Fumarato hidratases ou fumarases (FH) catalisam a reação estereoespecífica reversível de hidratação do fumarato em L-malato. Essas enzimas se apresentam em todas as classes de organismos, desde procariotos a eucariotos, e podem ser encontradas nas formas mitocondrial e citosólica. A enzima tem papel importante na produção de energia pois participa do ciclo do ácido cítrico, na resposta ao dano do DNA e como supressor tumoral. A fumarase humana (HsFH), que pertence à classe II, é codificada pelo gene 1q42.1, possui 467 aminoácidos em cada monômero com peso molecular de 50,2 kDa cada. Estudos associaram mutações no gene da FH com diversas doenças humanas como acidúria fumárica, leiomiomatoses de útero e pele (MCUL), que quando associadas com um agressivo carcinoma múltiplo de células é conhecido como leiomiomatose hereditária e câncer renal (HLRCC). Apesar da grande importância da fumarase humana no metabolismo energético, ainda há pouca informação em relação ao mecanismo catalítico adotado pela enzima e o efeito estrutural e cinético causado pelas mutações envolvidas com essas doenças. Diante disso, nosso trabalho utilizou uma abordagem híbrida que envolve a caracterização biofísica, bioquímica e estrutural da enzima HsFH, e seus mutantes: N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH e N373DHsFH, identificados em pacientes. Estudos cinéticos foram realizados em sete diferentes pHs e, pela primeira vez para fumarases, o ensaio foi realizado com os dois substratos presentes na mesma mistura reacional, confirmando a contribuição da reação reversa para a velocidade global da enzima. De acordo com os estudos de termoflúor a proteína é estabilizada em pHs alcalinos e através da ligação de compostos no sítio ativo. A estrutura da enzima HsFH nativa foi resolvida a 1,8 Å e identificou a presença de moléculas de HEPES complexadas na região C-terminal da enzima. Os estudos cinéticos demonstraram um aumento da eficiência catalítica na presença do HEPES, sugerindo um possível papel alostérico de seu sítio de ligação para a atividade catalítica. Foram determinadas as estruturas para os mutantes N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH e N373DHsFH. As mutações Q185R, E362Q, S365G e N373D foram identificadas no sítio ativo afetando diretamente a capacidade da proteína em ligar os substratos, enquanto que a mutação H180R foi localizada no sítio B, que conduz os substratos e produtos para dentro e fora do sítio ativo. Já a mutação K230R está localizada no domínio central, mas os resultados de termoflúor demonstram um efeito direto na capacidade da enzima em acomodar o substrato. A mutação N107T, localizada longe do sítio ativo foi a única que permaneceu ativa e teve seus parâmetros cinéticos residuais determinados. O presente trabalho contribui para o entendimento das bases estruturais que correlacionam mutações na HsFH, deficiência enzimática e patologia. / Fumarate hydratases or fumarases (FH) catalyze the reversible stereospecific hydration of fumarate to L-malate. They are present in all classes of organisms, from prokaryotes to eukaryotes, and can be found in the mitochondrial and cytosolic forms. The enzyme has an important role in energy production as part of the well-known Citric Acid Cycle, in DNA damage response and as tumor suppressor. Human fumarase (HsFH) belongs to class II and is encoded by 1q42.1 gene. HsFH is tetrameric and has 467 amino acids per monomer, with predicted molecular weight of 50.2 kDa. Several studies associated FH gene mutations with some human diseases such as fumaric aciduria, multiple cutaneous and uterine leiomyomatosis (MCUL), which when associated with an aggressive form of multiple cell carcinoma is known as hereditary leiomyomatosis and renal cancer (HLRCC) syndrome. Although the major role of HsFH in energetic metabolism, there are still little structural and kinetic information about the mutants involved in these diseases. Thus, this study aims, through a hybrid approach, composed by biophysics, biochemical and structural characterization of mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, G282VHsFH, E362QHsFH, S365GHsFH and N373DHsFH identified from patients. Steady-state kinetics studies were performed in seven different pHs and, for the first time, the contribution of both substrates was analyzed simultaneously in a single kinetic assay and allowed to quantify the contribution of the reverse reaction for kinetics. According to thermofluor studies, structural stability can be achieved at alkaline pHs and suggests that ligand binding can modulate the protein stability. HsFH crystal structure was solved at 1.8 Å resolution and identified HEPES molecules complexed with the enzyme C-terminal region. Kinetics studies with HEPES showed an increase of the catalytic efficiency and suggests that HEPES binding site might have an allosteric role. Crystal structures for the mutants N107THsFH, H180RHsFH, Q185RHsFH, K230RHsFH, E362QHsFH, S365GHsFH and N373DHsFH were determined. The mutations Q185R, E362Q, S365G and N373D were identified in the active site and affect the substrate binding capacity directly, while mutation H180R was localized in the B site, which conducts the substrates and products in and out the active site. The mutation K230R is localized in the central domain, but thermofluor results demonstrate a direct effect on the ability of the enzyme to accommodate the substrate. The N107T mutation located far from the active site was the only one that remained active and had its residual kinetic parameters determined. The present work contributes to the understanding of the structural bases that correlate mutations in HsFH, enzymatic deficiency and pathology.

