Das translationell kontrollierte Tumorprotein (TCTP) ist ein hochkonserviertes, ubiquitär in Eukaryonten vorkommendes Protein. Seine Bezeichnung geht auf die erstmalige Beschreibung in Tumorzellen zurück und weist zugleich auf seine besondere translationelle Regulation hin. Das zugehörige Gen wird als TPT1 bezeichnet und befindet sich beim Menschen auf dem langen Arm des Chromosom 13. Eine pathophysiologische Bedeutung für TCTP wurde bei Tumorerkrankungen, Erkrankungen des allergischen Formenkreises sowie bei Infektionen durch Parasiten beschrieben. Für diese Arbeit wurde zur Untersuchung grundlegender Regulationsphänomene die TCTP-mRNA des Kaninchens als geeignetes Modell ausgewählt. Es wurden die volllangen TCTP-mRNA1 und 2, die sich in der Länge ihrer 3'UTR unterscheiden, sowie Deletionsvarianten, denen die UTR-Abschnitte fehlen, kloniert. In Proteinbindungsstudien (Electromobility Shift Assays, UV-Crosslinking-Experimente, RNA-Affinitätschromatographie) wurden potentielle Bindungsfaktoren der TCTP-UTRs analysiert. Die an der mRNA des Kaninchens erarbeiteten Ergebnisse wurden durch Untersuchungen an humanen Melanomzellen ergänzt. In in vitro Translationsexperimenten wurde gezeigt, dass die Regulation der TCTP-mRNA durch ihre 5'UTR und 3'UTR2 vermittelt wird. In RNA-Bindungsstudien konnte eine Reihe potentieller Bindungsfaktoren der UTRs charakterisiert werden. Bei Verwendung von Extrakten aus verschiedenen Kaninchengeweben zeigten sich deutliche gewebsspezifische Unterschiede. Frühere Untersuchungen hatten gezeigt, dass es in der Melanomzelllinie MeWo bei der Ausbildung einer Chemoresistenz zu einer Expressionssteigerung des TCTP kommt. In einem ersten Schritt wurde der Beitrag von Transkription und Translation in vergleichenden Northern und Western Blot Analysen untersucht. Auf mRNA-Niveau findet man in den resistenten Zellen eine deutliche Steigerung der Expression im Vergleich zu den sensiblen Zellen. Der mRNA-Menge in den chemosensiblen Zellen steht eine vergleichsweise geringe Menge an Protein gegenüber. Folglich liegt die mRNA in diesen Zellen in einem inaktiven Zustand vor. Es konnten drei Cytoskelettproteine gamma-Actin, beta-Tubulin und alpha-Actinin als Bindungspartner der TCTP-3'UTR in den Melanomzellen identifiziert werden. Eine Bedeutung von TCTP für die Entstehung der Chemoresistenz lässt sich aufgrund seiner anti-apoptotischen Wirkung vermuten. Die Regulation der TCTP-Translation stellt bei durch Cytostatika hervorgerufener Hemmung der Transkription einen wichtigen Pathomechanismus in chemoresistenten Melanomzellen dar. / The translationally controlled tumor protein (TCTP) is a highly conserved protein expressed in all eukaryotic organisms. It was first described in tumor cells showing a special regulation of translation. The chromosomal localisation of the respective human gene TPT1 has been determined (13q14). TCTP has been implicated in cellular processes such as cell growth and apoptosis. Its medical importance has been shown in malignant transformation, allergic reactions and immunity against parasitic organisms. In order to investigate basic mechanisms of translational regulation the rabbit TCTP-mRNA was chosen due to its high homology to its human counterpart. The TPT1 gene is transcribed into two TCTP-mRNAs differing in the length of their 3'untranslated regions. These two mRNAs and variants missing the untranslated regions were cloned into expression vectors. In Electro mobility shift assays, UV-crosslinking assays and RNA affinity purification several TCTP-mRNA binding factors were characterised. Furthermore, the role of TCTP in human chemoresistant melanoma cells was investigated. In cell-free translation assays the importance of the 5'UTR and 3'UTR2 was shown. However, in wheat germ extracts the regulation of the TCTP-mRNA mediated by its 5'UTR is less important. In Electro mobility shift assays and UV-crosslinking assays with radiolabelled transcripts of the untranslated regions great variations in tissue-specific protein binding were found. Recently, TCTP had been implicated in the development of chemoresistance in the human melanoma cell line MeWo. As a first step, the contribution of transcriptional and translational regulation was analysed by comparing TCTP-expression in Northern and Western blot assays. Transcription of the TPT1 gene is increased in chemoresistant melanoma cells whereas translation is inhibited in those cells susceptible to chemotherapeutic agents. Three proteins, gamma-actin, beta-tubulin and alpha-actinin, were identified as factors binding to the TCTP-3'UTR in melanoma cells. For the interaction of these cytoskeleton components their ability to bind intracellular calcium ions could be of great importance. The role of TCTP in the development of chemoresistance can be explained by its anti-apoptotic function. In conclusion, the regulation of TCTP-translation when transcription is blocked by inhibitors of DNA-function is an important mechanism to overcome the effect of these anti-proliferative agents.

