Thesis advisor: Junona Moroianu / Some non melanoma skin cancers (NMSC) have been associated with human papillomavirus (HPV) pathogenesis, like epidermodysplasia verruciformis (EV) and squamous cell carcinoma (SCC). EV is a genetically inherited skin disease that develops when the individuals are infected with cutaneous HPV types belonging to the β-genus, especially types 5 and 8. Transgenic mouse lineages expressing all early genes of cutaneous HPV8 develop papillomas, dysplasias and SCC after UV irradiation and this correlates with enhanced HPV8 oncogenes expression. We have previously discovered that the nuclear localization of mucosal HPV16 E7 and HPV11 E7 proteins is mediated by their zinc-binding domain via a Ran-dependent pathway and independent of nuclear import receptors and that a patch of hydrophobic residues within the zinc-binding domain of HPV16 E7 and HPV11 E7 proteins is responsible for their nuclear import via hydrophobic interactions with FG nucleoporins. Here we investigated the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein using confocal microscopy to analyze the intracellular localization of EGFP-8E7, its subdomains and its mutants after transient transfections. We also investigated the nuclear import ability of GST-8E7, its subdomains and mutants using in vitro nuclear import assays in digitonin-permeabilized HeLa cells. In addition, we performed isolation assays to study the direct interaction between HPV8 E7 and two FG nucleoporins, Nup62 and Nup153 or the nuclear export receptor, CRM1. We found that the nuclear import of cutaneous HPV8 E7 is mediated by a nuclear localization signal (NLS) located within its zinc-binding domain. Furthermore, we determined that the hydrophobic residues within the 65LRLFV69 patch are responsible for the nuclear import and nuclear localization of HPV8 E7 via direct hydrophobic interactions with FG nucleoporins, Nup62 and Nup153, whereas the positively charged arginine 66 plays no significant role in the function of the NLS. In addition, we examined the nuclear export mechanism of cutaneous HPV8 E7 protein and showed that it has a leucine-rich nuclear export signal (NES) in its C-terminal domain that is recognized by the CRM1 nuclear export receptor. These studies are essential for understanding the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Non Melanoma Skin Cancer (NMSC) affects 3.3 million Americans each year and results from Ultra Violet Radiation (UVR) damage to DNA in the form of pyrimidine dimers and photoproducts [1]–[5]. Cells directly detect the damage and initiate apoptosis, cell cycle arrest, or DNA repair by modulating p53 and p21 levels [6]–[9]. Current methods of photoprotection include sunscreen, but controversy over safety of some active ingredients necessitates research into more natural alternatives [10]–[12]. In particular, 24 hour incubation with bovine milk sphingomyelin (BSM) has demonstrated photoprotective potential by reducing p21 and p53 levels in keratinocytes (KRTs) after UV radiation [13], [14]. This thesis aims to expand on past BSM research by exploring the mechanism for photoprotection. Normally, sphingomyelin (SM) is metabolically degraded to ceramide which then leads to cell apoptosis [6]. The goals of this thesis were to characterize a fluorescent SM (FSM) to assess changes in intracellular fluorescence distribution after various incubation and post-UV exposure times. FSM was deemed functionally equivalent to BSM by reducing levels of p21 after UV. Furthermore, quantification demonstrated that FSM trafficking and intracellular fluorescence were independent of continuous incubation time, warranting further investigation into shorter timepoints like 1 hour. Across several post-UV timepoints, the 1 hour incubation had a consistently higher average cytoplasmic mean gray value compared to 24 hour incubation. In addition, the no UV control was significantly lower compared to the 24 hour and 12 hour post-UV timepoints. No post-UV differences were observed for the 24 hour incubation, suggesting future work is necessary for the 1 hour incubation, which potentially streamlines future experiments. Two immunofluorescence stains for endogenous SM (lysenin) and ceramide were also optimized for preliminary fluorescence distribution studies and colocalization with FSM. Finally, a 3T3 fibroblast spheroid model was utilized as proof-of-concept for future 3D KRT cultures and depth of dye penetration quantification methods. These findings suggest FSM is an appropriate model for BSM trafficking, a shorter FSM incubation time could potentially be adopted in future studies, dual immunofluorescence staining for SM and ceramide is viable, and spheroids provide a promising model for future 3D KRT studies.