INTRODUÇÃO: O acometimento de artérias ou veias tronculares periféricas por neoplasias malignas é raro. Quando o tratamento cirúrgico é realizado sendo constatado o acometimento arterial pela neoplasia, a melhor conduta é a ressecção conjunta seguida de reconstrução arterial imediata, restabelecendo o eixo vascular e evitando a isquemia de tecidos nobres com suas possíveis conseqüências. OBJETIVOS: O objetivo desse trabalho é analisar os resultados do tratamento cirúrgico de pacientes portadores de neoplasias malignas, submetidos a ressecções tumoral e vascular associadas à reconstrução vascular, avaliando principalmente a morbidade, a mortalidade e a perviedade primária relacionadas às reconstruções arteriais. MÉTODOS: Foram acompanhados os pacientes portadores de neoplasias malignas em regiões cervical, abdominal ou extremidades inferiores, operados eletivamente no período de setembro de 1997 a setembro de 2004 no Hospital do Câncer A.C.Camargo em São Paulo, submetidos à ressecções tumoral e vascular associadas à reconstrução arterial (associada ou não à reconstrução venosa de segmento venoso ressecado no mesmo feixe). O seguimento vascular foi em ambiente ambulatorial, realizando-se exame clínico vascular e exames de imagem para o acompanhamento das reconstruções. Foram analisadas: as características clínicas dos pacientes, as reconstruções vasculares, as complicações vasculares e não vasculares precoces (até 30 dias); as complicações vasculares e não vasculares tardias (após 30 dias), a perviedade primária das reconstruções arteriais e a sobrevida dos pacientes. RESULTADOS: foram operados 36 pacientes, sendo divididos em grupos de acordo com a localização das neoplasias: Cervical (14), Extremidade (13) e Abdome (9). No grupo Cervical, foram realizadas 17 reconstruções (16 arteriais e 1 venosa) nos 14 pacientes, predominantemente com a veia safena. Houve uma oclusão sintomática precoce do enxerto carotídeo com seqüelas importantes e uma oclusão sintomática tardia de enxerto carotídeo sem seqüelas (esse paciente também apresentou oclusão de enxerto arterial para o membro superior sem isquemia grave). No grupo Extremidade, foram realizadas 23 reconstruções (13 arteriais e 10 venosas) nos 13 pacientes, predominantemente com a safena. Houve uma rotura precoce de enxerto arterial femoral, tratada por meio de ligadura e evoluindo sem seqüelas. No grupo Abdome, foram realizadas 13 reconstruções (9 arteriais e 4 venosas) nos 9 pacientes, predominantemente com prótese. Uma paciente apresentou oclusão tardia sintomática de ramo do enxerto aorto-bifemoral, sendo realizado enxerto femoral cruzado, evoluindo sem seqüelas. Não houve diferença estatística entre os índices de perviedade arterial primária entre os grupos (p=0,593). Em relação às reconstruções venosas, houve cinco oclusões sintomáticas. O tempo de seguimento mediano nos grupos Cervical, Extremidade e Abdome foi respectivamente de 11,5, 25 e 18 meses, sendo significantemente menor no grupo Cervical (p=0,034). Houve duas amputações de membro não relacionados às complicações dos enxertos. Não houve óbitos no período intra-hospitalar, sendo todos decorrentes da evolução da doença neoplásica (11 do grupo Cervical, 8 do Extremidade e 3 do Abdome). CONCLUSÕES: 1. as reconstruções arteriais associadas à ressecção de tumores malignos com acometimento arterial em segmentos cervical, abdominal ou extremidades inferiores podem ser realizadas com baixos índices de morbi-mortalidade; 2. não houve diferença entre os índices de perviedade primária das reconstruções arteriais realizadas nos grupos estudados / INTRODUCTION: Arteries or peripheral truncular veins are rarely affected by malignant neoplasias. When arteries affected by neoplasia are observed during surgical treatment, the best approach is in bloc resection followed immediately by arterial reconstruction to reestablish the vascular axis and avoid ischemia of important tissues with the possible consequences. OBJECTIVES: The objectives of this study were to analyze the surgical treatment results from