALS in common bean. The method was used to accumulate minor genes of ALS resistance into single genotypes. Four intermediate resistant landraces were used to develop a double cross population that was screened using a mixture of ALS races. Selection in F1 and F2 population was done on the basis of intermediate resistance (disease severity score 4.0-6.0), while selection from F3 population was based on resistance (disease severity score 1.0-3.0). Ten advanced F4 lines along with their parents were evaluated for ALS resistance. The F4 advanced lines had a significantly improved resistance to ALS compared to their parents. Hence the method was successful in accumulating minor genes for resistance thus showing significant breeding progress in breeding for durable resistance. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Made available in DSpace on 2016-06-02T19:32:01Z (GMT). No. of bitstreams: 1 4244.pdf: 1071959 bytes, checksum: 6ba82849d82a8316bf2d47e4273770cb (MD5) Previous issue date: 2011-03-02 / Euterpe edulis is a very important palm tree from the Brazilian Atlantic Forest due to its ecological and economic role. However, due to the indiscriminate overexploitation its populations has been reducing greatly. This study aimed to characterize the conservation status of a E. edulis population in a fragment of Atlantic Forest at Southern Bahia. We monitored this population along 5 running years in a 0.8 hectare. Environmental variables were also collected to test which factors could affect plant development and projection matrices analysis were performed to estimate the population growth rate and sensibility. The results showed that population s density is lower if compared with others studies. We verified a decrease in the seedling s recruitment over years, which were attributed to fragmentation side-effects, such as microclimate changes and / or reproductive isolation or inbreeding depression. We also observed that the low rate of transition from stages 1 to 2, represents a bottleneck for the population growth. These results emphasize the urgency of more long-term studies on the population ecology and genetic of E. edulis and also management strategies to promote the sustainability of such natural populations. / Euterpe edulis é uma das espécies de maior importância ecológica e econômica na Mata Atlântica brasileira. Apesar de a espécie possuir características que favorecem o manejo sustentável, a exploração predatória gerou um cenário de populações naturais severamente reduzidas. Este estudo procurou caracterizar o atual estado de conservação de uma população no sul da Bahia. Para tanto foi definido um bloco amostral 0,8 hectare, onde foi monitorada uma população de E. edulis, dividida em seis estágios de desenvolvimento. Foram geradas matrizes de projeção para verificar a tendência atual de conservação da população e variáveis ambientais foram analisadas para inferir sobre como tais fatores podem afetar o desenvolvimento da espécie. Os resultados mostraram que a densidade da população encontrada nesse estudo é menor que a relatada em outros estudos já realizados com a espécie. Verificou-se também a diminuição abrupta no recrutamento de plântulas a cada ano, o que foi atribuído a algum efeito colateral da fragmentação florestal, tal como alterações microclimáticas, e/ou efeitos do isolamento reprodutivo, como a depressão endogâmica. A lenta transição do estágio jovens 1 para jovens 2 se mostrou como principal gargalo ao crescimento populacional, o que sugere a necessidade de estudos sobre a redução na fecundidade. Ambientes com maior homogeneidade de dossel propiciaram maior crescimentos em jovens 2. Propõe-se que a produção e manutenção de mudas em viveiros até a idade de um ano, seguida de plantio em áreas próximas às bordas de clareiras, possa diminuir a mortalidade dos estágios iniciais e garantir maior êxito na perpetuação das populações de Euterpe edulis no sul da Bahia.

