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Dissertation to obtain the degree of Master in Chemical and Biochemical Engineering

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Complex systems, i.e. systems composed of a large set of elements interacting in a non-linear way, are constantly found all around us. In the last decades, different approaches have been proposed toward their understanding, one of the most interesting being the Complex Network perspective. This legacy of the 18th century mathematical concepts proposed by Leonhard Euler is still current, and more and more relevant in real-world problems. In recent years, it has been demonstrated that network-based representations can yield relevant knowledge about complex systems. In spite of that, several problems have been detected, mainly related to the degree of subjectivity involved in the creation and evaluation of such network structures. In this Thesis, we propose addressing these problems by means of different data mining techniques, thus obtaining a novel hybrid approximation intermingling complex networks and data mining. Results indicate that such techniques can be effectively used to i) enable the creation of novel network representations, ii) reduce the dimensionality of analyzed systems by pre-selecting the most important elements, iii) describe complex networks, and iv) assist in the analysis of different network topologies. The soundness of such approach is validated through different validation cases drawn from actual biomedical problems, e.g. the diagnosis of cancer from tissue analysis, or the study of the dynamics of the brain under different neurological disorders.

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The role of sexual or intrafamilial transmission of hepatitis C is controversial. A phylogenetic analysis was performed on the non-structural region 5B of the hepatitis C virus (NS5B-HCV). High percentages of homology (mean of 98.3%) were shown between the couples. Twenty (83.3%) of the 24 men but only two of the women (8.3%) reported having had sexually transmitted diseases during their lives. The risk factors for HCV acquisition were blood transfusion (10 couples), use of illegal injected drugs (17), use of inhalants (15), acupuncture (5) and tattoos (5). The shared use of personal hygiene items included toothbrushes between six couples (25%), razor blades between 16 (66.7%), nail clippers between 21 (87.5%) and manicure pliers between 14 (58.3%). The high degree of similarity of the hepatitis C virus genome supports the hypothesis of hepatitis C virus transmission between these couples. The shared use of personal hygiene items suggests the possibility of intrafamilial transmission of infection.

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RESUMO:O glicosilfosfatidilinositol (GPI) é um complexo glicolipídico utlizado por dezenas de proteínas, o qual medeia a sua ancoragem à superfície da célula. Proteínas de superfície celular ancoradas a GPI apresentam várias funções essenciais para a manutenção celular. A deficiência na síntese de GPI é o que caracteriza principalmente a deficiência hereditária em GPI, um grupo de doenças autossómicas raras que resultam de mutações nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensáveis para a biossíntese do GPI. Uma mutação pontual no motivo rico em GC -270 no promotor de PIGM impede a ligação do factor de transcrição (FT) Sp1 à sua sequência de reconhecimento, impondo a compactação da cromatina, associada à hipoacetilação de histonas, e consequentemente, impedindo a transcrição de PIGM. Desta forma, a adição da primeira manose ao GPI é comprometida, a síntese de GPI diminui assim como as proteínas ligadas a GPI à superficie das células. Pacientes com Deficiência Hereditária em GPI-associada a PIGM apresentam trombose e epilesia, e ausência de hemólise intravascular e anemia, sendo que estas duas últimas características definem a Hemoglobinúria Paroxística Nocturna (HPN), uma doença rara causada por mutações no gene PIGA. Embora a mutação que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficiência em GPI varia entre células do mesmo tecido e entre células de tecidos diferentes. Por exemplo nos granulócitos e linfócitos B a deficiência em GPI é muito acentuada mas nos linfócitos T, fibroblastos, plaquetas e eritrócitos é aproximadamente normal, daí a ausência de hemólise intravascular. Os eventos transcricionais que estão na base da expressão diferencial da âncora GPI nas células hematopoiéticas são desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os níveis de PIGM mRNA variam entre células primárias hematopoiéticas normais. Adicionalmente, a configuração dos nucleossomas no promotor de PIGM é mais compacta em células B do que em células eritróides e tal está correlacionado com os níveis de expressão de PIGM, isto é, inferior nas células B. A presença de vários motivos de ligação para o FT específico da linhagem megacariocítica-eritróide GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrição. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expressão de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrição restritamente eritróide, regula a transcrição de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigação do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrição de PIGM em ambas as células B e eritróide. Curiosamente, ao contrário do que acontece nas células B, em que a transcrição de PIGM requer a ligação do FT geral Sp1 ao motivo -270GC, nas células eritróides Sp1 regula a transcrição de PIGM ao ligar-se a montante e não ao motivo -270GC. Para além disso, demonstrou-se que Sp2 não é um regulador directo da transcrição de PIGM quer nas células B quer nas células eritróides. Estes resultados explicam a ausência de hemólise intravascular nos doentes com IGD associada a PIGM, uma das principais características que define a HPN. Por último, resultados preliminares mostraram que a repressão da transcrição de PIGM devida à mutação patogénica -270C>G está associada com a diminuição da frequência de interacções genómicas em cis entre PIGM e os seus genes “vizinhos”, sugerindo adicionalmente que a regulação de PIGM e desses genes é partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, específico-detecido, por meio de FTs genéricos e específicos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.

