Current ACS guidelines recommend routine screening for cancer (ACS, 2002). Motivation to adhere to guidelines may be different for individuals with and without a family history of melanoma (Jonna, et al., 1998). Prior research examining the relationship between family history and skin cancer detection behaviors (Berwick et al., 1996; Friedman et al., 1993; Oliveria et al., 1999) have failed to utilize a theoretical framework to derive hypotheses. The purpose of the present study was to examine the utility of Protection Motivation Theory (PMT) in explaining intentions to engage in skin cancer screening (SCS) and skin self-examination (SSE). In addition, the present study explored whether PMT variables explained the relationship between having a family history of melanoma and SCS/SSE intentions. The research design was cross-sectional with 101 participants in the positive family history group and 80 participants in the negative family history group. Using a standardized, self-report measure, participants were assessed on demographic characteristics, melanoma risk factors, PMT variables, family history, and SCS/SSE behaviors and intentions. Statistical analyses included descriptive statistics, chi square for categorical variables, t-tests for continuous variables, correlational analyses, and multiple regression analyses. The majority of participants (N = 181) were Caucasian (97%) and female (59%). As expected, findings indicated that greater perceived vulnerability, self-efficacy, and response efficacy were associated with greater SCS intentions (p greater or less than .0001). Additionally, greater self-efficacy and response efficacy were associated with greater SSE intention (p greater or less than .01). Additionally, positive family history individuals reported greater perceived vulnerability, greater self-efficacy, and less perceived severity than negative family history individuals (p greater or less than .01). Individuals with a family history of melanoma also had greater SCS intentions and were more likely to have a healthcare provider who recommended SCS. Finally, perceived vulnerability and self-efficacy partially mediated the relationship between group status and SCS intentions. The present study confirms and extends prior research on psychological factors associated with SCS/SSE intentions and on individuals with a family history of melanoma. Clinical implications and future directions are discussed.

In western societies, skin cancer incidence has increased dramatically over recent decades, due predominantly to increased sun exposure habits. Ultraviolet (UV) light exposure and individual light sensitivity of the skin constitute two important factors affecting the risk for skin cancer development. Individuals with a heightened propensity to get sunburnt have a higher risk for skin malignancies, and need to protect themselves more systematically from the sun. Individual UVlight sensitivity can be determined either by self-estimation of tendency to burn and tan, as in the Fitzpatrick’s classification, or by use of a phototest. Although phototesting constitutes a considerably more objective method, it is only sparsely used, chiefly due to financial and resource related factors, and is mainly limited to investigation of photodermatoses or dose-management in photo therapy. The general aim of this thesis was to develop and improve aspects of the phototest procedure in rder to broaden the utilisation of phototesting within the fields of research, clinical practice and skin cancer prevention. As a first step, a new phototesting technique, using a divergent UVB beam was evaluated. The principle of the method is to provoke a circular UVB-erythema in the skin, the diameter of which is related to the administered dose and thus the Minimal Erythema Dose (MED). In a test group of healthy subjects, naked eye reading by a trained observer resulted in a more exact, estimation of UVB-sensitivity, compared to traditional phototesting. Since the diffuse border of the provoked erythema was challenging for the untrained observer to read, the need for an objective, bio-engineering technique for test reading was clear. In this thesis, Laser Doppler perfusion imaging (LDPI) has been used. This data also enabled an objective description of doseresponse for the reaction, an outcome not possible in traditional testing. The divergent beam method was also shown to be useful as a model for evaluation of the effect of topically applied substances. In order to broaden the utilisation of phototests in general, a test procedure built on patient performed self-reading of skin