Made available in DSpace on 2014-10-09T12:42:40Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:16Z (GMT). No. of bitstreams: 1 05031.pdf: 4962096 bytes, checksum: 39c12c06c0063abb20c1c82005ecef33 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

A mucopolissacaridose tipo VI (MPS VI) ou Síndrome de Maroteaux-Lamy, é uma doença autossômica recessiva causada pela deficiência da enzima lisossomal Nacetilgalactosamina- 4-sulfatase (ARSB), a qual resulta no armazenamento lisossômico de dermatan sufato em vários tecidos e órgãos, dando origem a uma condição clínica de espectro variável, desde formas mais graves até formas mais atenuadas. O acúmulo de substrato não degradado causa um importante comprometimento ósseo, problemas respiratórios, baixa estatura e outros problemas, afetando os olhos, o coração e outros órgãos. Embora a síndrome de Maroteaux-Lamy não tenha uma incidência definida no Brasil, é reconhecido que, no nosso meio, é muito mais freqüente do que em outros países e regiões. Ela é particularmente freqüente no município de Monte Santo (Bahia), de aproximadamente 50.000 habitantes e onde já foram registrados 13 casos da doença. O diagnóstico é importante porque existe hoje um tratamento específico para a doença, a terapia de reposição enzimática (TRE), que vem mostrando bons resultados, especialmente quando iniciada em idade precoce. Descrevemos neste trabalho uma adaptação para microplacas da medida fluorimétrica da atividade de ARSB, e uma nova metodologia de análise molecular, ambas padronizados para sangue total impregnado em papel-filtro (STIPF). Essas técnicas foram desenvolvidas para incluir um teste de triagem neonatal para MPS VI, realizado nas amostras coletadas para o “teste do pezinho” nos neonatos do município de Monte Santo. Esses métodos permitem a detecção de pacientes com MPS VI e dos portadores da mutação específica que parece ser responsável pela alta incidência de MPS VI nessa localidade, uma vez que todos os pacientes lá diagnosticados apresentavam a mesma mutação (p.H178L) em homozigose. O trabalho foi desenvolvido em três etapas: na primeira foi realizada a padronização das técnicas em 100 amostras de STIPF; na segunda foi feito um teste-piloto com amostras de neonatos de Monte Santo, para avaliação das técnicas padronizadas e para o estudo de termoestabilidade em controles hígidos; na terceira foram analisadas amostras de STIPF de neonatos provenientes de Monte Santo pelos dois métodos (bioquímico e molecular). A padronização para realização da medida fluorimétrica da atividade enzimática de ARSB em microplacas indicou que o método é sensível, permitiu diferenciar os valores da população normal dos valores dos pacientes afetados e possibilitou a identificação segura de pacientes com MPS VI. Nas padronizações da análise molecular da mutação p.H178L em STIPF foi possível diferenciar os indivíduos normais, heterozigotos e homozigotos. Os resultados preliminares disponíveis indicam que o protocolo de triagem neonatal para MPS VI desenvolvido no presente trabalho poderá ser facilmente incorporado por laboratórios de referência, contribuindo para a detecção e tratamento precoce dos pacientes afetados por MPS VI. / Mucopolysaccharidosis type VI (MPS VI) or Maroteux-Lamy syndrome, is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme Nacetylgalactosamine- 4-sulfatase (ARSB), which results in lysosomal storage of dermatan sufate in various tissues and organs and leads to a variable clinical spectrum, including more severe and attenuated forms. The accumulation of undegraded substrate causes bone involvement, respiratory problems and short stature, among other signs and symptoms, affecting the eyes, heart and other organs. Although the Maroteaux-Lamy syndrome does not have a defined incidence in Brazil, it is recognized that in our environment it is much more frequent than in other countries and regions. It is particularly frequent in the municipality of Monte Santo (Bahia) approximately 50,000 inhabitants and where there have already been 13 cases of the disease. The diagnosis is important because today there is a specific treatment for the disease, enzyme replacement therapy (ERT) which has shown good results, especially when started at an early age. We describe herein the standardization of the microplate fluorometric method for the ARSB test and a new methodology of molecular analysis, both adapted for dried blood spots (DBS) samples. These techniques were developed for inclusion of MPS VI in the newborn screening program that already tests the neonates of the city of Monte Santo, Bahia, Brasil for metabolic diseases. The methods were developed to detect patients with MPS VI and also for carriers, once the disease seems to have a high incidence (around 1:5.000) at this location. Also, all patients that have already been diagnosed in this city presented the same mutation (p.H178L) in homozygosis. The study was conducted in three stages: in the first was performed in 100 DBS samples an standardization of the techniques; in the second was done a pilot test with samples of newborns of Monte Santo, for the evaluation of standardized techniques and for the thermostability study in healthy controls; in the third were analyzed newborns samples from Monte Santo for both biochemical and molecular methods. Standardization on microplate for fluorimetric enzyme activity of the ARSB showed the assay sensitivity, differentiating values between normal and affected and allowing a reliable detection of patients with MPS VI. On the standardization for molecular analysis in DBS it was possible to differentiate the results for normal individuals, heterozygous and affected for the mutation p.H178L. The preliminary results available indicate that the protocol of neonatal screening for MPS VI developed in this work can be easily incorporated by reference laboratories, contributing to the detection and premature treatment of MPS VI affected patients.