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Tissue engineering arises from the need to regenerate organs and tissues, requiring the development of scaffolds, which can provide an optimum environment for tissue growth. In this work, chitosan with different molecular weights was used to develop biodegradable 3D inverted colloidal crystals (ICC) structures for bone regeneration, exhibiting uniform pore size and interconnected network. Moreover, in vitro tests were conducted by studying the influence of the molecular weight in the degradation kinetics and mechanical properties. The production of ICC included four major stages: fabrication of microspheres; assembly into a cohesive structure, polymeric solution infiltration and microsphere removal. Chitosan’s degree of deacetylation was determined by infrared spectroscopy and molecular weight was obtained via capillary viscometry. In order to understand the effect of the molecular weight in ICC structures, the mass loss and mechanical properties were analyzed after degradation with lysozyme. Structure morphology observation before and after degradation was performed by scanning electron microscopy. Cellular adhesion and proliferation tests were carried out to evaluate ICC in vitro response. Overall, medium molecular weight ICC revealed the best balance in terms of mechanical properties, degradation rate, morphology and biological behaviour.

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INTRODUCTION: Listeria monocytogenes is a ubiquitous microorganism in nature and is responsible for listeriosis, an infectious disease caused by consumption of contaminated food. METHODS: Molecular characterization was performed on 19 strains of Listeria monocytogenes (serovars 1/2a, 1/2b, 4b and 4c), isolated from dairy products in Rio Grande do Sul, Brazil. The molecular techniques applied were random amplification of polymorphic DNA and restriction enzyme analysis. In addition to the molecular analysis, the antimicrobial resistance profile was determined. RESULTS: The strains studied showed a low degree of diversity. In relation to the antimicrobial resistance profile of those microorganisms from the samples analyzed, all of them were susceptible to the antimicrobials tested. CONCLUSIONS: The molecular techniques that were used presented good discriminatory power for the strains studied. Furthermore, all of the samples that were analyzed were susceptible to the antimicrobials tested.

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INTRODUCTION: Hepatitis B is common in Brazil, although there are regional differences regarding the degree of endemicity, the most frequent forms of transmission and the presence of different evolutive stages of chronic disease. The present study aimed to determine the clinical, demographic and epidemiological characteristics of patients chronically infected with hepatitis B virus (HBV) residing in the Ribeirão Preto region, southeastern Brazil. METHODS: A total of 529 medical records of individuals with HBV monoinfection were reviewed. RESULTS: More than 60% of the subjects were males, with a mean age of 38 years-old. The HBeAg-negative serological pattern was verified in 84.4% of the patients, among whom the risk of vertical/intrafamily transmission was 43.2% (p = 0.02). The consumption of alcohol in amounts exceeding 20g a day was observed in 21.3% of the subjects and was more frequent among men (33%) (p < 0.001). Among patients with cirrhosis, 54.1% were alcohol abusers (p = 0.04), all of them males. The presence of cirrhosis was more frequent in the HBeAg-positive group (24.4%) than in the HBeAg-negative group (10.2%) (p < 0.001). CONCLUSIONS: High proportions of HBV-infected subjects with an HBeAg-negative pattern were observed, with a higher risk of vertical/intrafamily transmission. Alcohol abuse was associated with male subjects and with cirrhosis of the liver in this group. A tendency toward an increase in the number of HBeAg-negative cases was observed over time.

