<p>High-latitude watersheds have been regarded as a carbon sink with soil carbon accumulating at low temperature. This sink is now believed to turn into a source, acting as positive feedback to climate warming. However, thawing permafrost soils would allow more water to percolate down to deeper soil layers where some of the carbon could be “consumed” in weathering and exported as bicarbonate to the sea. Using a hydrological mixing model showed that this could counterbalance the predicted positive feedback resulting from thawing soils.</p><p>Vegetation-covered riparian zones in headwater areas appear to have a significant role for the dissolved constituent fluxes. Higher concentrations of weathering products are found in taiga and tundra rivers with larger areas of forest and peat cover in the watershed. These landscape elements can thus be regarded as “hot spots” of river loading with dissolved constituents.</p><p>Comparing a regulated and an unregulated river tested the hypothesis that damming leads to a depletion of major elements also in oligotrophic river systems as a consequence of changes in landscape elements. A loss of upper soils and vegetation through inundation prevents the contact of surface waters with vegetated soil, and consequently reduces weathering fluxes. The hypothesis that the lower fluxes of dissolved silica (DSi) in the regulated river could also be explained by biological uptake was then tested using a model, and budget calculations indicate a significant reduction as a result of regulation. About 10% of this reduction can be attributed to the flooding of the fluvial corridor and the rest to diatom blooms in the reservoirs. A more detailed study of landscape elements for the headwaters of the river Luleälven showed that only 3% of the surface area has been inundated by reservoirs but ca. 37% of the deciduous forest. Such a significant loss of hot spots may indeed explain the observed lower DSi fluxes in the regulated watersheds of northern Sweden.</p>

High-latitude watersheds have been regarded as a carbon sink with soil carbon accumulating at low temperature. This sink is now believed to turn into a source, acting as positive feedback to climate warming. However, thawing permafrost soils would allow more water to percolate down to deeper soil layers where some of the carbon could be “consumed” in weathering and exported as bicarbonate to the sea. Using a hydrological mixing model showed that this could counterbalance the predicted positive feedback resulting from thawing soils. Vegetation-covered riparian zones in headwater areas appear to have a significant role for the dissolved constituent fluxes. Higher concentrations of weathering products are found in taiga and tundra rivers with larger areas of forest and peat cover in the watershed. These landscape elements can thus be regarded as “hot spots” of river loading with dissolved constituents. Comparing a regulated and an unregulated river tested the hypothesis that damming leads to a depletion of major elements also in oligotrophic river systems as a consequence of changes in landscape elements. A loss of upper soils and vegetation through inundation prevents the contact of surface waters with vegetated soil, and consequently reduces weathering fluxes. The hypothesis that the lower fluxes of dissolved silica (DSi) in the regulated river could also be explained by biological uptake was then tested using a model, and budget calculations indicate a significant reduction as a result of regulation. About 10% of this reduction can be attributed to the flooding of the fluvial corridor and the rest to diatom blooms in the reservoirs. A more detailed study of landscape elements for the headwaters of the river Luleälven showed that only 3% of the surface area has been inundated by reservoirs but ca. 37% of the deciduous forest. Such a significant loss of hot spots may indeed explain the observed lower DSi fluxes in the regulated watersheds of northern Sweden.

Bakgrund: Många patienter i palliativ vård rapporterar en minskad, eller förändrad, förmåga att känna smak och lukt. Smak-och luktförändringar (SoL-förändringar) kan innebära lidanden för den enskilde patienten, men kan även påverka näringsintag samt få negativa följder i det dagliga livet. Syfte: Syftet var att rapportera om cancerpatienters erfarenheter av smak- och luktförändringar och påverkan och hantering av dessa i det dagliga livet när de är i palliativ fas. Metod: Kvantitativ data samlades in från åtta deltagare, under tre mättillfällen per deltagare, via ett frågeformulär. Kvalitativ data beståendes av kommentarer till frågorna, redovisades som citat. Resultat: Det var fem huvudfynd som framkom i resultatet. Ett övergripande tema som kunde urskiljas var sårbarheten och lidandet i samband med SoL-förändringar.  Vidare framkom att SoL-förändringar rapporterades av samtliga deltagare, dock inte vid alla mättillfällen. En definitionssvårighet av SoL-förändringar uppmärksammades också bland svaren. Ytterligare var aptitnedsättningar lågt rapporterade bland deltagarna. Detta stod i kontrast till illamående som rapporterades av samtliga deltagare. Diskussion: Sårbarheten och lidandet som uppkom i samband med Sol-förändringar kunde ge en negativ påverkan på det dagliga livet. Drivkraften att tillgodogöra sig föda för sin överlevnad återges med insikten att det ofta inte är för njutningens skull som måltider intas.  Resultatet visade på vikten av att SoL-förändringar uppmärksammas inom den palliativa sjukvården.