La maladie de la tache noire affecte les fruits d’ananas mature, les rendant impropre à la consommation. Actuellement, aucune méthode de contrôle n'est disponible pour cette maladie. Une meilleure connaissance du pathosystème est nécessaire pour trouver des moyens de lutte efficaces.Des entretiens et échantillonnages menés auprès de producteurs durant l’hiver austral révèlent une prévalence de la maladie de 74%. Les champignons pathogènes appartiennent à plusieurs espèces : Fusarium ananatum (72% des isolats), Talaromyces stollii (21%), F. oxysporum (6%) et F. proliferatum (1%). Leur potentiel toxinogène a été déterminer, Les champignons du genre Fusarium ont produit des mycotoxines identifiées comme les fumonisines FB1, FB2 et la beauvericine. Sur un milieu de culture ananas, une concentration en beauvericine de 34959 µg kg-1 a été mesurée pour l’espèce F. proliferatum.Une méthode d’inoculation de Fusarium ananatum directement dans le parenchyme a permis de décrire la réponse du biochimique du fruit. La voie des phénylpropanoïdes est sollicitée, particulièrement avec l’élicitation du caffeoylisocitrate et du coumaroylisocitrate dans la zone infectée. Une comparaison des profils métaboliques montre que la réponse du fruit à une inoculation est plus importante chez le cultivar résistant ‘MD-2’ que chez le cultivar sensible ‘Queen’. La majorité des métabolites élicités par l’attaque sont déjà présents dans les fruits sains mature de la variété résistante. Le potentiel antifongique des composés phénoliques à était évalué. Les acides coumarique, caféoylquinique et férulique inhibent la croissance du mycélium à des concentrations similaires à celle trouvées dans les fruits infectés.Une approche par imagerie a permis de décrire l’anatomie des fruits des deux cultivars et notamment la fusion imparfaite des sépales et bractée chez ‘Queen’. Les nectaires et les parois carpellaires jouent un rôle clef dans le processus d'infection et de colonisation de Fusarium ananatum. / Fruiltet core rot (FCR) disease affects the fruits of mature pineapples. Current disease controls are not available. A deeper knowledge of the pathosystem is needed to find an effective means of control FCR.A diagnostic survey conducted with producers during the southern winter revealed a prevalence of the disease of 74%. Pathogenic fungi belong to several species: Fusarium ananatum (72% isolates), Talaromyces stollii (21%), F. oxysporum (6%) and F. proliferatum (1%). Their toxinogenic potential was determined. Fusarium fungi produced mycotoxins identified as fumonisins FB1, FB2 and beauvericin. On a pineapple culture medium, a concentration of beauvericine of 34959 μg kg-1 was measured for the species F. proliferatum.A method of inoculating Fusarium ananatum directly into the parenchyma allowed to describe the biochemical response of the fruit. The phenylpropanoids pathway is involved, particularly with the elicitation of caffeoylisocitrate and coumaroylisocitrate in the infected zone. A comparison of the metabolic profiles shows that the response to inoculation of resistant cultivar 'MD-2' is higher than in the sensitive cultivar 'Queen'. Most of the metabolites elicited by the attack are already present in healthy mature fruits of the resistant variety. The antifungal potential of the phenolic compounds was evaluated. Coumaric, caffeoylquinic and ferulic acids inhibit mycelial growth at concentrations similar to those found in infected fruits.An imaging approach allowed to describe the anatomy of the fruits of the two cultivars, to identify the key roles played by nectaries and carpel margins in the infection and colonization process of Fusarium ananatum.