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This Work Project investigates the determinants of reelection using data on the 278 Portuguese mainland municipalities for the period 1976-2009. We implement a logit fixed effect model to control for the municipalities’ unobserved characteristics that remain constant over time. Political variables, such as the vote share of the incumbent’s party in previous election, the number of mayor’s consecutive mandates and abstention rate, are found to be relevant in explaining incumbent’s reelection. Moreover, as to the mayor’s individual characteristics, age and education contribute to explain reelection prospects. We also provide weak evidence that a higher degree of fiscal autonomy increases political turnover and that the good economic prospects of the municipality positively affect reelection. Finally, the residents’ level of education and the size of the municipal population have an explanatory power on mayor’s reelection. We perform several robustness checks to confirm these results.

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INTRODUCTION: The aim of the present study was to identify the presence of Leishmania (Leishmania) chagasi infection in dogs in the City of Palmas, Tocantins, Brazil, using the PCR technique to list the hot spots of infected dogs in the city and associate their occurrence to significant environmental changes at capture sites. METHODS: DNA was extracted from blood of dogs, and the PCR were performed with primers RV1/RV2. After screening the population studied, the regions of the city that had the highest occurrence of canine infection were detected. These sites were visited, and ecological parameters denoting anthropogenic disturbance were evaluated. RESULTS: Some important features were listed in the regions visited, such as low urbanization, lack of public collection of sewage, limited garbage collection, vacant lots with tall vegetation, decaying organic matter, and, most importantly, the occurrence of stray dogs and poultry in homes. CONCLUSIONS: The methodology for screening the population was very efficient, especially in evaluating a large number of individuals in a short time, with a high degree of automation. The results indicate an association between the observed parameters and the occurrence of infection in dogs. The model presented in the city is ideal for studies of disease progression and expansion and for the evaluation of control measures adopted for canine VL.

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INTRODUCTION: Since the emergence of antiretroviral therapy, the survival of patients infected with human immunodeficiency virus has increased. Non-adherence to this therapy is directly related to treatment failure, which allows the emergence of resistant viral strains. METHODS: A retrospective descriptive study of the antiretroviral dispensing records of 229 patients from the Center for Health Care, University Hospital, Federal University of Juiz de Fora, Brazil, was conducted between January and December 2009. RESULTS: The study aimed to evaluate patient compliance and determine if there was an association between non-adherence and the therapy. Among these patients, 63.8% were men with an average age of 44.0 ± 9.9 years. The most used treatment was a combination of 2 nucleoside reverse transcriptase inhibitors with 1 non-nucleoside reverse transcriptase inhibitor (55.5%) or with 2 protease inhibitors (28.8%). It was found that patients taking lopinavir/ritonavir with zidovudine and lamivudine had a greater frequency of inadequate treatment than those taking atazanavir with zidovudine and lamivudine (85% and 83.3%, respectively). Moreover, when the combination of zidovudine/ lamivudine was used, the patients were less compliant (χ2 = 4.468, 1 degree of freedom, p = 0.035). CONCLUSIONS: The majority of patients failed to correctly adhere to their treatment; therefore, it is necessary to implement strategies that lead to improved compliance, thus ensuring therapeutic efficacy and increased patient survival.