Non-melanoma skin cancer (NMSC) is the most common malignant tumor, representing more than a third of all malignant tumors combined and the incidence is increasing every year. Ultraviolet (UV) radiation from the sun is the most dominant factor contributing to tumor initiation and progression. The condition is most prevalent in populations with lighter skin and older age. Current pharmaceutical molecular research targets the inhibition of the Epidermal Growth Factor Receptor (EGFR), a receptor which is commonly over-expressed or dysregulated in skin malignancies. This study evaluates the content and location of the damage marker p21 within keratinocytes that were incubated in sphingomyelin (SM) and later exposed to UV. Confocal microscopy and automated image processing provided the tools to assess large populations of keratinocytes in the effort to accurately identify the photoprotective qualities of sphingomyelin. Classification of individual cells into subpopulations yielded results suggesting SM may be involved in the inhibition of EGFR, and could potentially be a more naturally derived treatment.

No description available.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / A sociedade tem passado por um conjunto de transformaÃÃes em sua estrutura. Um dos aspectos mais interessantes dessa transformaÃÃo à observar como a juventude passa por tais mudanÃas de maneira rÃpida, mas nem sempre guiada pelo aspecto qualitativo desse processo. A juventude, principalmente da periferia, està sendo vÃtima de uma ordem social que se estrutura na Ãtica da desumanizaÃÃo. Tendo como base essa premissa, a presente pesquisa tem como objetivo compreender o papel formativo do Movimento Nacional dos Meninos e Meninas de Rua (MNMMR) na Comunidade do Lagamar atravÃs da perspectiva dos participantes, observando esse processo dentro de uma experiÃncia de construÃÃo da resiliÃncia e do empoderamento. Para compreender esse processo formativo (MACEDO, 2010), utilizei da construÃÃo das histÃrias de vida (DELORY-MOMBERGER, 2008; FERRAROTTI, 1996; NÃVOA & FINGER, 2010; PINEAU, 2006, 2012), de acordo com os procedimentos da entrevista narrativa (JOVCHELOVITCH & BAUER, 2002) buscando compor traÃos identitÃrios importantes do MNMMR do Cearà que acaba por completar 30 anos de histÃria em 2015. A categoria da resiliÃncia (ANTUNES, 2011; ASSIS, 2006; CYRULNIK, 2004, 2012; MELILLO & OJEDA, 2005; POLETTI, 2013; YUNES, 2003) e do empoderamento forjado no processo de lutas e conquistas sociais (FREIRE, 1983, 1986, 2006; LOBO, 2011; SAWAIA, 2008) ajudam a compreender esse processo de exclusÃo social e de tomada de consciÃncia crÃtica frente Ãs adversidades sociais. Nas histÃrias de vida, fica evidenciado a importÃncia social do MNMMR, tornando-se um movimento social importante para a Comunidade do Lagamar por atuar com os sujeitos em situaÃÃo de rua. A relevÃncia mostra que as dimensÃes de resiliÃncia e do empoderamento, podem servir de significados para a melhoria de uma formaÃÃo humana propiciada pelo prÃprio MNMMR. O presente trabalho investigativo traz à tona um diÃlogo formativo, que contempla tambÃm um processo educativo, uma aÃÃo educativa construÃda no seio da rua, tendo como protagonista a juventude da periferia da cidade de Fortaleza.

No description available.