tests (a traditional phototest and an irritant patch test) was evaluated. The reliability of these self-readings was shown to be substantial when compared to the control readings of a trained observer. Using the self-reporting procedure, phototesting was evaluated as a tool in primary prevention of skin cancer. The study focussed on sun habits and sun protection behaviour, and also on investigating the impact of different forms of presentation of the preventive information. Results showed significantly higher impact for a personally mediated preventive message than by letterform. For individuals with heightened UV-sensitivity the performance of a phototest led to a greater tendency to adopt sun protection behaviour than for subjects with a lower UV-sensitivity, suggesting that phototesting is a useful way to improve the outcome in terms of preventive behaviours for this group of susceptible, at-risk individuals. Divergent beam phototesting, patient-performed self-reading, and the application of phototesting in skin cancer prevention emerge as three novel, previously little investigated, aspects of phototesting, for which promising results could be demonstrated. / Under de senaste årtiondena har insjuknandet i hudcancer ökat dramatiskt i västvärlden, detta till stor del beroende på förändrade solvanor. Exponering för solens ultravioletta strålning (UVstrålning) samt den individuella ljuskänsligheten i huden utgör två viktiga faktorer av betydelse för uppkomsten av hudcancer. Individer med ökad benägenhet att bli rödbrända i solen löper också ökad risk för hudcancer av solexponering, och behöver således vara extra noga med att skydda sig mot solen. Hur känslig man är mot solljuset kan bedömas antingen genom självskattning (klassificering enligt Fitzpatrick), eller genom att använda ett ljustest. Det sistnämnda är en betydligt mer objektiv metod, men används ändå relativt sparsamt, sannolikt ofta beroende på brist på resurser, tid eller klinisk rutin. Det övergripande syftet med avhandlingen var att utveckla och förbättra aspekter på ljustestningsförfarandet med inriktning på att kunna bredda användningen av ljustest inom forskning, klinisk verksamhet och hudcancerprevention. Som ett första steg undersöktes och utvärderades en ny ljustestteknik, baserad på en divergent (spridd) UV-stråle. Genom att belysa huden med ett cirkulärt UV-ljusfält framkallas en cirkulär rodnad (erytem), där diametern på rodnaden står i relation till den individuella ljuskänsligheten i huden. I jämförelse med traditionell ljustestningsmetodik visade sig metoden resultera i en noggrannare uppskattning av ljuskänslighet, samt möjligheten att beskriva ett dos-responsförhållande inom det rodnade hudområdet. Eftersom kanten på den framkallade rodnaden tenderade att bli ganska diffust avgränsad framkom dock, med undantag för speciellt tränade avläsare, svårigheter att läsa av testet med enbart ögats hjälp. Av den anledningen krävdes mer objektiv, hudfysiologisk mätmetodik. I de genomförda studierna användes så kallad Laser Doppler perfusion imaging (LDPI) för detta. Förutom uppskattning av ljuskänsligheten testades den divergenta UV-strålen också som modell för skattning av anti-inflammatorisk effekt av ämnen som appliceras på huden, exempelvis cortison, och visade sig användbar för detta. I syfte att öka förutsättningarna för bredare användning av ljustest, genomfördes en studie där försökspersonerna själva fick avläsa ett traditionellt ljustest och rapportera in testresultatet. Resultaten jämfördes med avläsningar utförda av en kunnig avläsare, och visade på god tillförlitlighet. Slutligen, med hjälp av den beskrivna självavläsningsproceduren, undersöktes i en primärvårdspopulation, om ljustestning kan vara användbart för att förebygga hudcancer, med inriktning på att påverka individers solvanor, solskyddsbeteende och attityder gentemot solning. I studien jämfördes även olika modeller för att presentera ett preventionsbudskap, och där ett muntligt sådant, förmedlat vid ett läkarbesök, hade ett betydligt bättre genomslag än motsvarande, enbart skriftlig, information. För individer med hög ljuskänslighet bidrog ljustestet till ökat solskyddsbeteende, vilket indikerar att ljustest skulle kunna vara ett användbart verktyg i eftersträvan att förebygga hudcancer speciellt i denna grupp av individer med förhöjd hudcancerrisk. Sammanfattningsvis utgör ljustestning med divergent UV-stråle, självavläsning av ljustest samt användning av ljustest vid hudcancerprevention tre nya, tidigare sparsamt undersökta aspekter på ljustestning, för vilka den här avhandlingen visar lovande resultat.