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RESUMO: Background: Problemas de saúde mental são um grande problema clínico e social na República da Moldávia, representando uma quota significante de deficiência, sendo classificada no top cinco das dez linhas na hierarquia das condições. A taxa de incidência tem sido crescente na República da Moldávia, atingindo cerca de 15.000 por ano (14,655 em 2011), ou seja, 411,4 por 100 mil habitantes, e uma taxa de prevalência de 97.525 pessoas em 2011, ou seja, 2,737.9 por 100 mil habitantes. Sistema de atendimento psiquiátrico fornece serviços de saúde mental escassos a nível da comunidade, visando principalmente terapia hospitalar, centralizada, através de uma rede de três hospitais psiquiátricos, com 1.860 camas e 4 sanatórios psico- neurológicos com 1890 camas, assim alimentando-se a estigmatização do paciente. Objetivos: O objetivo deste estudo foi a avaliação das necessidades individuais dos beneficiários e do seu nível de autonomia dentro de cuidados residenciais, para o planeamento de reformas de saúde mental e desinstitucionalização na República da Moldávia. Este estudo foi encomendado pelo Ministério do Trabalho, Proteção Social e da Família e pelo Ministério da Saúde, com o apoio da Organização Mundial da Saúde, para determinar o cumprimento eficaz do artigo 19 da Convenção da ONU. O estudo tem os seguintes objetivos: Avaliar o nível de autonomia dos residentes nos hospitais psiquiátricos e sanatórios psico-neurológico, usando uma amostra representativa de 10 por ce nto do número total de pacientes/residentes e comparação cruzada; Para avaliar quatro sanatórios psico-neurológicos para adultos e três hospitais psiquiátricos; Para desenvolver recomendações para o planeamento da desinstitucionalização das pessoas com problemas de saúde mental e colocação na comunidade com base nos resultados do estudo. Metodologia e resultados: O estudo fez uso de duas ferramentas globais: questionário para a avaliação individual dos residentes do estabelecimento de saúde mental, e questionário de avaliação institucional. Todos os entrevistados foram divididos em quatro categorias conforme com o grau de dependência e preparação de viver de forma independente na comunidade. Apenas 1,2% dos entrevistados de PNHB eram totalmente dependentes de terceiros ou serviços especializados, tornando-se a categoria 4, que necessitam de cuidados e apoio contínuo. No PH esta categoria de pessoas é ausente. Conclusões: A condição dos entrevistados foi pior em PNBH que em PH. No entanto, ainda, aqueles que estão prontos para ser desinstitucionalizados correspondem com a maior parte dos entrevistados. Todos os hospitais tinham o consentimento do utente para admissão e tratamento, enquanto não houve consentimento qualquer em PNBH. É bastante óbvio que tanto os hospitais como também a sistema de assistência residencial não atingem a sua finalidade, o que significa que a maioria dos utentes pode ser desinstitucionalizados, sem qualquer terapia de suporte.------------------ABSTRACT: Background: Mental health problems are a major clinical and social issue in the Republic of Moldova,accounting for a significant share of disability and ranking in top five of the ten lines in the hierarchy of conditions. The incidence rate has been growing in the Republic of Moldova to reach approximately 15 thousand a year (14,655 in 2011), i.e. 411.4 per 100 thousand population, and a prevalence rate of 97,525 thousand people in 2011, i.e. 2,737.9 per 100 thousand population. Psychiatric care system provides for scanty mental health services at community level, aiming mainly at centralized hospital-based therapy through a network of three psychiatric hospitals tallying up 1,860 beds and 4 psycho-neurological boarding houses with 1,890 beds, thus fuelling up patient stigmatization. Objectives: The purpose of this study was to assess the individual needs of beneficiaries and their level of autonomy within residential care for the planning of mental health system reforms and deinstitutionalization in the Republic of Moldova. This study was commissioned by the Ministry of Labour, Social Protection and Family and by the Ministry of Health, with the World Health Organization support, to provide for effective enforcement of article 19 of the UN CRPD. The study pursued the following goals: To evaluate the level of autonomy of the psychiatric hospital and psycho-neurological boarding house residents by using a representative sample of 10 per cent of the total number of patients / residents and cross-comparison; To evaluate four psycho-neurological boarding houses for adults and three psychiatric hospitals; To develop recommendations for planning the deinstitutionalization of people with mental health problems and community placement based on the study findings.Methodology and results: The study made use of two global tools: questionnaire for individual assessment of mental health facility residents, and institutional assessment questionnaire. All interviewees were divided into four categories by one’s degree of dependence and readiness to live independently in the community. Only 1.2% of respondents from PNHB were fully dependent on a third party or specialized services, making up category 4, requiring continuous care and support. In PH this category of people is absent.Conclusions: The condition of respondents was worse in PNBH than in PH. However, yet, those ready to be deinstitutionalized accounted for most of respondents there. All hospitals had the resident’s consent to admission and treatment, whereas there was no consent in PNBH whatsoever. It is quite obvious that both the hospitals and residential care system do not achieve their intended purpose, meaning that the majority of residents may be deinstitutionalized without any support therapy.