The advances of high-throughput technologies, such as next-generation sequencing and microarrays, have rapidly improved the accessibility of molecular profiles in tumor samples. However, due to the immaturity of relevant theories, analyzing these data and systematically understanding the underlying mechanisms causing diseases, which are essential in the development of therapeutic applications, remain challenging. This dissertation attempts to clarify the effects of DNA copy number alterations (CNAs), which are known to be common mutations in genetic diseases, on steady- state gene expression values, time-course expression activities, and the effectiveness of targeted therapy. Assuming DNA copies operate as independent subsystems producing gene transcripts, queueing theory is applied to model the stochastic processes representing the arrival of transcription factors (TFs) and the departure of mRNA. The copy-number-gene-expression relationships are shown to be generally nonlinear. Based on the mRNA production rates of two transcription models, one corresponding to an unlimited state with prolific production and one corresponding to a restrictive state with limited production, the dynamic effects of CNAs on gene expression are analyzed. Simulations reveal that CNAs can alter the amplitudes of transcriptional bursting and transcriptional oscillation, suggesting the capability of CNAs to interfere with the regulatory signaling mechanism. With this finding, a string-structured Bayesian network that models a signaling pathway and incorporates the interference due to CNAs is proposed. Using mathematical induction, the upstream and downstream CNAs are found to have equal influence on drug effectiveness. Scoring functions for the detection of unfavorable CNAs in targeted therapy are consequently proposed. Rigorous experiments are keys to unraveling the etiology of genetic diseases such as cancer, and the proposed models can be applied to provide theory-supporting hypotheses for experimental design.

Thesis (M.Ost.)--Unitec New Zealand, 2008. / Coda (electronic version) title-page has 2009 date. Includes bibliographical references (leaves 38-47).

Introdução: A rede lisossômica é um complexo de vias metabólicas que influenciam processos como degradação de organelas danificadas ou senescentes, processamento de antígenos, reutilização de aminoácidos essenciais e, em última instância, um sistema de fundamental importância para a fisiologia celular normal. Nesse contexto, os lisossomos possuem uma relevância muito grande, uma vez que é nessa organela que ocorre a destruição das moléculas envolvidas em todos os processos citados acima. Entre as unidades operacionais nos lisossomos estão as hidrolases lisossômicas, que são mais de 50 enzimas com capacidade, em ambiente ácido, de realizar a quebra de substratos específicos. A GlcNAc-fosfotransferase é um complexo hexamérico (J2K2L2) residente na porção cis do complexo de Golgi que realiza a adição de resíduos de manose-6- fosfato nas cadeias de oligossacarídeos das hidrolases lisossômicas. Com ação subsequente, a enzima descobridora realiza a remoção da manose, expõe os resíduos de fosfato e possibilita que as hidrolases sejam reconhecidas pelos receptores de manose-6- fosfato e direcionadas aos compartimentos lisossomais. As subunidades J e K da GlcNAc-fosfotransferase são codificadas pelo gene GNPTAB, localizado no cromossomo 12, constituído de 21 éxons, e a subunidade L pelo gene GNPTG, localizado no cromossomo 16, constituído de 11 éxons. Alterações patogênicas em GNPTAB podem causar as doenças Mucolipidose II ou III alfa/beta e alterações em GNPTG causam a doença Mucolipidose III gama. O defeito genético leva à atividade residual, ou nula, da enzima que acaba por gerar o extravasamento das hidrolases lisossômicas ao meio extracelular e o acúmulo de substratos nos lisossomos. Objetivos: (1) caracterizar as alterações patogênicas em GNPTAB em um grupo de pacientes brasileiros não relacionados com Mucolipidose II ou III alfa/beta, e (2) definir um protocolo de pesquisa molecular para os pacientes brasileiros. Metodologia: É um estudo transversal, com amostragem por conveniência, e inclui pacientes com diagnóstico clínico e bioquímico de Mucolipidose II ou III. Foi extraído DNA genômico dos pacientes a partir de sangue obtido por punção venosa periférica. O gene GNPTAB foi sequenciado através da técnica de Sanger. As alterações do tipo troca de sentido foram analisadas pelos programas de Bioinformática Polyphen2, Sift e Consurf, e as preditas como patogênicas foram pesquisadas em alelos controles brasileiros e analisadas por estudos funcionais, quando possível. A geração de construtos das alterações p.Ser385Leu e c.3503_3504delTC foi realizada por mutagênese sítio-dirigida e a atividade residual destes foi avaliada 24 horas após expressão em células HEK. Para a análise financeira, valores atuais de equipamentos, reagente de biologia molecular e materiais plásticos foram utilizados para estimar o custo de uma extração de DNA, reação de PCR, purificação com PEG8000 e sequenciamento. Resultados: Foram incluídos 13 pacientes (ML II= 8; ML III= 5) e, adicionalmente, de uma mãe de paciente com diagnóstico clínico e bioquímico de