Made available in DSpace on 2014-12-17T14:13:52Z (GMT). No. of bitstreams: 1 RigobertoGC_DISSERT.pdf: 2462609 bytes, checksum: 1753126d41dc7df59645485b6308c3f5 (MD5) Previous issue date: 2011-05-30 / Background. Gaucher Disease (GD) is a hereditary lysosomal storage disorder characterized by the accumulation of glucosylceramide, mainly in the cells of the reticuloendothelial system, due to a deficiency of the enzyme acid &#946;-glucosidase (GBA). Diagnosis is usually based on measurement of GBA activity in peripheral leukocytes. The purpose of this study was to evaluate the ability of screening for GBA and chitotriosidase activity using Dried Blood Spots on Filter Paper (DBS-FP) to identify individuals at high risk for GD in high-risk populations such as that of Tabuleiro do Norte, a small town in Northeastern Brazil. Methods. Between June 1, 2007 and May 31, 2008, 740 consented residents and descendants of traditional families from Tabuleiro do Norte were submitted to screening with DBS-FP. Subjects with GBA activity <2.19 nmol/h/mL were referred to analysis of GBA and chitotriosidase activity in peripheral leukocytes and in plasma, respectively. Subjects at highest risk for GD (GBA activity in peripheral leukocytes <5.6 nmol/h/mg protein) were submitted to molecular analysis to confirm diagnosis. Results. Screening with DBS-FP identified 135 subjects (18.2%) with GBA activity <2.19 nmol/h/mL, 131 of whom remained in the study. In 10 of these (7.6%), GBA activity in leukocytes was 2.6 5.5 nmol/h/mg protein. Subsequent molecular analysis confirmed 6 cases of heterozygosity and 4 normals for GD. Conclusion. DBS-FP assay was shown to be an effective initial GD screening strategy for high-prevalence populations in developing regions. Diagnosis could not be established from GBA activity in leukocytes alone, but required confirmation with molecular analysis / A doen?a de Gaucher (DG) ? uma patologia de dep?sito de gordura nos lisossomos, de heran?a autoss?mica recessiva, caracterizada pelo ac?mulo do substrato glicosilceramida, principalmente nas c?lulas do sistema reticuloendotelial, em raz?o da defici?ncia da enzima &#946;-glicosidase ?cida (GBA). O diagn?stico, comumente, ? feito pela dosagem da atividade da GBA em leuc?citos perif?ricos. Tabuleiro do Norte (TN), Cear?, Brasil, ? um munic?pio com cerca de 28.000 habitantes com a preval?ncia da DG de 1:4.000 habitantes, possivelmente a mais elevada do Brasil. O objetivo da disserta??o ? avaliar o rastreamento para DG realizado em TN com base na an?lise das atividades enzim?ticas da GBA e da quitotriosidase em amostras Sangue Seco em Papel de Filtro (SSPF). Entre 01 de junho de 2007 a 31 de maio de 2008, 740 indiv?duos residentes e descendentes de fam?lias de TN participaram do rastreamento para DG a partir de amostras de SSPF. Indiv?duos com atividade GBA<2,19 nmol/h/mL foram selecionados para an?lise da atividade da GBA e da quitotriosidase em leuc?citos perif?ricos e no plasma, respectivamente. Os indiv?duos com maiores riscos de DG (atividade de GBA em leuc?citos perif?ricos <5,6 nmol/h/mg de prote?na) foram referenciados para an?lise molecular para confirma??o diagn?stica. A triagem com amostras de SSPF identificou 135 indiv?duos (18,2%) com atividade da GBA<2,19 nmol/h/mL, dos quais 131 permaneceram no estudo. Em dez destes (7,6%), a atividade da GBA em leuc?citos variou de 2,6-5,5 nmol/ h/mg de prote?na, considerados suspeitos da DG. A an?lise molecular subsequente revelou, entretanto, que se tratava de seis indiv?duos heterozigotos para a muta??o G377S e, em quatro deles, n?o foram identificadas muta??es da DG. A an?lise enzim?tica de amostras de SSPF mostrou ser uma estrat?gia eficaz de triagem da DG em popula??es com alto risco, mas a medida da atividade da GBA em leuc?citos deve ser realizada para confirma??o diagn?stica. O diagn?stico de DG em indiv?duos assintom?ticos n?o deve ser firmado baseando-se apenas na an?lise da atividade da GBA em leuc?citos, sendo necess?ria, tamb?m, a confirma??o diagn?stica pela an?lise molecular