OBJETIVO: A perda de heterozigosidade (LOH) é capaz de avaliar as alterações genéticas de lesões potencialmente malignas. Este ensaio avalia as regiões cromossômicas polimórficas que estão próximas ou na região dos oncogenes e genes supressores de tumor conhecidos. Os objetivos desta tese foram três principais: 1) Avaliar a frequência de perda de heterozigosidade de leucoplasias bucais com diferentes graus de severidade histopatológico em regiões cromossômicas próximas aos genes supressores de tumores. 2) Comparar e correlacionar o perfil de perda de heterozigosidade entre indivíduos da British Columbia (Canadá) e Rio Grande do Sul (Brasil). 3) Avaliar os danos ao DNA que podem ocorrer durante o processamento e armazenamento das amostras de tecido parafinado. MÉTODOS: Amostras de leucoplasia bucal (com e sem displasias), fixadas em formalina tamponada 10% e parafinadas, obtidas nos laboratório de patologia bucal do Canadá e do Brasil foram selecionadas e microdissectadas. Procedeu-se a extração de DNA, amplificação por PCR das seguintes regiões microssatélites: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) e 17p13.1 (tp53 e D17S786). Após o produto do PCR foi separado e visualizado em gel de poliacrilamida por autoradiografia. RESULTADOS: Observou-se uma forte correlação entre o perfil de perda de heterozigosidade entre indivíduos com leucoplasia bucal de ambos os países, independentemente da etnicidade. Além disso, pode-se notar que amostras de tecidos parafinados submetidos a mais de 24 horas de fixação em formalina tamponada 10% não serão, em sua maioria, boas amostras para análises de DNA. CONCLUSÃO: As lesões potencialmente malignas, provavelmente não são influenciadas em sua etiopatogênia pelas diferenças étnicas. O modelo de risco genético validado por Zhang e colaboradores (2012) parece ser aplicável em nossa comunidade, sendo necessário a sua validação, respeitando procedimentos técnicos padronizados. Ainda, vale ressaltar, que é imprescindível que a comunidade científica passe a adotar metodologias que preservem o material genético das peças dos bancos de tecidos parafinados, que são de inestimável valor para a pesquisa biomédica. / OBJECTIVE: Loss of heterozygosity (LOH) can evaluate genetic alterations of pre-malignant lesions. This assay evaluates the chromosomal polymorphic regions that are present in tumor suppressor genes and oncogenes. The main objectives of this thesis were: 1) Evaluate the frequency of LOH of oral leukoplakias with different histopathological degrees at chromosomal regions of tumor suppressor genes. 2) Compare the profile of LOH between individuals from British Columbia (Canada) and Rio Grande do Sul (Brazil). 3) Evaluate the DNA damage that may occur with FFPE (formalin-fixed paraffin-embedded) tissues. METHODS: FFPE samples of oral leukoplakia (with and without dysplasia), obtained in Canadian and Brazilian oral pathology laboratories were selected and microdissected. DNA extraction and PCR amplification of the following microsatellite regions were conducted: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) and 17p13.1 (tp53 and D17S786). PCR products were separated and visualized on polyacrylamide gel by autoradiography. RESULTS: A strong correlation between the LOH profile among individuals with oral leukoplakia from both countries was observed, regardless ethnicity. Furthermore, FFPE tissues subjected to more than 24 hours of fixation in 10% buffered formalin are not, generally, good samples for DNA analysis. CONCLUSION: Pre-malignant lesions etiopathogenesis may not be influenced by ethnicity. The genetic risk model validated by Zhang et al. (2012) seems to be applicable in our community, requiring its own validation, respecting standardized procedures. Still, it is important to emphasize that it is imperative that a scientific community adopts methodologies that preserve the genetic material FFPE tissues that are an invaluable resource for biomedical research.