In den letzen Jahren gab es durch epidemiologische und molekularbiologische Studien vermehrt Hinweise, dass kutane humane Papillomviren (HPV) ursächlich an der Entstehung nicht-melanozytärer Hauttumore (engl. NMSC) beteiligt sind. Ziel der vorliegenden Arbeit war die Identifizierung molekularer Mechanismen der viralen Proteine E6 und E7 kutaner HPV-Typen. Die E6 oder E7 Gene der verschiedenen HPV-Typen 1, 4, 5, 8, 20, 38 und RTRX7 wurden untersucht. Natürliche Wirtszellen dieser Viren, humane primäre Keratinozyten (HPK) der Haut, wurden mit rekombinanten, für E6 oder E7 kodierenden Retroviren infiziert. Die Analysen erfolgten in Monolayer-Kultur (undifferenzierte Keratinozyten) oder in organotypischen Hautmodellen (Induktion der Keratinozytendifferenzierung). Die Expression von E6 oder E7 führte in Monolayer-HPK zu einer Verlängerung der Lebensspanne und zu einer deutlich erhöhten Verdoppelungsrate. Eine Telomeraseaktivierung, die charakteristisch für immortale Zellen ist, wurde nur in HPV 8 E6 positiven HPK nachgewiesen. In organotypischen Hautmodellen induzierte das E7 Protein von HPV 1, 4 und 38 starke Veränderungen in der Differenzierung sowie eine Zunahme der Proliferation. Weiterhin wurde eine Aufhebung der normalen Zellzykluskontrolle in suprabasalen HPV 5 E7 oder HPV 8 E7 beobachtet. Hinweise auf ein starkes invasives Potential von E7-infizierten HPK wurden für HPV 8 E7 bestätigt und für HPV 4 E7, HPV 38 E7 und RTRX7 E7 erweitert. Molekulare Mechanismen der viralen Gene E6 und E7 kutaner HPV unterscheiden sich von mukosalen Typen. Das Mehrstufenmodell der Karzinogenese beinhaltet eine Reihe fundamentaler Zelltransformationen, die für eine Tumorgenese nötig sind. In dieser Arbeit beschriebene Mechanismen der Modulation der Zelldifferenzierung und Zellproliferation durch die kutanen HPV-Typen 4, 5, 8 und 38 können unter Umständen zur Induktion und Progression früher Stadien von Plattenepithelkarzinomen (SCC) beitragen. / In the last years epidemiologic and molecular biological studies accumulated increasing evidence that cutaneous human papillomaviruses are etiologically involved in the formation of non-melanoma skin cancer (NMSC). The presented work aims to identify the underlying molecular mechanisms of the viral proteins E6 and E7 of cutaneous HPV types. The E6 and E7 genes of the different HPV types 1, 4, 5, 8, 20, and RTRX7, which are in vivo associated with cutaneous benign or malignant lesions, were studied. Natural host cells of these viruses, human primary keratinocyts (HPK) of the skin, were infected with recombinant E6 and E7 encoding retroviruses. The following analyses were performed in monolayer culture (non-differentiated keratinocytes) or in organotypic skin culture (induction of keratinocyte differentiation). The expression of E6 and E7 elongated the life span of monolayer HPK and significantly increased the doubling rate. An activation of the telomerase, characteristic for immortalized cells, was only detected in HPV 8 E6 positive cells. In organotypic skin cultures E7 of HPV 1, 4 and 38 induced drastic changes in differentiation and proliferation. Additionally an impairment of the normal cell cycle control in suprabasale HPV 5 E7 and 8 E7 cultures was seen. Hints for a strong invasive potential of E7 infected HPK were proven for HPV 8 E7 and expanded to HPV 4 E7, HPV 38 E7 and RTRX E7. The viral E6 and E7 genes of cutaneous and mucosal HPV types exhibit different molecular mechanisms. The multistep model of carcinogenesis includes a series of fundamental cell transformations necessary for tumorigenesis. Mechanisms for the modulation of cell differentiation and proliferation by cutaneous HPV types 4, 5, 8 and 38 described in this work could potentially contribute to the induction and progression of early stages of squamous cell carcinoma.

No description available.

Genetic changes are hallmarks of cancer development involving the activation and/or inactivation of oncogenes and tumour suppressor genes, respectively. In non-melanoma skin cancer (NMSC) development, the initiation of genetic mutations results from exposure to solar ultraviolet radiation. Non-melanoma skin cancers are comprised of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Several related cutaneous lesions also exist, of which solar keratoses (SK) are widely accepted as a precursor dysplasia to SCC development. The study of recurrent genetic changes present within NMSC and SK should help reveal causative mutations in skin cancer development. Such analysis could also elucidate links in the genetic similarity of these dysplasia. The rapid screening of numerical changes in DNA sequence copy number throughout the entire genome has been made possible by the advent of comparative genomic hybridisation (CGH). This technique enables the identification of net gains and loss of genetic material within a tumour DNA sample. Chromosomal regions of recurrent gain or loss identify loci containing putative oncogenes and tumour suppressor genes, respectively with potential roles in NMSC tumourigenesis. Used in conjunction with tissue microdissection and universal degenerate PCR techniques this can enable the elucidation of aberrations in small histologically distinct regions of tumour. Such a technique can utilize archival material such as paraffin embedded tissue, which is the major source of neoplastic material available for cancer research. This study used the CGH technique to investigate aberrations in BCC, SCC and SK samples. The screening of copy number abnormalities (CNAs) in BCC revealed that although these tumours were close to diploid and generally genetically stable, they did contain several recurrent aberrations. The loss of genetic material at 9q was identified in a third of BCC tumours studied. This is characteristic of inactivation of the PTCH tumour suppressor gene, a known attribute in some sporadic BCC development. Validation of this loss was performed via loss of heterozygosity, demonstrating good concordance with the CGH data. In addition the over-representation of the 6p chromosome arm was revealed in 47% of biopsies. This novel CNA is also commonly observed in other cutaneous neoplasias, including Merkel cell carcinoma and malignant melanoma. This suggests a possible common mechanism in development and or promotion in these cutaneous dysplasias, the mechanisms of which have yet to be clearly defined. In contrast to BCC, numerical genetic aberrations in SCC and SK were much more frequent. Several regions of recurrent gain were commonly shared between both dysplasias including gain of 3q, 4p, 5p, 8q, 9q, 14q, 17p, 17q and 20q. Common chromosomal regions of loss included 3p, 8p, 9p, 11p, 13q and 17p. In addition loss of chromosome 18 was significantly observed in SCC in comparison to SK, a possible defining event in SK progression to SCC. The identification of shared genetic aberrations suggests a clonal and genetic relationship between the two lesions. This information further supports the notion for re-classification of SK to an SCC in situ or superficial SCC. Finally, the CNAs detected have been similarly observed in other squamous cell-derived tumours, for example cervical and head and neck SCC. This provides further evidence to common mechanisms involved in the initiation, development and progression of SCC neoplasia. This study has identified a number of recurrent chromosomal regions, some of which are novel in NMSC development. The further delineation of these loci should provide additional evidence of their significance and degree of involvement in NMSC tumourigenesis. The identification of the cancer-causing genes mapped to these loci will further demarcate the genetic mechanisms of BCC and SCC progression. An understanding of the events involved in skin cancer formation and progression should shed additional light on molecular targets for diagnostics, management and therapeutic treatment.