Title from PDF of title page. / Document formatted into pages; contains 117 pages. / Thesis (Ph.D.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT Current ACS guidelines recommend routine screening for cancer (ACS, 2002). Motivation to adhere to guidelines may be different for individuals with and without a family history of melanoma (Jonna, et al., 1998). Prior research examining the relationship between family history and skin cancer detection behaviors (Berwick et al., 1996; Friedman et al., 1993; Oliveria et al., 1999) have failed to utilize a theoretical framework to derive hypotheses. The purpose of the present study was to examine the utility of Protection Motivation Theory (PMT) in explaining intentions to engage in skin cancer screening (SCS) and skin self-examination (SSE). In addition, the present study explored whether PMT variables explained the relationship between having a family history of melanoma and SCS/SSE intentions. / ABSTRACT: The research design was cross-sectional with 101 participants in the positive family history group and 80 participants in the negative family history group. Using a standardized, self-report measure, participants were assessed on demographic characteristics, melanoma risk factors, PMT variables, family history, and SCS/SSE behaviors and intentions. Statistical analyses included descriptive statistics, chi square for categorical variables, t-tests for continuous variables, correlational analyses, and multiple regression analyses. The majority of participants (N = 181) were Caucasian (97%) and female (59%). As expected, findings indicated that greater perceived vulnerability, self-efficacy, and response efficacy were associated with greater SCS intentions (p greater or less than .0001). Additionally, greater self-efficacy and response efficacy were associated with greater SSE intention (p greater or less than .01). / ABSTRACT: Additionally, positive family history individuals reported greater perceived vulnerability, greater self-efficacy, and less perceived severity than negative family history individuals (p greater or less than .01). Individuals with a family history of melanoma also had greater SCS intentions and were more likely to have a healthcare provider who recommended SCS. Finally, perceived vulnerability and self-efficacy partially mediated the relationship between group status and SCS intentions. The present study confirms and extends prior research on psychological factors associated with SCS/SSE intentions and on individuals with a family history of melanoma. Clinical implications and future directions are discussed. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

In the last fifteen years, a great deal of effort has been put forth, worldwide, for investigating and enhancing various aspects of optical coherence tomography (OCT). This thesis begins with a description of the technique of OCT, and an analysis of its underlying theory. The design and construction of an OCT system is described, with particular emphasis on a novel delay scanning method, and novel signal processing. Application of OCT to non-destructive characterisation of seeds, examination of skin lesions, measurement of fluid flow, and refractive index determination, are then demonstrated. Two technological enhancements to OCT are presented in this thesis. The first, an extended-range Fourier domain optical delay line (FDODL), extends the scan range of the traditional FDODL by a factor of almost 9, by scanning the galvanometer mirror around the region of zero tilt-angle. Polarisation optics are used to prevent light coupling back into the interferometer after only a single pass through the FDODL. A non-coplanar version of the FDODL is also presented, which overcomes the losses associated with the polarisation-based design, but trades off scan range to do so. Both versions of FDODL demonstrated excellent linearity and scan uniformity. The second technology presented here, bifocal optical coherence refractometry (BOCR), affords OCT the ability to measure refractive indices within turbid media. It achieves this by generating two confocal gates within the sample. From knowledge of the system parameters, and measurements of the confocal gate separation, the refractive index within the medium is evaluated to within ±0.01. Refractive index mapping is then demonstrated in a number of turbid samples. Three other applications of OCT are also demonstrated in this thesis. The first is the use of OCT to measure full thickness in lupin seeds. Although OCT could not penetrate the entire thickness of the hull, it is demonstrated that the variation in thickness of the two layers observed with OCT, explained 81% of the variation in thickness of the entire hull measured under a SEM. OCT was then applied, for what is believed to be the first time, in a large scale seed screening program. The second application is a preliminary investigation of the suitability of OCT to aid in the diagnosis of skin lesions. Although our system did not possess sufficient positioning accuracy to enable a direct one-to-one comparison between OCT and histology, a number of correspondences between OCT and histology images were demonstrated. The final application of OCT demonstrated here is a novel phase-locked-loop based demodulation scheme, to perform Doppler OCT. This demodulation scheme demonstrated a dynamic range of 98dB, a velocity range of ±20mm/s, and velocity resolution of 0.5mm/s. Using this system, laminar flow was demonstrated in milk flowing through a capillary tube.