ML II. A análise molecular identificou seis alterações patogênicas novas, as c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly) e p.Try1111*. A análise de bioinformática das alterações do tipo troca de sentido as caracterizaram como prejudiciais para a função da proteína e os resíduos 76 e 385 como estrutural e funcional, respectivamente, além de ambos como altamente conservados entre as espécies. A análise funcional dos mutantes p.Ser385Leu e c.3503_3504delTC identificaram atividades residuais de 1,5% e nula, respectivamente. Também foram identificadas outras seis alterações patogênicas previamente descritas. A alteração c.3503_3504delTC foi a que apresentou a maior frequência (40%, n= 10/25 alelos), seguido pela p.Ile403The (12%, n=3/25 alelos). Quanto às relações genótipo-fenótipo, sete pacientes com ML II possuem genótipos combinados de alterações do tipo mudança de fase de leitura e sem sentido, enquanto que os cincos pacientes com ML III alfa/beta apresentam pelo menos uma alteração do tipo troca de sentido, o que evidencia a relação entre alterações que impactam a funcionalidade da proteína e fenótipos mais graves. A análise retrospectiva definiu o protocolo 1.0 que finalizaria o diagnóstico com um custo médio de R$ 338,45, em uma amostra de 25 pacientes. A análise prospectiva do protocolo 2.0, sobre a mesma amostra de pacientes, indicou que o mesmo finalizaria o diagnóstico com o custo médio de R$ 299,80, uma economia de 25%. Discussão/Conclusão: As novas alterações patogênicas descritas nesse trabalho confirmam a alta heterogeneidade alélica do gene GNPTAB. A análise funcional da alteração p.Ser385Leu confirma sua patogenicidade, que está de acordo com o fenótipo ML II do paciente, e evidencia a necessidade de mais estudos a fim de constatar o motivo desse resíduo ser importante para a proteína. A síntese dos protocolos demonstrou ser uma estratégia interessante e economicamente importante, uma vez que diminui os gastos envolvidos para finalizar o diagnóstico molecular. / Introduction: The lysosomal network is a complex of metabolic ways that influence processes like damaged or senescent organelles degradation, antigen processing, essential amino acid reutilization, and, in the last instance, it is important for normal cell physiology. In this context, lysosomes have a great relevance since it is in this organelle that the destruction of the molecules involved in all the processes mentioned above occurs. The operational units in the lysosomes are the lysosomal hydrolases that are more than 50 enzymes with capacity, in an acid environment, to breakdown specific substrates. GlcNAc-phosphotransferase is a hexameric complex (J2K2L2) located in the cis portion of the Golgi complex that performs the addition of mannose-6-phosphate residues in oligosaccharides chains on lysosomal hydrolases. In a subsequent way, the uncovering enzyme removes the mannose residues, exposes the phosphate residues, and enables the recognition of hydrolases by mannose-6-phosphate receptors. The J and K subunits are codified by GNPTAB gene, which is located in chromosome 12 and consists of 21 exons, and the L subunit, encoded by GNPTG gene, that is located in chromosome 16 and consists of 11 exons. The consequences of pathogenic alterations in GNPTAB are Mucolipidosis II or III alpha/beta diseases and alterations in the GNPTG are Mucolipidosis III gamma disease. The genetic defect leads to residual or absent activity of enzyme which ultimately generates an overflow of lysosomal hydrolases to the extracellular environment and accumulation of substrates in lysosomes. Objectives: (1) to characterize, by sequencing of the GNPTAB gene, the pathogenic alterations in a group of unrelated Brazilian patients with Mucolipidosis II or III alpha/beta, and (2) to define a molecular research protocol for Brazilian patients. Methodology: It is a crosssectional study with convenience sampling, and it includes patients with biochemical and clinical diagnosis of Mucolipidosis II or III. The DNA was amplified by PCR technique and sequencing by Sanger technique. All patients in the present study had all exons amplified. The missense alterations were analyzed by Polyphen2, Sift, and ConSurf softwares, and the alterations predicted as pathogenic were studied through research in Brazilian control alleles. The p.Ser385Leu and c.3503_3504delTC were evaluated by site-direct mutagenesis and the residual activity was evaluated 24 hours after expression in HEK cells, through radioactive assays. For cost-price analysis, current values for equipment, molecular biology reagents, and plastic materials were utilized to estimate the cost of DNA extraction, PCR reaction, PEG8000 purification, and sequencing. Results: Of the 13 patients, 8 were clinically diagnosed with Mucolipidosis II and 5 with Mucolipidosis III alpha/beta and, additionally, a mother of one patient with biochemical and clinical diagnosis of Mucolipidosis II was also analyzed. The DNA analysis identified six novel pathogenic alterations in GNPTAB: c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly), and p.Tyr1111*. The bioinformatics analysis of missense alterations were characterized as damaging for protein function, and residues 