No description available.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-07-19T14:11:29Z No. of bitstreams: 1 marilhotadeudornelas.pdf: 12344431 bytes, checksum: 54c1201792fc2e6934f063da77401a72 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-09T12:10:59Z (GMT) No. of bitstreams: 1 marilhotadeudornelas.pdf: 12344431 bytes, checksum: 54c1201792fc2e6934f063da77401a72 (MD5) / Made available in DSpace on 2017-08-09T12:10:59Z (GMT). No. of bitstreams: 1 marilhotadeudornelas.pdf: 12344431 bytes, checksum: 54c1201792fc2e6934f063da77401a72 (MD5) Previous issue date: 2009-03-26 / O câncer de pele é o mais frequente tipo de câncer humano e mostra aumento de sua incidência nos últimos anos, o que o torna um crescente problema para a saúde pública mundial. Em muitos casos, antes do surgimento do carcinoma, instala-se uma lesão precursora, - a Ceratose Actínica, que pode evoluir para Carcinoma Espinocelular. Considerando o aumento de novos casos e recidivas frequentes, estudos tem sido intensificados no sentido de determinar os parâmetros com significado prognóstico na predição daqueles tumores que terão comportamento mais agressivo, que poderão recorrer e evoluir com metástases. O objetivo deste estudo foi avaliar a expressão dos marcadores de proliferação celular (PCNA, Ki-67) e apoptose (p53, Bcl-2), em portadores de Carcinoma Espinocelular e Ceratose Actínica. Foram estudadas amostras de 30 pacientes, submetidos à cirurgia reparadora, sendo dez portadores do Carcinoma Espinocelular, dez de Ceratose Actínica e dez indivíduos livres de lesões submetidos à blefaroplastia. A proteína p53 foi expressa em todos os casos estudados, com padrões quantitativos diferentes. O Bcl-2 foi expresso em baixa intensidade em seis casos de Ceratose Actínica, também baixa nas peles de blefaroplastia e negativo nos casos de Carcinoma Espinocelular. O PCNA exibiu expressão intensa em todas as amostras estudadas. O Ki-67 apresentou expressão variável nos casos de carcinoma e de ceratose e negativo na pele de pálpebra. A intensidade da expressão de PCNA, Ki-67, p53 e Bcl-2, associada a outros indicadores, tais como a análise histopatológica e a apresentação clínica, fornece informações sobre a provável progressão, levando a um diagnóstico mais seguro da neoplasia. / The skin cancer is the most frequent type of human cancer and shows an increase in its incidence in recent years, making it an important public health problem worldwide. In many cases before the onset of carcinoma install a precursor lesion, actinic keratosis, which can develop into squamous cell carcinoma. Considering the increase of new cases and relapses frequent, studies have been intensified in order to determine the parameters with prognostic significance in predicting those tumors which have more aggressive behavior, and can develop metastases. The objective was to evaluate the expression of markers of cell proliferation (PCNA, Ki-67) and apoptosis (p53,Bc1-2) in patients with squamous cell carcinoma and actinic keratosis. We studied samples from 30 patients, ten patients of squamous cell carcinoma, ten with actinic keratosis and ten samples free of lesions underwent blepharoplasty. The p53 protein was expressed in all cases with different quantitative patterns. The Bcl-2 was expressed at low intensity in six cases of actinic keratosis in the skins of blepharoplasty and negative in cases of squamous cell carcinoma. The PCNA showed intense expression in all samples. The Ki-67 showed variable expression in cases of keratosis and carcinoma and negative in the skin of eyelid. In conclusion the intensity of expression of PCNA, Ki-67, P53 and Bcl-2, associated with other indicators such as histopathological analysis and clinical presentation, provides information on the likely progression, leading to a more secure diagnosis of malignancy.