The purpose of this study was to examine the trends, attrition and retention rates of National Merit Finalists at the University of Central Florida between the years of 1997 to 2005. This study was intended to provide information for higher education practitioners, faculty, and administrators to help them better understand the expectations and current trends of National Merit Finalists. The problem was to determine how to increase recruitment and retention while decreasing the attrition rates of these highly desirable students. The importance of this study includes identifying trends that may aid in future recruitment efforts for National Merit Finalists; finding the causes of dissatisfaction towards the University among these students; and identifying specific areas in which to alleviate those dissatisfactions. The results will hopefully provide insight into specific recruitment, services, and programming options for these students. The study examined data that was collected from the University of Central Florida's Burnett Honors College database known as FileMaker 8.0. The data examined characteristics such as grade point averages (high school and college); valedictorian and salutatorian status; test scores (SAT and ACT); Honors in the Major (undergraduate thesis) students; Honors and university status (withdrawn, probation, removed, disqualified, enrolled, graduated); Honors college attrition; university attrition; ethnicity; gender ratios; majors; and, prestigious scholarships awarded in college (such as the Rhodes, Truman, Marshall). The actual size of the sample was one hundred ninety-eight National Merit Finalists. Data was also collected from a survey given to all University of Central Florida National Merit Finalists. Descriptive statistics were reported for each of the components examined. This data examined the types of scholarship packages that National Merit Finalists were offered; the reasons students chose the University of Central Florida over other universities; the college recruitment process; hours studied for the PSAT; siblings; perceptions on being a National Merit Finalist; the number of times students changed their majors; job status; transportation; computer attainment; disabilities; and the potential disadvantages of being labeled as a National Merit Finalist. The data could be utilized to examine the trends of our National Merit Finalists, in order to see what is working and what is not in terms or recruitment and retention; and also to further examine what these students want from their institutions. Findings indicated that problems exist in regard to the following: the recruitment of female and minority National Merit Finalists; males historically score higher on the SAT than females; decreasing the attrition rates of this population at the University of Central Florida; the majority of National Merit Finalists at the University of Central Florida come from Florida; the majority of National Merit Finalists at the University of Central Florida do not tend to be high school salutatorians or valedictorians; high school counselors seem to be the least effective tool for recruiting National Merit Finalists at the University of Central Florida; and the majority of National Merit Finalists at the University of Central Florida did not study at all for the PSAT test. However, the University of Central Florida is extremely competitive with other institutions of higher education with regard to scholarship packages. Results also revealed the following: the SAT is a more widely accepted tool for determining NMSC status as opposed to the ACT; the majority of National Merit Finalists have a GPA between 3.600 and 3.999 at the University of Central Florida; the University of Central Florida is succeeding in making its National Merit Finalists feel special during the recruitment process; the most influential reason that National Merit Finalists are choosing UCF is based upon the financial scholarship packages they are offered; and the majority of National Merit Finalists at the University of Central Florida do not feel that there are disadvantages toward being labeled as such. This data provides a basis for further research on National Merit Finalists trends, attrition, and retention. Practical considerations are revealed in the data that will influence future recruitment methods and lead to higher retention rates and increased student satisfaction. Several other recommendations are made to conduct further research studies on the trends, attrition, and retention rates of National Merit Finalists.