76 and 385 as structural and functional, respectively, and both as highly conserved among the species. The functional analysis of mutants p.Ser385Leu and c.3503_3504delTC showed the residual activity of GlcNAcphosphotransferase of 1.5% and 0%, respectively. Six others pathogenic alterations previously described were also identified. The alteration c.3503_3504delTC showed the highest frequency (40%, n=10/25 alleles) followed by p.Ile403The (12%, n=3/25 alleles). The retrospective analysis defined the 1.0 protocol that finalized the molecular diagnosis at the cost of R$ 338,45 per sample, in a group of 25 patients. The prospective analysis of 2.0 protocol, in the same patients, indicated that it would finalize diagnosis at the cost of R$ 299,80 per sample, a saving of 25%. Discussion/Conclusion: The novel pathogenic alterations described confirm the high allelic heterogeneity of GNPTAB gene. In the genotype-phenotype relationship, 7 patients with Mucolipidosis II have combined genotype of frameshift or nonsense alterations, or both, and 5 Mucolipidosis III alpha/beta patients have at least one missense alteration, that shows the correlation between alterations that cause impact on the protein function and severe phenotype. The functional analysis of alteration p.Ser385Leu confirms its pathogenicity and makes evident the need of more studies in order to determine the reason this residue is so important for protein function. Protocol synthesis proves to be an interesting and economically important strategy, once it decreases costs to conclude the molecular diagnosis.

Schizophrenia is often a debilitating psychiatric disorder, characterised by both positive and negative symptoms, and cognitive and psychosocial impairments. The established disorder has been associated with a number of brain abnormalities, however it is at present unknown whether these brain changes occur prior to onset of schizophrenia, or in unaffected relatives with a familial vulnerability to develop the disorder, or only in those at high risk that go on to develop the disorder. Furthermore, most studies have been conducted cross-sectionally, which may have obscured subtle longitudinal changes in familial high risk individuals, and these studies tend to have focused on localized cortical gray matter , and thus it is unclear whether they affect different cortical parameters differentially. Prospective familial high risk studies utilizing surface based MRI programmes provide a good method to investigate this. In the Edinburgh High Risk Study, structural magnetic resonance imaging (MRI) scans of 150 young individuals at familial high risk of schizophrenia, 34 patients with first-episode schizophrenia and 36 matched controls were obtained. Of the high risk participants with scans suitable for analysis, 17 developed schizophrenia after the scans were taken, whilst 57 experienced isolated or sub-clinical psychotic symptoms, and 70 remained well. We used Freesurfer to extract volumetric and surface-based measurements of several cortical and localized sub-cortical regions with the aim of assessing whether any alterations found were present in all those at high risk, or selectively in the high risk cohort based on future clinical outcome, or only in those experiencing their first-episode of psychosis. It was found that those experiencing their first episode of schizophrenia exhibited significantly more widespread brain alterations compared to those at high risk or controls, both on a more global cortical level and in more localized regions of the cortex, with cortical thickness being generally thinner than in the other groups, and cortical surface area and gyrification increased compared to the other groups. An increased global surface area was also shared with the HR[ill] group, suggesting that this could be a marker that is predictive of future transition to psychosis. Within the high risk cohort, some brain alterations seemed to present as general vulnerability markers, specifically in the temporal lobe at baseline, whilst longitudinally both localized and global cortical alterations distinguished the high risk cohort from the control group, and a different developmental trajectory of the hippocampus was also found. These findings show that some brain alterations may be more accurately characterized as general vulnerability markers of the disorder, whilst some are specifically present in patients who have experienced their first episode of schizophrenia, whilst some also occur before disorder onset in those at high risk that go on to develop schizophrenia. The findings have some clinical implications, as they suggest that it is possible to assess who at high risk will go on to develop schizophrenia based on brain structural alterations. This may provide clinicians with an early window of opportunity for intervention, as it has been found that early intervention may improve patient's prognosis. The findings also have important implications for the understanding of the underlying eitology of schizophrenia, as they suggest that some of these alterations are present before illness onset, and not associated with medication effects, thus potentially lying on the causal path of developing schizophrenia.