patients with malignant neoplasias who underwent tumor and vascular resection associated with vascular reconstruction and, in particular, to evaluate morbidity, mortality and primary patency relating to arterial reconstruction. METHODS: Patients with malignant neoplasias in the cervical, abdominal or lower extremity regions who underwent elective surgery between September 1997 and September 2004 at Hospital do Câncer A.C. Camargo, São Paulo, were followed up. These patients underwent tumor and vascular resections associated with arterial reconstruction (with or without reconstruction of the venous segment resected in the same bundle). The vascular follow-up was in an outpatient environment, through clinical vascular examination and imaging examinations to monitor the reconstructions. The patients\' clinical characteristics, vascular reconstructions, early vascular and non-vascular complications (within 30 days), late vascular and non-vascular complications (beyond 30 days), primary patency of arterial reconstructions and survival were analyzed. RESULTS: Thirty-six patients underwent surgery and were grouped according to neoplasia location: Cervical (14), Extremity (13) and Abdomen (9). In the Cervical group, 17 reconstructions were performed (16 arterial and 1 venous) in 14 patients, predominantly using the saphenous vein. There were one early symptomatic occlusion of the carotid graft with significant sequelae and one late symptomatic occlusion of the carotid graft without sequelae (this patient also presented arterial graft occlusion to the upper limb, without severe ischemia). In the Extremity group, 23 reconstructions were performed (13 arterial and 10 venous) in 13 patients, predominantly using the saphenous vein. There was one early rupture of a femoral arterial graft, with treatment by means of ligature and evolution without sequelae. In the Abdomen group, 13 reconstructions were performed (9 arterial and 4 venous) in 9 patients, predominantly using a prosthesis. One patient presented a late symptomatic occlusion in a branch of the aortobifemoral graft, for which a femoral crossover graft was performed, which evolved without sequelae. There was no statistical difference in primary arterial patency rates between the groups (p=0,593). Five symptomatic occlusions relating to venous reconstructions occurred. The median follow-up for the Cervical, Extremity and Abdomen groups were 11,5, 25 and 18 months, respectively: significantly shorter in the Cervical group (p=0,034). There were two limb amputations, unrelated to graft complications. There were no deaths while in hospital. Deaths occurred only as a result of neoplastic disease evolution (11 Cervical, 8 Extremity and 3 Abdomen patients). CONCLUSIONS: 1. arterial reconstructions associated with resection of malignant tumors affecting arteries in the cervical, abdominal or lower extremity segments can be performed with low morbidity-mortality rates; 2. there is no difference between primary patency rates of the arterial reconstructions performed, regarding the groups studied

The rare disease pulmonary arterial hypertension (PAH) results in high blood pressure in the lung caused by narrowing of lung arteries. Genes causative in PAH were discovered through family studies and very often harbour rare variants. However, the genetic cause in heritable (31%) and idiopathic (79%) PAH cases is not yet known but are speculated to be caused by rare variants. Advances in high-throughput sequencing (HTS) technologies made it possible to detect variants in 98% of the human genome. A drop in sequencing costs made it feasible to sequence 10,000 individuals including 1,250 subjects diagnosed with PAH and relatives as part of the NIHR Bioresource - Rare (BR-RD) disease study. This large cohort allows the genome-wide identification of rare variants to discover novel causative genes associated with PAH in a case-control study to advance our understanding of the underlying aetiology. In the first part of my thesis, I establish a phenotype capture system that allows