Cardiovascular disease is the leading cause of death in the United States, and coronary artery disease (CAD) kills over 370,000 people annually. There are available therapies that offer a temporary solution; however, only a heart transplant can fully resolve heart failure, and donor organ shortages severely limit this therapy. C-kit+ human cardiac stem cells (hCSCs) offers a viable alternative therapy to treat cardiovascular disease by replacing damaged cardiac tissue; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation has limited the efficacy of stem cell therapy. Tissue engineering solutions offer potential tools to overcome current limitations of stem cell therapy. Materials derived from natural sources such as keratin from human hair offers innate cellular compatibility, bioactivity, and low immunogenicity. Keratin proteins extracted using oxidative chemistry known as keratose (KOS) have shown therapeutic potential in a wide range of applications including cardiac regeneration. My studies utilize KOS hydrogels to modulate c-kit+ hCSC differentiation, and explore the capability of differentiated cells to regenerate vascular tissue. In the first Chapter, we reviewed literature relevant to keratin-based biomaterials and their biomedical applications, the use of stem cells in cardiovascular research, and the differentiation of vascular smooth muscle cells (VSMCs). The section on biomedical applications of keratin biomaterials focuses on the oxidized form of keratin known as keratose (KOS), because this was the material used for our research. Since we planned to use this material in conjunction with c-kit+ hCSCs, we also briefly explored the use of stem cells in cardiovascular research. Additionally, we examined some key signaling pathways, developmental origins, and the cell phenotype of VSMCs for reasons that will become clear after observing results from chapters 2 and 3. Based upon our review of the literature, KOS biomaterials and c-kit+ hCSCs were determined to be promising as a combined therapeutic for the regeneration of cardiac tissue. Research in Chapter 2 focused on characterizing the effects of KOS hydrogel on c-kit+ hCSC cell viability, proliferation, morphology, and differentiation. Results demonstrated that KOS hydrogels could maintain hCSC viability without any observable toxic effects, but it modulated cell size, proliferation, and differentiation compared to standard tissue culture polystyrene cell culture (TCPS). KOS hydrogel produced gene and protein expression consistent with a VSMC phenotype. Further, we also observed novel "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. Results from this study lead us to question what signaling pathways might be responsible for the apparent VSMC differentiation pattern we observed on KOS hydrogels. Research in Chapter 3 explored the time course of VSMC differentiation, cell contractility, inhibition of VSMC differentiation, and measured protein expression of transforming growth factor beta 1 (TGF-β1) and its associated peptides for hCSCs cultured on KOS hydrogels, tissue culture polystyrene, and collagen hydrogels. A review of VSMC differentiation signaling pathways informed our decision to investigate the role of TGF-β1 in VSMC differentiation. Results demonstrated that KOS hydrogel differentiated hCSCs significantly increased expression for all three vascular smooth muscle (VSM) markers compared to TCPS differentiated cells. Additionally, KOS differentiated hCSCs were significantly more contractile than cells differentiated on TCPS. Recombinant human (rh) TGF-β1 was able to induce VSM differentiation on TCPS. VSM differentiation was successfully inhibited using TGF-β NABs and A83-01. Enzyme-Linked Immunosorbent Assay (ELISA) analysis revealed that both TCPS and KOS hydrogel differentiated cells produced TGF-β1, with higher levels being measured at early time points on TCPS and later time points on KOS hydrogels. Results from supplementing rhTGF-β1 to TCPS and KOS hydrogels revealed that KOS seems to interact with TGF-β to a greater extent than TCPS. Western blot results revealed that latency TGFβ binding protein (LTBP-1) and latency associated peptide (LAP) had elevated levels early during differentiation. Further, the levels of LTBP-1 and LAP were higher on KOS differentiated hCSCs than TCPS hCSCs. This study reaffirms previous results of a VSM phenotype observed on KOS hydrogels, and provides convincing evidence for TGF-β1 inducing VSM differentiation on KOS hydrogels. Additionally, results from ELISA and western blot provide evidence that KOS plays a direct role in this pathway via interactions with TGF-β]1 and its associated proteins LTBP-1 and LAP. Results from chapter 2 and 3 offered significant evidence that our cells exhibited a VSMC phenotype, and that TGF-β1 signaling was a key contributor for the observed phenotype, but we still needed an animal model to explore the therapeutic potential of our putative VSMCs. Research in Chapter 4 investigated a disease model to test the ability of KOS hydrogel differentiated cells to regenerate vascular tissue. To measure vascular regenerative capability, we selected a murine model of critical limb ischemia (CLI). CLI was induced in 3 groups (n=15/group) of adult mixed gender NSG mice by excising the femoral artery and vein, and then treated the mice with either PBS (termed as PBS-treated), Cells differentiated on TCPS (termed as Cells from TCPS), or KOS hydrogel-derived VSMCs (termed as Cells from KOS). Blood perfusion of the hind limbs was measured immediately before and after surgery, then 14, and 28 days after surgery using Laser Doppler analysis. Tissue vascularization, cell engraftment, and skeletal muscle regeneration were measured using immunohistochemistry, 1,1'-Dioctadecyl3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiL) vessel painting, and hematoxylin and eosin (HandE) pathohistological staining. During the 4-week period, both cell treatment groups showed significant increases in blood perfusion compared to the PBS-treated