[Truncated abstract] Melanoma cell adhesion molecule (MCAM) is highly expressed in more than 70% of metastatic melanoma and is correlated with invasive potential. However, the specific contribution MCAM makes to invasion and metastasis in melanoma is not clear. In this study, I have demonstrated that transfection of MCAM into MCAM-negative melanoma and CHO cells leads to changes in cell shape, and the modulation of cell-to-cell and cell-matrix interactions. MCAM positive cells were slower to spread on collagen type I, collagen type IV and laminin 1 than MCAM negative cells, although these differences were not apparent on vitronectin, fibronectin and laminin 10. In contrast, MCAM expression had little effect on cell adhesion to any of the matrices tested. MCAM positive (compared to negative) cells also showed morphological changes and a rearrangement of the actin cytoskeleton when plated on a matrix containing laminin 5. Taken together, these data suggest that MCAM expression modulates β1-integrinmediated spreading on matrix, but has little effect on αvβ3-mediated cell-matrix interactions. As this study provided little evidence to suggest that MCAM transfection altered β1 integrin expression levels on melanoma cells, it is proposed that a competitive interaction between the cytoplasmic domains of MCAM and β1 integrin may affect mature focal adhesion assembly. MCAM expression in melanoma cells was also associated with decreased cell movement over matrix into a scratch-wound site and an increased tendency to form cell cords on Matrigel. These two assays gauge the propensity of a cell to engage in cell-cell versus cell-matrix interactions, and suggest that MCAM positive cells favour cell-cell adhesion. Interestingly, MCAM transfection was also associated with an increased ability of melanoma cells to migrate through a basement membrane towards a chemoattractant. ... Analysis of the intracellular domain of MCAM revealed the presence of tyrosine and dileucine endocytosis signals. Interestingly, disruption of these two motifs did not seem to impair the internalization of MCAM from the cell surface. The di-leucine motif, however, was necessary for the recycling of MCAM back to the surface following endocytosis. Lastly, MCAM was found to exists as dimers within the cell membrane in the absence of ligand, although the exact location of the dimerization motif is not yet clearly defined. Collectively, findings from my study suggest: MCAM expression in melanoma cells facilitates cell-cell interactions, whilst concomitantly modulating cell-matrix interactions. MCAM transfection also leads to enhanced migration of melanoma cells through a basement membrane. Thus, MCAM expression may increase the ability of melanoma cells to migrate as a collective, a feature of highly invasive cancer. The intracellular domain of MCAM interacts with ApxL2, a novel member of the Shroom family of actin-binding proteins. It is likely that ApxL2 links a proportion of MCAM within the cell to the actin cytoskeleton, contributing to cell shape determination and other processes, such as migration. MCAM exists as dimers on the cell surface and is internalized at least partially by a clathrin-mediated mechanism.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-10-13T15:45:24Z No. of bitstreams: 2 Tese - Daniela Cristina Vinhal - 2016.pdf: 4607774 bytes, checksum: 60991eb50ad135a43a5bb84902551bba (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-10-14T19:58:28Z (GMT) No. of bitstreams: 2 Tese - Daniela Cristina Vinhal - 2016.pdf: 4607774 bytes, checksum: 60991eb50ad135a43a5bb84902551bba (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-14T19:58:28Z (GMT). No. of bitstreams: 2 Tese - Daniela Cristina Vinhal - 2016.pdf: 4607774 bytes, checksum: 60991eb50ad135a43a5bb84902551bba (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-30 / In Brazil, the estimates for the year 2016 point to approximately 596,000 new cases of skin cancer, being mostly related to exposure to ultraviolet radiation. According to current consensus, such damage can be minimized by prophylactic use of sunscreens. This way, this study