research nurses to record clinical measurements and other patient related information of PAH patients recruited to the NIHR BR-RD study. The implemented extensions provide a programmatic data transfer and an automated data release pipeline for analysis ready data. The second part is dedicated to the discovery of novel disease genes in PAH. I focus on one well characterised PAH disease gene to establish variant filter strategies to enrich for rare disease causing variants. I apply these filter strategies to all known PAH disease genes and describe the phenotypic differences based on clinically relevant values. Genome-wide results from different filter strategies are tested for association with PAH. I describe the findings of the rare variant association tests and provide a detailed interrogation of two novel disease genes. The last part describes the data characteristics of variant information, available non SQL (NoSQL) implementations and evaluates the suitability and scalability of distributed compute frameworks to store and analyse population scale variation data. Based on the evaluation, I implement a variant analysis platform that incrementally merges samples, annotates variants and enables the analysis of 10,000 individuals in minutes. An incremental design for variant merging and annotation has not been described before. Using the framework, I develop a quality score to reduce technical variation and other biases. The result from the rare variant association test is compared with traditional methods.

藥品的行銷由於受到衛生署相關法令的嚴格規範，藥商通常只能透過醫師、藥師將產品資訊適時揭露給病患，無論直接訴諸媒體對患者行銷；因此，藥商行銷服務的目標客戶，一般而言僅限於醫師及藥師。 罕見疾病患為數極少，參與臨床診斷研究的醫師甚少，而治療用藥劑量的調整與投予又相當複雜，因此，患者往往需要有額外的衛教護理人員協助，才能提高其對治療藥物的醫囑性及回診率，以確保臨床的治療成效。 本研究以4C架構為方法，從病患的觀點出發，依行銷交換理論的架構，從外顯單位效益成本、資訊搜尋成本、道德危機成本、專屬陷入成本等四大因素，對罕見疾病用藥衛教護理行銷略，進行深入的分析與探討。分析結果如下： 衛教護理行銷略可適當縮減銷售團隊的規模，可進一步減少人事成本的支出，大幅降低行銷總成本，並提高產品的競爭優勢。透過衛教護理行銷略可提升產品價值曲線與客戶鏈相結合，提高整體產品總效益，藉以拉高競爭者的進入障礙。衛教護理為患者提供個人化的服務，協助患者學習注射藥物、記錄用藥情形、提醒回診日期，讓患者體驗到服務的感動與生命受到尊重的感覺，進而對衛教護理系統產生絕對的依賴性，擴大患者的專屬陷入成本，達成差異化策略行銷的目標。 謹以本研究的發現，期能為罕見疾病藥品經營者提供可行的差異化策略建議，強化產品永續經營的競爭優勢。 / Marketing of drugs due to the strict regulations of the Department of Health Act, drug manufacturers usually only through physicians, pharmacists product information to timely disclose to patients with or without direct recourse to the media marketing of patients; Therefore, the target customers of the pharmaceutical companies marketing services. Generally limited to physicians and pharmacists. Minimal number of rare diseases, and is involved in the clinical diagnosis of physician rarely, coupled with the adjustment of the treatment dose is quite complicated, therefore, patients often need to have additional health education nurses to assist in order to improve their doctor's advice and return consultation rate to ensure the clinical outcome of the treatment. In this study, the 4C framework for the method, from the viewpoint of patients, nursing care education marketing strategy on rare diseases, in-depth analysis and discussion. The results are as follows: Nursing care education appropriate to reduce the size of the sales team, personnel costs, dramatically further reducing the total cost of marketing and improve the product competitive advantage. This strategy enhances product value curve and the combination with the customer chain, to improve the total effectiveness of the overall product in order to build up the entry barriers for competitors. To provide personalized service for patients, help them learn to inject able drugs, record prescriptions and remind the date of the return visit, to enable patients to experience the feelings and life of service by the feeling of respect, and thus on the nursing care education system is absolutely dependent to increase the patient's specific holdup cost to achieve the goal of the differentiation strategy marketing. Rise to the findings of this study, of drugs for rare diseases operators to provide viable differentiation strategy recommendations to strengthen the sustainable competitive advantage in the product.