O quimioterápico doxorrubicina, pertencente à família das antraciclinas, é um dos agentes antineoplásicos mais utilizados na medicina veterinária, no tratamento de uma variedade de sarcomas e carcinomas. No entanto, o uso da doxorrubicina está associado à presença de toxicidade, envolvendo principalmente anormalidades hematológicas, gastrointestinais e cardiovasculares, podendo induzir uma cardiomiopatia irreversível. O objetivo deste trabalho foi avaliar estas manifestações tóxicas, bem como as alterações hematológicas, bioquímicas e eletrocardiográficas em 25 cães com diferentes neoplasias tratados com doxorrubicina (30 mg/m² a cada 21 dias por três doses). Além disso, buscou-se avaliar as troponinas cardíacas I e T como marcadores cardíacos para a detecção precoce de cardiomiopatia induzida pela quimioterapia. Amostras de sangue foram coletadas antes de cada administração de doxorrubicina, sete dias após (nadir) e um mês depois do término do tratamento. As toxicidades hematológicas e gastrointestinais foram as alterações mais frequentes, ocorrendo em 22 (88%) e 19 (76%) cães, respectivamente. Neutropenia severa ocorreu em 25% dos casos, enquanto que não foram observados casos de trombocitopenia severa. Os sinais mais comumente observados foram vômito, anorexia e diarréia, os quais foram responsáveis, em parte, pelo aparecimento de hipoalbuminemia e reduções nas concentrações séricas de potássio, cálcio e proteína total. Observou-se também aumento nas concentrações séricas de proteína C reativa o que, assim como a diminuição da albumina, deve ser consequência de uma resposta de fase aguda induzida pela administração da doxorrubicina. Sepse foi incomum, ocorrendo em menos de 2% dos casos, e nenhum animal veio a óbito por complicações da quimioterapia. Sinais clínicos de cardiomiopatia não foram observados, porém aumentos nas troponinas cardíacas I e T ocorreram em 18 (72%) e 5 (20%) cães, respectivamente. Todos os cães com altas concentrações de troponina cardíaca T também apresentavam altas concentrações de troponina cardíaca I. Arritmias foram detectadas em 15 (60%) animais, mas não houve correlação com o aumento das troponinas cardíacas séricas e a presença de arritmias. No entanto, os cães que desenvolveram complexos atriais e ventriculares prematuros no eletrocardiograma apresentaram altas concentrações das troponinas. Este estudo demonstrou a ocorrência de alterações hematológicas e gastrointestinais toleráveis em cães recebendo doxorrubicina como único agente quimioterápico. Além disso, as concentrações séricas de troponinas cardíacas I e T podem ser utilizadas na detecção precoce de lesão miocárdica induzida pela doxorrubicina em cães. / The anthracicline chemotherapic doxorubicin is one of the anticancer agents most used in veterinary medicine to treat a variety of sarcomas and carcinomas. However, doxorubicin is associated with several toxicities, including hematological, gastrointestinal and cardiac abnormalities, and it can induce cardiomyopathy in dogs. The purpose of this study was to evaluate these toxic manifestations, as well as hematological, biochemical and electrocardiographic alterations in 25 dogs with spontaneously occurring malignant tumors treated with doxorubicin (30 mg/m² each 21 days during three doses). Moreover, cardiac troponins I (cTnI) and T (cTnT) were evaluated to determine cardiac-specific markers for predict cardiomyopathy-induced by chemotherapy. Blood samples were collected before each doxorubicin administration, seven days after each dose (nadir) and one month after the end of therapy. Hematological and gastrointestinal toxicities were the most frequent abnormalities observed and occurred in 22 (88%) and 19 (76%) dogs, respectively. Severe neutropenia occurred in 25% of the animals, while severe thrombocytopenia was not observed. Vomiting, anorexia and diarrhea occurred frequently, and were responsible, in part, for the presence of hypoalbuminemia and decreases in potassium, calcium and total protein serum concentrations. Increases in C reactive protein were also observed, wich together with decreases in albumin, may be due to acute phase response induced by doxorubicin. The frequency of sepsis was less than 2% and any animal died because of therapy complications. Signs of cardiomyopathy were not observed, however, increases in serum concentrations of cTnI and cTnT were found in 18 (72%) and 5 (20%) dogs, respectively. All dogs with high cTnT had high cTnI concentrations. Arrhythmias were detected in 15 (60%) animals, and it was not correlated with increases in cardiac troponins. Those dogs that developed atrial and ventricular premature complexes in the electrocardiogram showed increases in cTnI concentrations. In conclusion, doxorubicin used as single agent produced tolerable hematological and gastrointestinal toxicosis. Furthermore, cTnI and cTnT measurement can be used for early detection of myocardial injury caused by doxorubicin chemotherapy in dogs.