proposes the planning, synthesis, structural characterization and toxicological assessment of a new candidate compound to organic sunscreen, LQFM184 (20). The structural planning was based both on molecular similarity in chemical action triazine derivatives (10, 11 and 13), cinnamic (4) and LQFM048 (14), with the starting compound (E)-2-cyano-3-(4-hydroxy-3-methoxyphenyl)acrylamide (18) which was produced from vanillin. In addition, LQFM184 (20) compound has molecular weight 729.65 Daltons and the synthetic route proposal consisted of two stages, 53% overall yield, which were performed under the scope of green chemistry. All the synthesized compounds were characterized by techniques of nuclear magnetic resonance, infrared, mass, ultraviolet and fluorescence. From the spectrophotometric absorption technique it was identified that LQFM184 (20) compound had two peaks of maximum absorption (λmax.) 306 nm in the region of UVB and 344 nm for UVA and molar absorptivity (ε) whose values were above 104 M-1 cm-1. As to the technique of cyclic voltammetry LQFM184 (20) compound was stable considering the applied potential and when under an atmosphere of synthetic air showed a weight loss of ~14% in the range of 250°C, characterized as a stable compound at high temperatures. From the in vitro evaluation by the spectrophotometric method described by Mansur it presented Solar Protection Factor (SPF) equal to 3.69 for the UVB. Moreover, the compound does not promote irritation or serious damage when in contact with rabbit cornea cells SIRC and was classified in category 4 of the GHS system as OECD 423, low toxicity. At the end of this work it is possible to observe that the proposed synthetic route to obtain the LQFM184 (20) compound was effective, given the obtained high yields, laborious and low cost steps as well as promising profile sunscreen. / No Brasil, as estimativas para o ano de 2016 apontam para a ocorrência de, aproximadamente, 596 mil casos novos de câncer de pele, sendo em sua maioria relacionados com a exposição à radiação ultravioleta. Segundo consenso atual, tais danos podem ser minimizados através do uso profilático de protetores solares. Neste sentido, o presente trabalho propôs o planejamento, a síntese, a caracterização estrutural e a avaliação toxicológica de um novo composto candidato a fotoprotetor orgânico, o LQFM184 (20). O planejamento estrutural baseou-se tanto na semelhança molecular quanto na ação química de derivados triazínicos (10, 11 e 13), cinâmicos (4) e do LQFM048 (14), tendo como partida o composto (E)-2-ciano-3-(4-hidroxi-3-metoxifenil)acrilamida (18) que foi produzido a partir da vanilina. O composto LQFM184 (20) apresentou peso molecular de 729,65 Daltons e a rota sintética proposta constituiu-se por três etapas com rendimento global de 53%, das quais foram realizadas sob o âmbito da química verde. Todos os compostos sintetizados foram caracterizados através das técnicas de ressonância magnética nuclear, infravermelho, massas, ultravioleta e fluorescência. O composto LQFM184 (20) apresentou dois picos de absorção máxima (λmáx.), sendo 298 nm na região da UVB e 334 nm na UVA, além de coeficiente de absortividade molar (ε) acima de 104 M-1 cm-1. Quanto à voltametria cíclica o composto LQFM184 (20) manteve-se estável frente aos potenciais aplicados e quando exposto à atmosfera de ar sintético apresentou uma perda de massa de ~14% na faixa de 254°C, caracterizando-o estável sob temperaturas elevadas, visto que seu ponto de fusão ocorre em 147ºC. A partir da avaliação in vitro pelo método espectrofotométrico descrito por Mansur o mesmo apresentou Fator de Proteção Solar (FPS) igual a 3,69 para a UVB. Além disso, o composto não promove irritação ocular ou lesões oculares graves quando em contato com células de córnea de coelho SIRC e foi classificado na categoria 4 do sistema GHS conforme OECD 423, de baixa toxicidade. Ao término deste trabalho podemos observar que a rota sintética proposta para a obtenção do composto LQFM184 (20) se mostrou eficaz, face aos altos rendimentos obtidos, etapas pouco laboriosas e de baixo custo, bem como promissor perfil fotoprotetor.