Norfloxacin For Hepatopulmonary Syndrome: A Pilot Study of a Rare Disease Samir Gupta, Masters of Science, 2008 Graduate Department of Health Policy, Management and Evaluation University of Toronto Introduction: Hepatopulmonary Syndrome is a rare disease characterized by abnormal gas-exchange and a poor prognosis, with no known effective medical therapy. A rat model and preliminary human data suggest that this disease may be caused by intestinal bacterial overgrowth, systemic endotoxemia and increased nitric oxide. Methods: We conducted a pilot crossover randomized controlled trial of norfloxacin versus placebo over four weeks, in seven subjects with HPS or a milder condition called pre-HPS, with a primary outcome of alveolar-arterial oxygen gradient (AaDO2). Results: There was no trend toward improved AaDO2, this outcome and other intermediate outcomes were highly variable, and results suggested that a longer treatment course might be necessary. We identified multiple obstacles to recruitment. Conclusion: We believe that a full-scale study of norfloxacin therapy for HPS will require 1) a six-month therapeutic period, 2) more specific HPS diagnostic criteria for clinical and study populations, and 3) creative recruitment maneuvers.

Norfloxacin For Hepatopulmonary Syndrome: A Pilot Study of a Rare Disease Samir Gupta, Masters of Science, 2008 Graduate Department of Health Policy, Management and Evaluation University of Toronto Introduction: Hepatopulmonary Syndrome is a rare disease characterized by abnormal gas-exchange and a poor prognosis, with no known effective medical therapy. A rat model and preliminary human data suggest that this disease may be caused by intestinal bacterial overgrowth, systemic endotoxemia and increased nitric oxide. Methods: We conducted a pilot crossover randomized controlled trial of norfloxacin versus placebo over four weeks, in seven subjects with HPS or a milder condition called pre-HPS, with a primary outcome of alveolar-arterial oxygen gradient (AaDO2). Results: There was no trend toward improved AaDO2, this outcome and other intermediate outcomes were highly variable, and results suggested that a longer treatment course might be necessary. We identified multiple obstacles to recruitment. Conclusion: We believe that a full-scale study of norfloxacin therapy for HPS will require 1) a six-month therapeutic period, 2) more specific HPS diagnostic criteria for clinical and study populations, and 3) creative recruitment maneuvers.