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-30T18:02:27Z No. of bitstreams: 1 Marcelo Magalhaes Silva Estudo de alterações genéticas....pdf: 2168493 bytes, checksum: 48c50f165408a4314aa5ad8de3c72ca0 (MD5) / Made available in DSpace on 2012-07-30T18:02:27Z (GMT). No. of bitstreams: 1 Marcelo Magalhaes Silva Estudo de alterações genéticas....pdf: 2168493 bytes, checksum: 48c50f165408a4314aa5ad8de3c72ca0 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A leucemia/linfoma de células T do adulto (ATL) é uma severa doença linfoproliferativa de células T CD4+ associada ao HTLV-1. Por apresentar diferentes manifestações clínicas, essa neoplasia pode ser classificada em cinco formas: aguda, crônica, smoldering, linfomatosa e tumoral primária de pele. Embora alguns trabalhos venham estudando o processo oncogênico mediado pelo HTLV-1, diversos fatores responsáveis pelo desenvolvimento da ATL ainda permanecem desconhecidos. Este estudo teve como objetivo a investigação de alterações genéticas em células ATL (mutações pontuais em genes supressores de tumor e alterações microssatélites) e sua associação com a evolução clínica da doença e sobrevida dos pacientes. A presença de mutações pontuais nos supressores de tumor TP53, p15INK4B e p16INK4A foram avaliadas em 31 pacientes com diferentes formas da ATL (16 agudos, dez crônicos e cinco smoldering) por análise de seqüenciamento de DNA. Cinco pacientes (16%) apresentaram mutações pontuais no gene TP53, sendo que quatro dentre os mesmos foram classificados com a forma aguda. A presença de mutações nos genes avaliados foi associada com pior prognóstico em pacientes com a forma aguda. Em um dos pacientes incluídos neste trabalho (forma aguda) foi verificada a presença de alteração no éxon 2 do gene p16INK4A. Mutações pontuais não foram detectadas no gene p15INK4B em nenhum dos pacientes incluídos. Os marcadores D10S190, D10S191, D11S1391 e D18S21 foram utilizados para a análise de alterações microssatélites por metodologia semi-automatizada. Dentre os 25 pacientes ATL avaliados (seis agudos, oito crônicos, dez smoldering e um linfomatoso), sete apresentaram alterações microssatélites. Três desses pacientes apresentaram instabilidade (MSI), três pacientes apresentaram perda de heterozigosidade (LOH) e em um paciente foi verificado ambas as alterações. Na Bahia, mutações pontuais em TP53 foram detectadas principalmente na forma aguda da ATL e parece estar associada com pior prognóstico. Além disso, de acordo com nossos conhecimentos, este é o primeiro estudo a descrever tanto MSI com LOH em pacientes portando formas crônica e smoldering da ATL. / Adult T-cell leukemia/lymphoma (ATL) is a severe CD4+ lymphoproliferative disease associated to human T-cell lymphotropic virus type 1 (HTLV-1). ATL has different clinical manifestations and is classified in five clinical forms: acute, chronic, smoldering, lymphoma and primary cutaneous tumoral. Although the mechanisms of oncogenesis of the HTLV-1 have been investigated, the factors related to ATL development are still unknown. The goal of this study was to investigate genetic alterations in ATL cells (point mutations in tumor suppressor genes and microsatellite alterations) and their association with the clinical evolution of the disease and survival. The presence of point mutations in TP53, p15INK4B e p16INK4A were evaluated in 31 ATL patients (16 acute, ten chronic and five smoldering) by direct sequencing. Five of them (16%) had TP53 point mutations, four of them with the acute form of ATL. The presence of point mutations in this gene was associated to poor prognosis in acute patients. Only one case (acute form) has an alteration in the exon 2 of the p16INK4A gene. No point mutations of the p15INK4B were found in the patients included. The markers D10S190, D10S191, D11S1391 and D18S21 were considered for the microsatellite analysis using a semiautomated technique. From the twenty-five ATL cases included (six acute, eight chronic, ten smoldering and one lymphoma), seven showed microsatellite alteration. Among them, three patients had microsatellite instability (MSI), three had loss of heterozygosity and one patient presented both alterations. In Bahia, point mutations in TP53 were detected mainly in acute form of ATL and were associated to poor prognosis. The presence of MSI and LOH in the smoldering and chronic forms of ATL were demonstrated for the first time in this study.