Objetivo: Avaliar por meio da Tomografia Computadorizada singleslice, alterações nas estruturas do espaço mastigador, por disseminação loco-regional das neoplasias malignas originárias da loja tonsilar, trígono retromolar, seio maxilar e nasofaringe; correlacionar a presença de trismo com os achados tomográficos e dimensões do tumor. Material e métodos: foram selecionados prontuários de pacientes portadores de tumores malignos envolvendo as 4 regiões a serem estudadas, junto aos arquivos do Hospital Heliópolis. Foram excluídos os pacientes submetidos a terapêutica antineoplásica prévia ou com doenças inflamatórias ou infeciosas locais. Os prontuários foram revisados por um único examinador, onde foram coletadas informações relativas a idade, gênero, localização do tumor primário, tempo de evolução da doença, estadiamento do tumor e presença ou ausência de trismo. Após a administração do meio de contraste iodado, o protocolo de aquisição de imagens tomográficas foi de cortes axiais de 3mm de espessura, aquisições coronais foram feitas quando necessário. A análise das imagens foi feita por dois examinadores médicos, residentes do 3° de Radiologia médica, em momentos distintos, sem o conhecimento prévio das informações clínicas. Na avaliação do EM, os achados foram transcritos para uma planilha específica, considerando a presença ou ausência dos seguintes itens: simetria com o EM contralateral, obliteração do plano gorduroso, do trígono retromolar e do espaço faríngeo, edema e/ou atrofia dos músculos pterigoideos medial e lateral, destruição do ramo da mandíbula. Resultados: O trismo esteve presente em 10 pacientes estudados. Na associação entre dimensão do tumor e trismo, observamos que 90% dos pacientes apresentam tamanho T4, enquanto essa porcentagem é de 43% nos pacientes sem trismo. Analisando-se em termos de razão de chances, verificou-se que pacientes com tumores T4 apresentam uma chance de trismo de 11,6 vezes maior que as demais dimensões (T0 a T3). As neoplasias da loja tonsilar e trígono retromolar perfizeram 95% das neoplasias avaliadas. Foram encontrados apenas 3 casos de neoplasia da nasofaringe, e em nenhum deles observamos a presença de trismo, e não foi encontrado nenhum caso de neoplasia de seio maxilar. Relacionando os achados tomográficos com o grupo de pacientes com trismo, observamos que 60% dos pacientes com trismo apresentaram edema e/ou atrofia dos músculos pterigoideos na TC, enquanto que essa porcentagem foi de 21,8% nos pacientes sem trismo. Em relação às dimensões do tumor e sua relação com os achados das imagens observamos associação apenas entre tumores T4 e detecção de edema e/ou atrofia dos músculos pterigoideos, neste item pacientes com edema e/ou atrofia dos músculos pterigoideos apresentam 5,4 vezes ais chance de apresentarem trismo. Conclusões: Observamos na TC as seguintes alterações no EM: obliteração do plano gorduroso em 69,2 % dos pacientes, assimetria em 27,7%, edema/atrofia em 26,2 % dos pacientes e destruição óssea em 20%. Os graus de concordância inter examinadores foram variáveis. Encontramos associação apenas entre pacientes com trismo e edema/atrofia dos músculos pterigoideos. Neste mesmo item foi observado associação com tumores de dimensão T4. Não encontramos associação do trismo com os demais itens avaliados na TC. / Objective: Evaluate by computed tomography (CT) singleslice changes in the structures of the masticator space, for loco-regional dissemination of malignancies originating in the tonsil, retromolar area, maxillary sinus and nasopharynx to correlate the CT findings with trismus and the tumor dimensions. Material and Methods: The medical records of patients with malignant tumors involving the four regions to be studied were selected from the archives of the Heliopolis Hospital. Patients submited previously to antineoplastic therapy or with local inflammatory or infectious diseases were excluded. The medical records were reviewed by a single examiner, where information was collected on age, gender, tumor location, disease evolution, tumor stage, and the presence or absence of trismus. After administration of iodinated contrast media, the protocol the image acquisition was axial