INTRODUÇÃO: O acometimento de artérias ou veias tronculares periféricas por neoplasias malignas é raro. Quando o tratamento cirúrgico é realizado sendo constatado o acometimento arterial pela neoplasia, a melhor conduta é a ressecção conjunta seguida de reconstrução arterial imediata, restabelecendo o eixo vascular e evitando a isquemia de tecidos nobres com suas possíveis conseqüências. OBJETIVOS: O objetivo desse trabalho é analisar os resultados do tratamento cirúrgico de pacientes portadores de neoplasias malignas, submetidos a ressecções tumoral e vascular associadas à reconstrução vascular, avaliando principalmente a morbidade, a mortalidade e a perviedade primária relacionadas às reconstruções arteriais. MÉTODOS: Foram acompanhados os pacientes portadores de neoplasias malignas em regiões cervical, abdominal ou extremidades inferiores, operados eletivamente no período de setembro de 1997 a setembro de 2004 no Hospital do Câncer A.C.Camargo em São Paulo, submetidos à ressecções tumoral e vascular associadas à reconstrução arterial (associada ou não à reconstrução venosa de segmento venoso ressecado no mesmo feixe). O seguimento vascular foi em ambiente ambulatorial, realizando-se exame clínico vascular e exames de imagem para o acompanhamento das reconstruções. Foram analisadas: as características clínicas dos pacientes, as reconstruções vasculares, as complicações vasculares e não vasculares precoces (até 30 dias); as complicações vasculares e não vasculares tardias (após 30 dias), a perviedade primária das reconstruções arteriais e a sobrevida dos pacientes. RESULTADOS: foram operados 36 pacientes, sendo divididos em grupos de acordo com a localização das neoplasias: Cervical (14), Extremidade (13) e Abdome (9). No grupo Cervical, foram realizadas 17 reconstruções (16 arteriais e 1 venosa) nos 14 pacientes, predominantemente com a veia safena. Houve uma oclusão sintomática precoce do enxerto carotídeo com seqüelas importantes e uma oclusão sintomática tardia de enxerto carotídeo sem seqüelas (esse paciente também apresentou oclusão de enxerto arterial para o membro superior sem isquemia grave). No grupo Extremidade, foram realizadas 23 reconstruções (13 arteriais e 10 venosas) nos 13 pacientes, predominantemente com a safena. Houve uma rotura precoce de enxerto arterial femoral, tratada por meio de ligadura e evoluindo sem seqüelas. No grupo Abdome, foram realizadas 13 reconstruções (9 arteriais e 4 venosas) nos 9 pacientes, predominantemente com prótese. Uma paciente apresentou oclusão tardia sintomática de ramo do enxerto aorto-bifemoral, sendo realizado enxerto femoral cruzado, evoluindo sem seqüelas. Não houve diferença estatística entre os índices de perviedade arterial primária entre os grupos (p=0,593). Em relação às reconstruções venosas, houve cinco oclusões sintomáticas. O tempo de seguimento mediano nos grupos Cervical, Extremidade e Abdome foi respectivamente de 11,5, 25 e 18 meses, sendo significantemente menor no grupo Cervical (p=0,034). Houve duas amputações de membro não relacionados às complicações dos enxertos. Não houve óbitos no período intra-hospitalar, sendo todos decorrentes da evolução da doença neoplásica (11 do grupo Cervical, 8 do Extremidade e 3 do Abdome). CONCLUSÕES: 1. as reconstruções arteriais associadas à ressecção de tumores malignos com acometimento arterial em segmentos cervical, abdominal ou extremidades inferiores podem ser realizadas com baixos índices de morbi-mortalidade; 2. não houve diferença entre os índices de perviedade primária das reconstruções arteriais realizadas nos grupos estudados / INTRODUCTION: Arteries or peripheral truncular veins are rarely affected by malignant neoplasias. When arteries affected by neoplasia are observed during surgical treatment, the best approach is in bloc resection followed immediately by arterial reconstruction to reestablish the vascular axis and avoid ischemia of important tissues with the possible consequences. OBJECTIVES: The objectives of this study were to analyze the surgical treatment results from patients with malignant neoplasias who underwent tumor and vascular resection associated with vascular reconstruction and, in particular, to evaluate morbidity, mortality and primary patency relating to arterial reconstruction. METHODS: Patients with malignant neoplasias in the cervical, abdominal or lower extremity regions who underwent elective surgery between September 1997 and September 2004 at Hospital do Câncer A.C. Camargo, São Paulo, were followed up. These patients underwent tumor and vascular resections associated with arterial reconstruction (with or without reconstruction of the venous segment resected in the same bundle). The vascular follow-up was in an outpatient environment, through clinical vascular examination and imaging examinations to monitor the reconstructions. The patients\' clinical characteristics, vascular reconstructions, early vascular and non-vascular complications (within 30 days), late vascular and non-vascular complications (beyond 30 days), primary patency of arterial reconstructions and survival were analyzed. RESULTS: Thirty-six patients underwent surgery and were grouped according to neoplasia location: Cervical (14), Extremity (13) and Abdomen (9). In the Cervical