Introdução: A rede lisossômica é um complexo de vias metabólicas que influenciam processos como degradação de organelas danificadas ou senescentes, processamento de antígenos, reutilização de aminoácidos essenciais e, em última instância, um sistema de fundamental importância para a fisiologia celular normal. Nesse contexto, os lisossomos possuem uma relevância muito grande, uma vez que é nessa organela que ocorre a destruição das moléculas envolvidas em todos os processos citados acima. Entre as unidades operacionais nos lisossomos estão as hidrolases lisossômicas, que são mais de 50 enzimas com capacidade, em ambiente ácido, de realizar a quebra de substratos específicos. A GlcNAc-fosfotransferase é um complexo hexamérico (J2K2L2) residente na porção cis do complexo de Golgi que realiza a adição de resíduos de manose-6- fosfato nas cadeias de oligossacarídeos das hidrolases lisossômicas. Com ação subsequente, a enzima descobridora realiza a remoção da manose, expõe os resíduos de fosfato e possibilita que as hidrolases sejam reconhecidas pelos receptores de manose-6- fosfato e direcionadas aos compartimentos lisossomais. As subunidades J e K da GlcNAc-fosfotransferase são codificadas pelo gene GNPTAB, localizado no cromossomo 12, constituído de 21 éxons, e a subunidade L pelo gene GNPTG, localizado no cromossomo 16, constituído de 11 éxons. Alterações patogênicas em GNPTAB podem causar as doenças Mucolipidose II ou III alfa/beta e alterações em GNPTG causam a doença Mucolipidose III gama. O defeito genético leva à atividade residual, ou nula, da enzima que acaba por gerar o extravasamento das hidrolases lisossômicas ao meio extracelular e o acúmulo de substratos nos lisossomos. Objetivos: (1) caracterizar as alterações patogênicas em GNPTAB em um grupo de pacientes brasileiros não relacionados com Mucolipidose II ou III alfa/beta, e (2) definir um protocolo de pesquisa molecular para os pacientes brasileiros. Metodologia: É um estudo transversal, com amostragem por conveniência, e inclui pacientes com diagnóstico clínico e bioquímico de Mucolipidose II ou III. Foi extraído DNA genômico dos pacientes a partir de sangue obtido por punção venosa periférica. O gene GNPTAB foi sequenciado através da técnica de Sanger. As alterações do tipo troca de sentido foram analisadas pelos programas de Bioinformática Polyphen2, Sift e Consurf, e as preditas como patogênicas foram pesquisadas em alelos controles brasileiros e analisadas por estudos funcionais, quando possível. A geração de construtos das alterações p.Ser385Leu e c.3503_3504delTC foi realizada por mutagênese sítio-dirigida e a atividade residual destes foi avaliada 24 horas após expressão em células HEK. Para a análise financeira, valores atuais de equipamentos, reagente de biologia molecular e materiais plásticos foram utilizados para estimar o custo de uma extração de DNA, reação de PCR, purificação com PEG8000 e sequenciamento. Resultados: Foram incluídos 13 pacientes (ML II= 8; ML III= 5) e, adicionalmente, de uma mãe de paciente com diagnóstico clínico e bioquímico de ML II. A análise molecular identificou seis alterações patogênicas novas, as c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly) e p.Try1111*. A análise de bioinformática das alterações do tipo troca de sentido as caracterizaram como prejudiciais para a função da proteína e os resíduos 76 e 385 como estrutural e funcional, respectivamente, além de ambos como altamente conservados entre as espécies. A análise funcional dos mutantes p.Ser385Leu e c.3503_3504delTC identificaram atividades residuais de 1,5% e nula, respectivamente. Também foram identificadas outras seis alterações patogênicas previamente descritas. A alteração c.3503_3504delTC foi a que apresentou a maior frequência (40%, n= 10/25 alelos), seguido pela p.Ile403The (12%, n=3/25 alelos). Quanto às relações genótipo-fenótipo, sete pacientes com ML II possuem genótipos combinados de alterações do tipo mudança de fase de leitura e sem sentido, enquanto que os cincos pacientes com ML III alfa/beta apresentam pelo menos uma alteração do tipo troca de sentido, o que evidencia a relação entre alterações que impactam a funcionalidade da proteína e fenótipos mais graves. A análise retrospectiva definiu o protocolo 1.0 que finalizaria o diagnóstico com um custo médio de R$ 338,45, em uma amostra de 25 pacientes. A análise prospectiva do protocolo 2.0, sobre a mesma amostra de pacientes, indicou que o mesmo finalizaria o diagnóstico com o custo médio de R$ 299,80, uma economia de 25%. Discussão/Conclusão: As novas alterações patogênicas descritas nesse trabalho confirmam a alta heterogeneidade alélica do gene GNPTAB. A análise funcional da alteração p.Ser385Leu confirma sua patogenicidade, que está de acordo com o fenótipo ML II do paciente, e evidencia a necessidade de mais estudos a fim de constatar o motivo desse resíduo ser importante para a proteína. A síntese dos protocolos demonstrou