slices 3mm, coronal acquisitions were made when necessary. The image analysis of the MS by two medical examiner residents in their 3rd year of Medical Radiology, at distinct moments , without prior knowledge of the clinical information. In the evaluation of the MS, the findings were transcribed to a specific worksheet, considering the presence or absence of the following items: symmetry with the contralateral MS, obliteration of the fat plane of the retromolar trigone and the pharyngeal space, edema, and/or the atrophy of the medial and lateral pterygoid muscles, and destruction of the mandibular ramus. Results: According to the adopted criteria for trismus in this study, the presence of trismus was observed in 10 patients. The association between tumor size and trismus shows that 90% of patients present size T4, while this percentage is 43% in patients without trismus. Analyzing in terms of odds ratios, the study showed that patients with T4 sized tumors had a chance of developing trismus 11.6 times greater than patients with T0 to T3 sized tumors. The neoplasms of tonsillar crypts and retromolar trigone aggregated 95% of the neoplasms evaluated. Only 5% of the cases accounted for neoplasms of nasopharynx, in which none of them presented the existence of trismus. No cases of neoplasm of maxillary sinus was found. Correlating the CT findings with the group of patients with trismus, the observation was made that 60% of patients with trismus showed edema and/or pterygoid muscle atrophy on CT, whereas this percentage was 21.8% in patients without trismus. Regarding the dimensions of the tumor and its relationship with the image findings, the detection of edema and/or atrophy of the pterygoid muscles was observed only in association with T4 sized tumors. In this instance, patients with edema and/or atrophy of the pterygoid muscles present a 5,4 times greater chance of having trismus. Conclusions: Observed in the following changes in TC IN: obliteration of fat plane in 69.2% of patients, 27.7% asymmetry, edema / atrophy in 26.2% of patients and 20% in bone destruction. The degree of inter examiners were variable. Association was found only among patients with trismus and edema / atrophy of the pterygoid muscles. In this same item was observed association with tumor size T4. We found no association of trismus with other items valued at TC.

Malignant glioma have poor prognosis resulting mainly from high level of cell proliferation and invasion and resistance to conventional therapy. Identification of novel targets that are critical elements in gliomagenesis may help improve therapeutic outcome. Using genome-wide explorations of a comprehensive glioma specimen population, we identified whole gain of chromosome 19 as one of the major chromosomal aberrations in high grade glioma that correlates to patients' outcomes. Our analysis revealed for the first time NOTCH3 as one of the most significant gene amplifications mapped to chromosome 19. This amplification is associated with worse outcome compared to tumors with non-amplified locus. NOTCH signaling pathway is essential for cell proliferation, stem cell maintenance and differentiation and its deregulation has been reported in several human cancers. NOTCHs are key positive regulators of cell-cell interactions, angiogenesis, cell adhesion and stem cell niche development which have been shown to play critical roles in gliomagenesis and glioma drug resistance. Our objective is to determine NOTCH3 molecular roles in glioma pathogenesis and aggressiveness. Here we show for the first time that NOTCH3 plays a role in glioma cell proliferation, cell migration, invasion and apoptosis. We also found a NOTCH3 glioma addiction phenomenon. Therefore, our study uncovers, for the first time, the prognostic value and the oncogenic function of NOTCH3 in gliomagenesis and supports NOTCH3 as a promising target of therapy in high grade glioma. Our studies allow the identification of a subset of population that may benefit from GSI-based therapies. This may lead to the design of novel strategies to improve therapeutic outcome of patients with glioma by establishing medical and scientific basis for personalized medicine.

