Cancer remains one of the most common causes for death in the world today. Researchers are continuously trying to improve old, and develop new, methods in order to strife this global problem. Much research is being made trying to find new specific biomarkers that can be used to detect and diagnose cancer in an early stage. One candidate protein for possible future use as a biomarker is tripeptidyl-peptidase II (TPPII) which has previously been shown to be up-regulated in Burkitt´s lymphoma. This paper focuses on the expression of TPPII on an mRNA-level to see if there is any difference between expression in human leucocytes from patients with a leukemia diagnosis and a healthy volunteer, in order to evaluate if the expression of TPPII have any future use as a biomarker. Patient samples were analyzed using real time qPCR, to study the expression of mRNA, and Western blot, in order to correlate the mRNA findings with protein expression. Three different cell lines with different characteristics regarding expression and function of TPPII were also used to validate the methods used and for comparison with the patient samples analyzed. A difference in expression of mRNA were seen between the different patient samples, both individually and between larger groups of samples with the same diagnosis, indicating a large individual variation, thus making future use in a clinical setting difficult. However, seeing as only a few samples were analyzed in this study, more research must be done in order to draw any final conclusions.

Alzheimer's disease is a progressive neurodegenerative disorder of the central nervous system. One pathological characteristic is excessive neuronal loss in specific regions of the brain. Among the areas most severely affected are the basal forebrain cholinergic neurons and their projection regions, the hippocampus and cortex. Neurotrophic factors, particularly the neurotrophins nerve growth factor and brain-derived neurotrophic factor, play an important role in the development, regulation and survival of basal forebrain cholinergic neurons. Furthermore, brain-derived neurotrophic factor regulates the function of hippocampal and cortical neurons. Neurotrophins are synthesized in hippocampus and cortex and retrogradely transported to the basal forebrain. Decreased levels of neurotrophic factors are suspected to be involved in the neurodegenerative changes observed in Alzheimer's disease. We examined autopsied parietal cortex, hippocampus and nucleus basalis of Meynert samples from age- and gender-matched Alzheimer's diseased and neuro logically non-impaired individuals using the quantitative technique of competitive RT-PCR. We also examined parietal cortex samples by Western blotting. We demonstrate a 3.4-fold decrease in brain-derived neurotrophic factor mRNA levels in the parietal cortex of patients with Alzheimer's disease compared to controls (p < 0.004) but fail to observe changes in BDNF protein levels in that brain region. We also demonstrate, for the first time, BDNF mRNA in the nucleus basalis of Meynert and report an age-related decline in the levels of BDNF mRNA in both control and AD samples. Using the competitive RT-PCR technique we fail to observe differences in BDNF mRNA levels in the hippocampus between AD and control subjects, conflicting with previous in situ hybridization studies and RNase protection assays. A decrease in brain-derived neurotrophic factor synthesis could have detrimental effects on hippocampal, cortical and basal forebrain cholinergic neurons and may account for their selective vulnerability in Alzheimer's disease. / Thesis / Master of Science (MSc)

名古屋大学博士学位論文 学位の種類 : 医学博士(論文) 学位授与年月日:平成4年9月22日 大野元嗣氏の博士論文として提出された

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-03-08T16:58:52Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Juliana Gusmão de Araujo.pdf: 5234646 bytes, checksum: 87f79f1087a57c64407eda5226cb557f (MD5) / Made available in DSpace on 2017-03-08T16:58:52Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Juliana Gusmão de Araujo.pdf: 5234646 bytes, checksum: 87f79f1087a57c64407eda5226cb557f (MD5) Previous issue date: 2012-12-26 / O colesteatoma adquirido, mesmo com os conhecimentos acumulados desde sua primeira descrição, ainda se mantém como um problema de saúde pública distante de ser solucionado. O entendimento mais profundo da patogênese do colesteatoma é de extrema importância visto que a natureza desta lesão é destrutiva e causadora de complicações potencialmente graves. Apesar das teorias propostas e das várias proteínas terem sido identificados no colesteatoma, a verdadeira etiopatogenia da doença ainda carece de investigações. Objetivo: Identificar biomarcadores do colesteatoma adquirido utilizando a plataforma proteômica. Casuística e Métodos: Foram coletadas amostras de colesteatoma e também fragmento de pele da região retroauricular de 12 indivíduos submetidos a cirurgia para remoção do colesteatoma. As amostras foram armazenadas em solução salina e mantidas a -20ºC até pesagem tecidual e extração das proteínas. Eletroforese bidimensional foi realizada, os géis foram corados com nitrato de prata e suas imagens digitalizadas. Todas as análises foram realizadas em triplicatas. Os peptídeos extraídos após a digestão do spots foram levados à espectrometria de massa e os espectros obtidos foram analisados usando o algoritmo Mascot utilizando os bancos de dados de proteína do NCBI e SwissProt. Resultados: Dos 393 spots identificados na análise do extrato proteico de colesteatoma adquirido, apenas 10 estavam dentro dos parâmetros estatísticos aceitáveis pelo algoritmo Mascot. As principais proteínas detectadas no colesteatoma adquirido foram a cadeia beta do fibrinogênio, proteína da matriz extracelular 2, actina citoplasmática 1, heparan sulfato glucosamina 3-O-sulfotransferase 3A1, fator de necrose tumoral alfa induzido proteína 8-like 1, Stanniocalcina-2, lisofosfolipase eosinofílica e OFUT1.Conclusão: Foram identificadas proteínas envolvidas com a migração celular, regulação da apoptose, vias de sinalização, hiperproliferação celular, cicatrização e processos inflamatórios. Pudemos, desta maneira, traçar um perfil proteômico do colesteatoma adquirido. / The acquired cholesteatoma, even with all the knowledge accumulated since its first description, still remains a public health problem, far from being solved. A deeper understanding of its pathogenesis is extremely important since it is a destructive lesion that might cause potentially serious complications. Several proteins have been identified in cholesteatoma and a few theories were described, however the true etiology of the disease still needs investigation. Objective: Identify acquired cholesteatoma biomarkers using proteomics platform. Patients and methods: Cholesteatoma samples were collected and also a skin fragment of the surgical incision of twelve patients undergoing surgery for cholesteatoma removal. The samples were stored in saline solution and kept at -20 ° C until weighing tissue and proteins extraction. Two-dimensional electrophoresis was conducted, the gels were stained with silver nitrate and their images were digitized. All analyzes were performed in triplicate. The peptides extracted after spots digestion were taken to mass spectrometry and the spectra obtained were analyzed using the Mascot algorithm comparing databases of the NCBI and SwissProt protein. Results: Of the 393 spots identified in the analysis of protein extracts of acquired cholesteatoma, only 10 were within acceptable statistical parameters by Mascot algorithm. The proteins detected in acquired cholesteatoma were fibrinogen beta chain, extracellular matrix protein 2, actin cytoplasmic 1, heparan sulfate glucosamine 3-O-sulfotransferase 3A1, tumor necrosis factor alpha 8 induced proteinlike 1, stanniocalcin-2, eosinophil lysophospholipase and OFUT1. Conclusion: Proteins involved in cell migration, regulation of apoptosis, signaling pathways, cellular proliferation, wound healing and inflammatory processes were identified. We were able to draw a proteomic profile of acquired cholesteatoma.