ser uma estratégia interessante e economicamente importante, uma vez que diminui os gastos envolvidos para finalizar o diagnóstico molecular. / Introduction: The lysosomal network is a complex of metabolic ways that influence processes like damaged or senescent organelles degradation, antigen processing, essential amino acid reutilization, and, in the last instance, it is important for normal cell physiology. In this context, lysosomes have a great relevance since it is in this organelle that the destruction of the molecules involved in all the processes mentioned above occurs. The operational units in the lysosomes are the lysosomal hydrolases that are more than 50 enzymes with capacity, in an acid environment, to breakdown specific substrates. GlcNAc-phosphotransferase is a hexameric complex (J2K2L2) located in the cis portion of the Golgi complex that performs the addition of mannose-6-phosphate residues in oligosaccharides chains on lysosomal hydrolases. In a subsequent way, the uncovering enzyme removes the mannose residues, exposes the phosphate residues, and enables the recognition of hydrolases by mannose-6-phosphate receptors. The J and K subunits are codified by GNPTAB gene, which is located in chromosome 12 and consists of 21 exons, and the L subunit, encoded by GNPTG gene, that is located in chromosome 16 and consists of 11 exons. The consequences of pathogenic alterations in GNPTAB are Mucolipidosis II or III alpha/beta diseases and alterations in the GNPTG are Mucolipidosis III gamma disease. The genetic defect leads to residual or absent activity of enzyme which ultimately generates an overflow of lysosomal hydrolases to the extracellular environment and accumulation of substrates in lysosomes. Objectives: (1) to characterize, by sequencing of the GNPTAB gene, the pathogenic alterations in a group of unrelated Brazilian patients with Mucolipidosis II or III alpha/beta, and (2) to define a molecular research protocol for Brazilian patients. Methodology: It is a crosssectional study with convenience sampling, and it includes patients with biochemical and clinical diagnosis of Mucolipidosis II or III. The DNA was amplified by PCR technique and sequencing by Sanger technique. All patients in the present study had all exons amplified. The missense alterations were analyzed by Polyphen2, Sift, and ConSurf softwares, and the alterations predicted as pathogenic were studied through research in Brazilian control alleles. The p.Ser385Leu and c.3503_3504delTC were evaluated by site-direct mutagenesis and the residual activity was evaluated 24 hours after expression in HEK cells, through radioactive assays. For cost-price analysis, current values for equipment, molecular biology reagents, and plastic materials were utilized to estimate the cost of DNA extraction, PCR reaction, PEG8000 purification, and sequencing. Results: Of the 13 patients, 8 were clinically diagnosed with Mucolipidosis II and 5 with Mucolipidosis III alpha/beta and, additionally, a mother of one patient with biochemical and clinical diagnosis of Mucolipidosis II was also analyzed. The DNA analysis identified six novel pathogenic alterations in GNPTAB: c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly), and p.Tyr1111*. The bioinformatics analysis of missense alterations were characterized as damaging for protein function, and residues 76 and 385 as structural and functional, respectively, and both as highly conserved among the species. The functional analysis of mutants p.Ser385Leu and c.3503_3504delTC showed the residual activity of GlcNAcphosphotransferase of 1.5% and 0%, respectively. Six others pathogenic alterations previously described were also identified. The alteration c.3503_3504delTC showed the highest frequency (40%, n=10/25 alleles) followed by p.Ile403The (12%, n=3/25 alleles). The retrospective analysis defined the 1.0 protocol that finalized the molecular diagnosis at the cost of R$ 338,45 per sample, in a group of 25 patients. The prospective analysis of 2.0 protocol, in the same patients, indicated that it would finalize diagnosis at the cost of R$ 299,80 per sample, a saving of 25%. Discussion/Conclusion: The novel pathogenic alterations described confirm the high allelic heterogeneity of GNPTAB gene. In the genotype-phenotype relationship, 7 patients with Mucolipidosis II have combined genotype of frameshift or nonsense alterations, or both, and 5 Mucolipidosis III alpha/beta patients have at least one missense alteration, that shows the correlation between alterations that cause impact on the protein function and severe phenotype. The functional analysis of alteration p.Ser385Leu confirms its pathogenicity and makes evident the need of more studies in order to determine the reason this residue is so important for protein function. Protocol synthesis proves to be an interesting and economically important strategy, once it decreases costs to conclude the molecular diagnosis.