control, and at day 28 the Cells from KOS group had significantly better blood flow than the Cells from TCPS group. Additionally, the Cells from KOS group demonstrated a significant increase in the ratio of DiL positive vessels, capillary density, and a greater density of small diameter arterioles compared to the PBS-treated group. Further, both cell-treated groups had similar levels of engraftment into the host tissue. We conclude that Cells from KOS therapy increases blood perfusion in an NSG model of CLI, but does not lead to increased cell engraftment compared to other cell based therapies. Overall, the results from this dissertation demonstrated that KOS hydrogels produce VSMC differentiation from c-kit+ hCSCs mediated by TGF-β1 signaling, and that the differentiated cells are able to increase blood perfusion in a CLI model by increasing capillary density, suggesting enhanced angiogenesis. Future studies should explore potential protein-protein interactions between KOS, TGF-β1 and its associated proteins. Additionally, we should plan animal studies that examine the efficacy of our cells to regenerate cardiac tissue following an acute myocardial infarction (AMI). / PHD

Für die Entstehung nicht-melanozytärer Hauttumore sind mehrere Risikofaktoren verantwortlich: UV-Exposition, Pigmentierung, Alter, Immunsuppression und möglicherweise Humane Papillomviren (HPV). Die molekularen Mechanismen der Tumorgenese des kutanen Plattenepithelkarzinoms (SCC) sowie der Präkanzerose Aktinische Keratose (AK) sind nur lückenhaft bekannt. Fokus dieser Arbeit ist die Untersuchung von SCC-Genexpressionsprofilen sowie der Einfluss kutaner HPV-Typen während der Karzinogenese bei immunkompetenten und immunsupprimierten, organtransplantierten Patienten. Durch Genexpressionsanalyse kutaner SCC, AK und normaler Haut konnten 118 differenziell exprimierte Gene in SCC mittels cDNA-Microarrays identifiziert werden. Bestätigt wurde die Expression von 11 aus 13 ausgewählten Genen (85%) mittels quantitativer real-time RT-PCR (qPCR), dabei konnte eine Korrelation der Genexpression mit der Progression der AK zum SCC für 3 Gene nachgewiesen werden. Dazu zählen das Gen Metalloproteinase-1, kodierend für ein Enzym, das in den Umbau von extrazellulärer Matrix involviert ist, das Protoonkogen RAB31 und das Tenascin-C (Tn-C) kodierende Gen Tn-C. Tn-C war im SCC-Gewebe an der Invasionsfront in Basalzellen sowie Keratinozyten im Stratum papillare und retikulare als Protein nachweisbar, nicht aber in normaler Haut. Die im Rahmen dieser Arbeit erstmalig nachgewiesene 2243 bp-Spleißvariante von Tn-C könnte aufgrund der primären Expression in SCC–Gewebe als diagnostischer Marker für SCC dienen. Diese Daten zeigen, dass simultane, multifaktorielle Dysregulationen von Genexpression und DNA-Reparatur, Zellzyklus und Proliferation, proteolytischen Enzymen und Adhäsionsmolekülen in SCC vorliegen. Ferner wurde die Expression von HPV in SCC und damit der kausale Zusammenhang einer HPV-Infektion mit der Hauttumorgenese untersucht. Das Infektionsmuster von SCC-Gewebe und normaler Haut mit spezifischen HPV-Typen erfolgte durch den Nachweis typenspezifischer HPV-DNA. Virale E6/E7-mRNA-Transkripte der kutanen HPV-Typen 8, 9 und 15 wurden in AK und SCC nachgewiesen. Dagegen konnten in HPV-DNA positiver, gesunder Haut oder Warzen keine HPV-Transkripte gefunden werden. Die Variantenanalyse des offenen Leserahmens von E6 identifizierte eine einzelne, bislang nicht beschriebene Punktmutation mit nicht bekannter Veränderung der Proteinstruktur. Die virale Aktivität der Onkogene E6 und E7 einiger kutaner Typen in AK und SCC weisen auf eine mögliche Rolle von HPV bei der kutanen Hautkarzinogenese hin. / During development of non-melanoma skin cancer, several risk factors are involved: UV-exposition, pigmentation, age, and potentially human papilloma virus (HPV). The molecular mechanisms underlying tumourgenesis in squamous cell carcinoma (SCC) and its pre-cancerosis actinic keratosis (AK) are not fully understood. In this study, the gene expression profile and HPV-infection status were analysed in SCC from immunocompetent and organ transplanted, immunocompromised patients.By global transcriptome analysis from cutaneous SCC, AK and healthy skin, 118 genes were identified differentially expressed in a cDNA-microarray. The expression of 11 out of 13 selected genes (85%) was investigated by real-time RT-PCR (qPCR) and the expression of three genes remarkably induced in SCC correlated with the progression to AK until SCC. These genes encoded for Metalloproteinase-1, which is involved in the remodelling of extracellular matrix, and the protooncogene RAB31 and Tenascin-C (Tn-C). Tn-C protein is expressed in SCC-tissue at the invasion front in basal cells and in keratinocytes in the Stratum papillare and retikulare, but not in healthy skin. This study, the 2243 bp Tn-C-specific splice-variant has for the first time detected in SCC, but not in normal skin. Thus it might serve as diagnostic marker of SCC progression. The data of the transcriptome analysis indicates that a simultaneous dysregulation of oncogene expression and DNA-repair, cell-cycle and proliferation, proteolysis and adhesion molecules exists in SCC. Additionally, the expression of HPV in SCC and thus the causal relationship between HPV-infection and tumourgenesis of SCC in immunocompromised patients was investigated. The HPV-infection pattern in SCC-tissue and normal skin was assessed by detection of DNA from cutaneous HPV-types. Viral E6/E7-mRNA-transcripts of the cutaneous HPV-types 8, 9, 15 were expressed selectively in AK and SCC. In contrast, no HPV-specific mRNA was present in HPV-DNA positive normal skin. The analysis of the open reading frame from the respective E6-protein genes unravelled one single pointmutation, which is not been characterized so far in terms of e.g. its impact on protein structure. The viral activity of the oncogenes E6 and E7 of cutaneous HPV-types indicates a potential function of HPV in the tumourgenesis of SCC in immunocompromised individuals.