While the etiology of ovarian cancer (OC) is not completely understood, evidence suggests that chronic inflammation may promote malignant transformation. However, familial history remains the strongest risk factor for developing OC and is associated with germline BRCA1 mutations, such as the 185delAG mutation. Normal human ovarian surface epithelial cells expressing the 185delAG mutant, BRAT, exhibit molecular and pathological changes that may contribute to OC oncogenesis. In the current study, I sought to determine whether BRAT could promote an inflammatory phenotype by investigating BRAT's impact on the expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Using a culture model system of normal human ovarian surface epithelial (OSE) cells with and without the BRCA1 185delAG frameshift mutation, BRAT, I investigated BRAT's role in IL-1β expression. OSE cells stably expressing the 185delAG mutation and ovarian surface epithelial cells with endogenous 185delAG were analyzed for differential target gene expression by real time PCR, western blot, ELISA, luciferase reporter and siRNA assays. Normal and malignant breast epithelial cell lines transiently expressing BRAT were also evaluated by real time PCR to determine whether BRAT-induced IL-1β expression is tissue specific. BRAT-expressing OSE cells exhibited enhanced IL-1β mRNA and protein expression. However, expression of BRAT in all breast cell lines failed to significantly alter IL-1β expression levels so that BRAT-mediated IL-1β expression promoting a chronic inflammatory phenotype conducive to malignant transformation may be limited to the ovary. Secondly, since OSE cells expressing the BRCA1 185delAG mutation have increased levels of IL-1β that may contribute to malignant transformation, in a pilot study, I sought to assess whether elevated urinary levels of IL-1β are associated with OC as well as compare urinary IL-1β levels with clinical parameters. Urinary and serum levels of IL-1β were analyzed by ELISA and biostatistical analysis from a patient cohort consisting of healthy women (N=10), women with ovarian benign disease (N=23), women with OC (N=32), women with other benign gynecological conditions (N=22), and women with other gynecological cancers (N=6). Urinary IL-1β levels were elevated in patients with ovarian benign disease and a first degree family history of ovarian and/or breast cancer. Urinary IL-1β levels were also correlated with increased body mass index. Urinary and serum IL-1β levels were increased in ovarian benign and OC patient samples supporting the theory of elevated urinary IL-1β being associated with cancer progression. Lastly, I sought to begin early molecular characterization of BRCA1 185delAG to better understand its role in ovarian transformation. I isolated 185delAG protein expressed in E. coli and utilized web tools to analyze the amino acid sequence to determine the molecular and structural characteristics. The study results showed the predicted BRCA1 185delAG protein product is an ordered, self-aggregating, alpha helical protein structurally and molecularly distinct from wild-type BRCA1. The BRCA1 185delAG amino acid sequence contained domains with resemblance to the Peptidase M20 family. Isolation of the BRCA1 185delAG protein product will allow for further protein analysis to better understand its' oncogeneic functions; as well as, elucidate the mechanism of tissue-specific BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

Every year approximately 2800 Swedes are diagnosed with malignant melanoma, the form of cancer that is most rapidly increasing in incidence in the Western world. The earlier we can identify and diagnose a malignant melanoma, the better is the prognosis. In Sweden, 155 000 benign naevi, harmless skin tumours or moles, are surgically excised each year, many of them because melanoma cannot be dismissed by non-invasive methods. The excisions result in substantial medical costs and cause unrest and suffering of the individual patient. For untrained physicians, it is often difficult to make an accurate diagnosis of melanoma, thus a tool that could help to strengthen the diagnosis of suspected melanomas would be highly valuable. This thesis describes the development and assessment of a non-invasive method for early skin cancer detection. Using near infrared (NIR) and skin impedance