A mucopolissacaridose tipo VI (MPS VI) ou Síndrome de Maroteaux-Lamy, é uma doença autossômica recessiva causada pela deficiência da enzima lisossomal Nacetilgalactosamina- 4-sulfatase (ARSB), a qual resulta no armazenamento lisossômico de dermatan sufato em vários tecidos e órgãos, dando origem a uma condição clínica de espectro variável, desde formas mais graves até formas mais atenuadas. O acúmulo de substrato não degradado causa um importante comprometimento ósseo, problemas respiratórios, baixa estatura e outros problemas, afetando os olhos, o coração e outros órgãos. Embora a síndrome de Maroteaux-Lamy não tenha uma incidência definida no Brasil, é reconhecido que, no nosso meio, é muito mais freqüente do que em outros países e regiões. Ela é particularmente freqüente no município de Monte Santo (Bahia), de aproximadamente 50.000 habitantes e onde já foram registrados 13 casos da doença. O diagnóstico é importante porque existe hoje um tratamento específico para a doença, a terapia de reposição enzimática (TRE), que vem mostrando bons resultados, especialmente quando iniciada em idade precoce. Descrevemos neste trabalho uma adaptação para microplacas da medida fluorimétrica da atividade de ARSB, e uma nova metodologia de análise molecular, ambas padronizados para sangue total impregnado em papel-filtro (STIPF). Essas técnicas foram desenvolvidas para incluir um teste de triagem neonatal para MPS VI, realizado nas amostras coletadas para o “teste do pezinho” nos neonatos do município de Monte Santo. Esses métodos permitem a detecção de pacientes com MPS VI e dos portadores da mutação específica que parece ser responsável pela alta incidência de MPS VI nessa localidade, uma vez que todos os pacientes lá diagnosticados apresentavam a mesma mutação (p.H178L) em homozigose. O trabalho foi desenvolvido em três etapas: na primeira foi realizada a padronização das técnicas em 100 amostras de STIPF; na segunda foi feito um teste-piloto com amostras de neonatos de Monte Santo, para avaliação das técnicas padronizadas e para o estudo de termoestabilidade em controles hígidos; na terceira foram analisadas amostras de STIPF de neonatos provenientes de Monte Santo pelos dois métodos (bioquímico e molecular). A padronização para realização da medida fluorimétrica da atividade enzimática de ARSB em microplacas indicou que o método é sensível, permitiu diferenciar os valores da população normal dos valores dos pacientes afetados e possibilitou a identificação segura de pacientes com MPS VI. Nas padronizações da análise molecular da mutação p.H178L em STIPF foi possível diferenciar os indivíduos normais, heterozigotos e homozigotos. Os resultados preliminares disponíveis indicam que o protocolo de triagem neonatal para MPS VI desenvolvido no presente trabalho poderá ser facilmente incorporado por laboratórios de referência, contribuindo para a detecção e tratamento precoce dos pacientes afetados por MPS VI. / Mucopolysaccharidosis type VI (MPS VI) or Maroteux-Lamy syndrome, is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme Nacetylgalactosamine- 4-sulfatase (ARSB), which results in lysosomal storage of dermatan sufate in various tissues and organs and leads to a variable clinical spectrum, including more severe and attenuated forms. The accumulation of undegraded substrate causes bone involvement, respiratory problems and short stature, among other signs and symptoms, affecting the eyes, heart and other organs. Although the Maroteaux-Lamy syndrome does not have a defined incidence in Brazil, it is recognized that in our environment it is much more frequent than in other countries and regions. It is particularly frequent in the municipality of Monte Santo (Bahia) approximately 50,000 inhabitants and where there have already been 13 cases