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INTRODUCTION:The need for studies that describe the resistance patterns in populations of Aedes aegypti (Linnaeus) in function of their region of origin justified this research, which aimed to characterize the resistance to temephos and to obtain information on esterase activity in populations of Aedes aegypticollected in municipalities of the State of Paraíba.METHODS:Resistance to temephos was evaluated and characterized from the diagnostic dose of 0.352mg i.a./L and multiple concentrations that caused mortalities between 5% and 99%. Electrophoresis of isoenzymes was used to verify the patterns of esterase activity among populations of the vector.RESULTS:All populations of Aedes aegypti were resistant to temephos, presenting a resistance rate (RR) greater than 20. The greatest lethal dose 50% of the sample (CL50) was found for the municipality of Lagoa Seca, approximately forty-one times the value of CL50 for the Rockefeller population. The populations characterized as resistant showed two to six regions of α and β-esterase, called EST-1 to EST-6, while the susceptible population was only seen in one region of activity.CONCLUSIONS:Aedes aegyptiis widely distributed and shows a high degree of resistance to temephos in all municipalities studied. In all cases, esterases are involved in the metabolism and, consequently, in the resistance to temephos.

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Strong consolidation is one of the most evident developments of banking markets around the world in recent decades. This change is raising questions on how and to what an extent competition is affected by the expansion of the largest banks. The aim of the present study is to measure the degree of competition in the Portuguese commercial banking market in the long-run, during the period ranging from1960 to 2013, by using the non-structural model developed by Panzar and Rosse. The main findings are that the Portuguese banking system, despite the legal restrictions in place, operated mostly in a market with some degree of competition and, at some points in time, presented some interesting competitive features. More recently, it has evolved into functioning as a cartel.

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INTRODUCTION: The present study investigated the prevalence of two single-nucleotide polymorphisms (SNPs) in the Toll-like receptor 3 (TLR3) gene in patients infected with hepatitis B virus (HBV) and hepatitis C virus (HCV). METHODS: Samples collected from HCV (n = 74) and HBV (n = 35) carriers were subjected to quantitative real-time PCR (qPCR) to detect the presence of the SNPs rs5743305 and rs3775291 in TLR3 and to measure the following biomarkers: alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and prothrombin time (PT). A healthy control group was investigated and consisted of 299 HCV- and HBV-seronegative individuals. RESULTS: No significant differences in allele, genotype and haplotype frequencies were observed between the investigated groups, and no association was observed between the polymorphisms and histopathological results. Nevertheless, genotypes TA/AA (rs5743305) and GG (rs3775291) appear to be associated with higher levels of ALT (p<0.01), AST (p<0.05) and PT (p<0.05). In addition, genotypes TT (rs5743305; p<0.05) and GG (rs3775291; p<0.05) were associated with higher GGT levels. CONCLUSIONS: This genetic analysis revealed the absence of an association between the polymorphisms investigated and susceptibility to HBV and HCV infection; however, these polymorphisms might be associated with a greater degree of biliary damage during the course of HCV infection.

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INTRODUCTION: This study aimed to evaluate spasticity in human T-lymphotropic virus type 1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients before and after physical therapy using the International Classification of Functioning, Disability and Health (ICF). METHODS: Nine subjects underwent physical therapy. Spasticity was evaluated using the Modified Ashworth Scale. The obtained scores were converted into ICF body functions scores. RESULTS: The majority of subjects had a high degree of spasticity in the quadriceps muscles. According to the ICF codes, the spasticity decreased after 20 sessions of physical therapy. CONCLUSIONS: The ICF was effective in evaluating spasticity in HAM/TSP patients.