spectroscopy, healthy and diseased skin of various subjects was examined to develop a new instrument for detecting malignant melanoma. Due to the complex nature of skin and the numerous variables involved, the spectroscopic data were analysed multivariately using Principal Component Analysis (PCA) and partial leas square discriminant analysis (PLS-DA). The reproducibility of the measurements was determined by calculating Scatter Values (SVs), and the significance of separations between overlapping groups in score plots was determined by calculating intra-model distances. The studies indicate that combining skin impedance and NIR spectroscopy measurements adds value, therefore a new probe-head for simultaneous NIR and skin impedance measurements was introduced. Using both spectroscopic techniques it was possible to separate healthy skin at one body location from healthy skin at another location due to the differences in skin characteristics at various body locations. In addition, statistically significant differences between overlapping groups of both age and gender in score plots were detected. However, the differences in skin characteristics at different body locations had stronger effects on the measurements than both age and gender. Intake of coffee and alcohol prior to measurement did not significantly influence the outcome data. Measurements on dysplastic naevi were significantly separated in a score plot and the influence of diseased skin was stronger than that of body location. This was confirmed in a study where measurements were performed on 12 malignant melanomas, 19 dysplastic naevi and 19 benign naevi. The malignant melanomas were significantly separated from both dysplastic naevi and benign naevi. Overall, the presented findings show that the instrument we have developed provides fast, reproducible measurements, capable of distinguishing malignant melanoma from dysplastic naevi and benign naevi non-invasively with 83% sensitivity and 95% specificity. Thus, the results are highly promising and the instrument appears to have high potential diagnostic utility.

Coeliac disease (CD) is considered to be the result of a complex interplay of intrinsic (genetic) factors and variable extrinsic (environmental) factors. The complex background of CD explains its wide spectrum of clinical manifestations. For a very long time CD was considered more or less a disease of childhood, which was extremely rare in adults. Nowadays we know that CD is one of the most common food intolerance disorders. An epidemiological study of CD in a geographically defined area of Sweden (Paper1) showed a prevalence of 95.5/ 100 000 inhabitants. Among the associated diseases an especially high incidence of associated thyroid disease, 10.8% was observed. In a fifteen-year cohort follow up study of all CD-patients residing in the counties of Örebro and Linköping (Paper 2) the total mortality was increased with 38% (SMR 1.38 95% C.I. 0.31-0.83). This was mainly explained by a 48% increased death rate in ischemic heart disease, significant in patients over 65 years (SMR 1.58 95% C.I. 1.00-2.06). However, there was a 47 % lower risk of all malignancies (SIR 0.53 95% C.I. 0.31-0.83). A cohort of 22 consecutively biopsy-proven adult CD patients (Paper 3), were followed in respect of antibody titres from diagnosis and after 1, 3, 6, and 12 months on a gluten free diet (GFD). All antibody titres fell sharply within one month. Thus excluding a CD diagnosis serologically on a patient who has initiated a GFD by herself is not to recommend. In another cohort with CD patients (Paper IV) who were diagnosed 8-12 years earlier recommended and who were recommended, the reliability of diet history, serological and biochemical markers to predict the appearance of the small intestinal mucosa were analysed (Paper IV). The history of a strict GFD gave a predictive value of 88% of a mucosa in remission. The values of serological tests (AGA, EmA and tTG) to predict a mucosa in remission were 93% for all. In CD patients in remission gastro-intestinal symptoms were evaluated with the GSRS questionnaire. Subjects with CD reported significantly more GI-symptoms than a general population sample (p<0.01). This was particularly true for women with CD who scored worse than female controls .By contrast men with CD reported no more symptoms than male controls.