of the disease. The diagnosis is important because today there is a specific treatment for the disease, enzyme replacement therapy (ERT) which has shown good results, especially when started at an early age. We describe herein the standardization of the microplate fluorometric method for the ARSB test and a new methodology of molecular analysis, both adapted for dried blood spots (DBS) samples. These techniques were developed for inclusion of MPS VI in the newborn screening program that already tests the neonates of the city of Monte Santo, Bahia, Brasil for metabolic diseases. The methods were developed to detect patients with MPS VI and also for carriers, once the disease seems to have a high incidence (around 1:5.000) at this location. Also, all patients that have already been diagnosed in this city presented the same mutation (p.H178L) in homozygosis. The study was conducted in three stages: in the first was performed in 100 DBS samples an standardization of the techniques; in the second was done a pilot test with samples of newborns of Monte Santo, for the evaluation of standardized techniques and for the thermostability study in healthy controls; in the third were analyzed newborns samples from Monte Santo for both biochemical and molecular methods. Standardization on microplate for fluorimetric enzyme activity of the ARSB showed the assay sensitivity, differentiating values between normal and affected and allowing a reliable detection of patients with MPS VI. On the standardization for molecular analysis in DBS it was possible to differentiate the results for normal individuals, heterozygous and affected for the mutation p.H178L. The preliminary results available indicate that the protocol of neonatal screening for MPS VI developed in this work can be easily incorporated by reference laboratories, contributing to the detection and premature treatment of MPS VI affected patients.

A pesquisa realizada na área do Sítio Arqueológico Célia Maria, localizado na região da Bacia do Rio Santo Anastácio (afluente da Bacia do Paraná), Estado de São Paulo, demonstrou uma ocupação guarani pré-colonial. A cerâmica foi datada por termoluminescência, situando a ocupação em torno de 450±60 anos atrás. Este trabalho traz dados referentes à cerâmica identificada na área do Sítio Célia Maria, que também apresentou três manchas pretas, lítico polido e possível estrutura de combustão. A partir da análise tecno-tipológica da cerâmica, demonstramos a sequência dos gestos técnicos para a produção dos artefatos, assim como reconstituímos graficamente a forma de vasos, a partir de fragmentos de bordas. Essas reconstituições possibilitaram inferir a forma e a função dos antigos recipientes guarani. Os dados espaciais somados às características dos atributos cerâmicos do Sítio Célia Maria foram confrontados com outras pesquisas disponíveis que versaram sobre a presença dos guarani na Bacia do Paraná e Paranapanema. / The research conducted in the archaeological site area Célia Maria, located in the River Basin Santo Anastácio (tributary of the Paraná Basin), State of São Paulo, demonstrated a precolonial Guarani occupation. The pottery was dated by thermoluminescence, standing occupation around 450±60 years ago. This work has data concerning pottery indentified in Site Célia Maria, who also had three black spots, polished lytic and as possible combustion structure. From the techno-typological ceramics analysis, we demonstrated the gestures sequence to the production of technical artifacts, as well as graphically reconstitute the form of vessels, from fragments of edges. These reconstructions allowed inferring the shape and function of the ancient Guarani containers. The data added to the spatial characteristics of ceramics tributes of Site Célia Maria were compared with other available research that focused on the presence of the Guarani in the Basin of Paraná and Paranapanema.