group, 17 reconstructions were performed (16 arterial and 1 venous) in 14 patients, predominantly using the saphenous vein. There were one early symptomatic occlusion of the carotid graft with significant sequelae and one late symptomatic occlusion of the carotid graft without sequelae (this patient also presented arterial graft occlusion to the upper limb, without severe ischemia). In the Extremity group, 23 reconstructions were performed (13 arterial and 10 venous) in 13 patients, predominantly using the saphenous vein. There was one early rupture of a femoral arterial graft, with treatment by means of ligature and evolution without sequelae. In the Abdomen group, 13 reconstructions were performed (9 arterial and 4 venous) in 9 patients, predominantly using a prosthesis. One patient presented a late symptomatic occlusion in a branch of the aortobifemoral graft, for which a femoral crossover graft was performed, which evolved without sequelae. There was no statistical difference in primary arterial patency rates between the groups (p=0,593). Five symptomatic occlusions relating to venous reconstructions occurred. The median follow-up for the Cervical, Extremity and Abdomen groups were 11,5, 25 and 18 months, respectively: significantly shorter in the Cervical group (p=0,034). There were two limb amputations, unrelated to graft complications. There were no deaths while in hospital. Deaths occurred only as a result of neoplastic disease evolution (11 Cervical, 8 Extremity and 3 Abdomen patients). CONCLUSIONS: 1. arterial reconstructions associated with resection of malignant tumors affecting arteries in the cervical, abdominal or lower extremity segments can be performed with low morbidity-mortality rates; 2. there is no difference between primary patency rates of the arterial reconstructions performed, regarding the groups studied

I introduce the concept of Territory Building – a social media application approach that intends to guide and support sufferers of rare diseases in the process of “making sense” of online information. Similar to approaches that emphasize the sharing of web resources – such as social bookmarking sites - user interaction in regards to web resources is at the centre of this concept. The idea of Territory Building is built on a geographical metaphor, guiding a community in assembling a “virtual territory”, consisting of existing web resources related to the community’s domain of interest, along with user-generated information pertaining to these resources. As users build up this Territory, claiming new resources for it, charting out the place of this information in the context of the existing Territory, finally colonizing these resources by interacting, discussing, rating the information, they participte in a process that should help both the invidual and the community as a whole in “making sense” of this information. The “added value” generated through the contributions and engagement of the userbase as a whole becomes more accessible to the individual, and individuals in turn have a more immediate means of contributing to the existing knowledge. Paying close attention to the needs of the intended target group, I designed and implemented a prototype web-application based on the Territory Building approach, and deployed it for use by persons suffering from the rare disease Trigeminal Neuralgia. I observed how this audience made use of the approach, and how implementation details were received. 80 users registered in the prototype system, 31 web resources were contributed by users. The findings suggest that the Territory Building concept can address a genuine need in regards to assembling and engaging with available online information, and that patients are interested in making use of this approach for the purpose of disseminating information resources to their peers. Lessons learned also indicate that the approach can potentially support those affected by rare diseases in “making sense” of the information available to them, and that means of accessing and creating user-generated knowledge in the context of existing web-resources can increase the potential of users to benefit from the collaborative knowledge-generating processes of their peers.

This diploma thesis describes the issue of rare diseases in terms of their essence and the situation in the Czech Republic nowadays. As a disease model was chosen cystic fibrosis. The main goal of this work is to quantify the cost of treatment of cystic fibrosis for three consecutive years and to analyse their structure in the cohorts of patients.

My thesis is focused on Czech Association for Rare Diseases funding. Primary objective was to create fundraising strategy for this organization. This strategy is now financed mainly by pharmaceutical companies, which causes conflict of interests. Secondary objective was to propose suitable marketing communication strategy. To achieve these goals, it was neccessary to clarify theoretical knowledge of nonprofit institutions first. In practical part of my thesis I select companies, which, I believe, could profit from cooperation with Czech Association for Rare Diseases most. There are also few ideas stated about communications and PR improvement of Czech Association for Rare Diseases.