The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin. Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis. After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique. <b>Keywords:</b>single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras

The purpose of this thesis is to describe what it is like to suffer from malignant lymphoma and to highlight the care given to these patients. A reflective lifeworld approach, founded in phenomenological philosophy, has been used. Data have been collected using interviews and have been analyzed using essence-seeking analysis. The results are founded upon three empirical studies and a philosophical excursus. The results are presented in four sections. The thesis describes how patients with malignant lymphoma live in limbo characterized by existential uncertainties, partly caused by the mortal threat of the disease and by failings in the actions of the healthcare staff. Patients fear dying when suffering from malignant lymphoma, regardless of whether the disease is a genuine medical threat to their life. Thus, there is a substantial need for existential support for these patients. However, the results show that deficiencies in existential support can lead to patients feeling objectified, which in turn increases their existential uncertainties. Care that is solely directed towards the physiological body and excludes the human as a subject can be experienced as a disparagement. Care that includes the patients’ lifeworld provides alleviated suffering and a possibility for the patients themselves to take an active part in the health process. Despite the healthcare staff’s genuine ambition to alleviate the suffering, patients’ existential questions are met with a degree of conflict; on the one hand they are a natural part of healthcare, and on the other the questions are of such character that they are not part of professional healthcare. A healthcare culture that does not fully acknowledge the importance of existential questions appears to be one of the greatest obstacles to a holistic healthcare approach. Furthermore, there appears to be a lack of shared strategies among the healthcare staff when meeting the patients’ existential questions. A conflict arises in an unnecessary battle between biology and existence, which in turn increases the patients’ existential insecurities. Medical knowledge is insufficient in caring for patients with malignant lymphoma. An understanding of caring science is needed in order for the care to become caring and able to meet the needs as described by patients with malignant lymphoma. Healthcare staff most be provided with sufficient support to meet the patients’ existential questions. The organization of healthcare is characterised by being a culture in which existential questions are not given sufficient attention. It seems that healthcare staff give priority to medical/technical tasks rather than conversations of existential character.

BACKGROUND: Malignant and paramalignant pleural effusions are important complications of many malignancies. The two main management options debated in the literature are: 1) insertion of an indwelling pleural catheter (IPC) to achieve chronic drainage of the effusion, or 2) hospitalization with tube thoracostomy and subsequent chemical pleurodesis (CP) with talc or doxycycline to prevent fluid reaccumulation. We aimed to describe a large series of patients with malignant pleural effusions managed with an IPC, identify and validate factors identified in the literature as predictors of spontaneous pleurodesis in the IPC group and compare the group managed with IPC to patients managed with CP. METHODS: We designed a retrospective cohort study comparing patients with malignant and paramalignant pleural effusions managed either with CP between March 1, 2003 and February 28, 2006 or IPC insertion between May 1, 2006 and April 1, 2009. The CP group was identified through the prescription of talc or doxycycline and the IPC group from the IPC clinic database. Data were collected from paper and electronic records and from the Government of Ontario. RESULTS: We identified 193 consecutive patients with an ECOG performance status of less than 4 (ECOG less than 4 means that the patient is not completely disabled and confined to bed or chair) having undergone IPC insertion and 168 who were managed with CP. None of the variables we tested were significant predictors of spontaneous pleurodesis in the IPC group. Pleural effusion control rates at 6 months were higher in the IPC group than in the CP group (52.7% vs 34.0%, p<0.01) but the rate of freedom from pleural effusion at 180 days and catheter removal at 90 days was not significantly different (25.8% in the IPC group and 34.0% in the CP group p=0.14). Patients in the IPC group had a significantly longer median survival (148 days measured from the date of catheter insertion vs 133 days in the CP group, log-rank p<0.05). CONCLUSION: We found an intriguing possible survival benefit favouring management of malignant or paramalignant effusions with an IPC. Given possible biases due to the design of this study and uncertain explanatory mechanism, this needs to be confirmed in a randomized controlled trial. Quality of life, an important measure of success for these palliative procedures, should also be measured.