O mecanismo de interferência por RNA (RNAi) é um evento de silenciamento gênico através da degradação do RNA mensageiro. Desta forma, a administração de siRNA (molécula efetora do RNAi) é uma terapia promissora para o tratamento de diversas doenças como o câncer. Porém, para a sua efetiva aplicação terapêutica é necessário o desenvolvimento de sistemas de liberação capazes de liberar o siRNA nas células alvo, promover a sua internalização celular e evitar a sua degradação. Dentre os sistemas de liberação, os de liberação localizada, como os sistemas de geleificação in situ, apresentam vantagens sobre administração sistêmica. Formulações fluidas precursoras (FFP) que formam sistemas líquido cristalinos viscosos in situ podem ser obtidas a partir de lipídeos anfifílicos que absorvem água do meio e se rearranjam. Neste contexto, a presente pesquisa teve como objetivo avaliar o gel formado in situ a partir da FFP (G-FFP), composta por monoglicerídeos (MO), polietilenoimina (PEI), propilenoglicol (PG) e tampão tris, como sistema de liberação localizada de siRNA na terapia do câncer de pele. Os resultados mostraram que o G-FFP é uma mistura de fase cúbica e fase hexagonal. O G-FFP liberou o siRNA de maneira sustentada e complexado ao PEI. A FFP pode ser esterilizada por filtração em membrana e foi capaz de complexar altas concentrações de siRNA (15 mM) e de proteger o siRNA da degradação. A citotoxicidade foi dependente da concentração de FFP e esta quando complexada com siRNA teve a toxicidade diminuída. O siRNA liberado do G-FFP foi internalizado pelas células A431, FaDu, HeLa, A549, WM35/DLC2-GFP e MCF-7/DLC2-GFP. Além disto, siRNAs específicos liberados pelo G-FFP foram capazes de reduzir a expressão da proteína Firefly luciferase em células HeLa e FaDu, pórem não foram capazes de reduzir a expressão do receptor do fator de crescimento epidérmico (EGFR) nas células A431, HeLa, A549 e FaDu. A redução da expressão de EGFR em células A549 foi observado quando a terapia com siRNA foi combinada com internalização fotoquímica. Destes resultados, pode-se inferir que a transfecção celular do siRNA e o silenciamento gênico promovido por ele foi dependente tanto do tipo de linhagem celular como do alvo desejado. Os estudos in vivo mostraram que ocorre a formação de gel intratumoral e após 3 dias da administração intratumoral da FFP contendo siRNA específico para EGFR houve redução de 30% no tamanho dos tumores comparados aos tumores tratados com FFP sem siRNA. Diante destes resultados, pode-se concluir que o sistema desenvolvido tem potencial como sistema de liberação localizada de siRNA quando aplicado subcutaneamente ou intratumoral, uma vez que complexa o siRNA, promove a sua internalização celular e o siRNA liberado no citoplasma das células pode reduzir a expressão de proteínas de interesse. / RNA interference (RNAi) is a mechanism in which small interfering RNA molecules (siRNA) inhibit gene expression, by causing the messenger RNA degradation. Thus, siRNA is a promising therapy for the treatment of several diseases such as cancer. However, the development of delivery systems able to protect the siRNA from degradation and promote its cell uptake is essential for therapeutic use of siRNA. Among the delivery systems, the localized delivery system such as in situ gelling delivery system, have advantages over systemic administration. Precursor fluid formulations (FFP), which forms in situ viscous liquid crystalline systems, can be obtained from amphiphilic lipids that absorb water from the environment and self-assembling. In this context, the present study aimed to evaluate the in situ gel formed from the FFP (G-FFP), composed of monoglycerides (MO), polyethyleneimine (PEI), propylene glycol (PG) and Tris buffer, as localized delivery system for siRNA in skin cancer therapy. The results showed that the G-FFP is a mixture of cubic and hexagonal phase. The G-FFP sustained release of siRNA and the siRNA is released complexed with PEI. The FFP can be sterilized by membrane filtration at 0.22 ?m. FFP was able to complex high siRNA concentration (15 mM) and protect the siRNA from degradation. The cytotoxicity was dependent on the FFP concentration, when FFP was complexed with siRNA it was observed a decreased in toxicity. The siRNA released from G-FFP was uptake by A431, FaDu, HeLa, A549, WM35/DLC2-GFP and MCF-7/DLC2-GFP cells. In addition, specific siRNAs released from G-FFP were able to reduce the expression of Firefly luciferase in HeLa and FaDu cells, but they were unable to reduce the expression of the epidermal growth factor receptor (EGFR) in A431, HeLa, A549 and FaDu cells. The reduction of expression of EGFR in A549 cells was observed when siRNA therapy was combined with photochemical internalization. From these results, can be inferred that the efficiency of siRNA transfection and knockdown was dependent on both the type of cell line and the desired target protein. In vivo studies showed that the gel was formed in situ after intratumoral injection. 3 days after intratumoral administration of FFP containing EGFR specific siRNA, 30% of reduction in the tumors size compared to tumors treated with FFP without siRNA was observed. Based on these results, could be concluded that the developed system is a potential siRNA delivery system when administered subcutaneously and intratumoral, because it was able to complex siRNA, promoted its cell uptake and the siRNA released into the cytoplasm of the cell may reduce the expression of target proteins

A Terapia Fotodinâmica (TFD) é uma modalidade de tratamento de câncer relativamente nova e promissora. Baseia-se na utilização de uma substância fotossensibilizante e luz para provocar um dano seletivo ao tecido alvo, sendo que a seletividade ao órgão/tecido deve ser reconhecida como uma das vantagens no tratamento do câncer. Atualmente, o desenvolvimento de sistemas de liberação e promotores de absorção visando a otimização da liberação tópica de fotossensibilizadores, apresenta-se como um promissor e inexplorado campo de pesquisa na TFD do câncer de pele. Sistemas de liberação compostos por monoleína, um lipídeo polar biocompatível, e água são capazes de aumentar a permeação de fármacos na pele, além de serem capazes de controlar a liberação dos mesmos. No presente estudo foram desenvolvidas e caracterizadas nanodispersões de cristais líquidos como sistemas de liberação para os fotossensibilizadores Protoporfirina IX (PpIX) e Ftalocianinas de Zinco (ZnPc) e Cloro Alumínio (ClAlPc), objetivando otimizar a penetração cutânea destes na epiderme. As nanodispersões foram avaliadas em relação a sua eficiência de encapsulação, estabilidade física e química dos fotosensibilizadores veiculados, bem como a permeação e retenção cutânea in vitro e in vivo por microscopia de fluorescência. Estudos de pré-tratamento foram realizados, objetivando-se verificar o efeito promotor de absorção cutânea das nanodispersões desenvolvidas. Os experimentos de eficiência de encapsulação e estabilidade mostraram que as nanodispersões são um sistema de liberação adequado a aplicação tópica de fotosensibilizadores. Os experimentos in vitro mostraram que as nanodispersões aumentaram a penetração cutânea da PpIX e ClAlPc no estrato córneo em, respectivamente, 3,4 e 11,7 vezes, quando comparadas a formulação controle. Na epiderme mais derme sem estrato córneo, os aumentos promovidos pelas nanodispersões foram de 6,5 e 9,7 vezes para a PpIX e ClAlPc. No experimento in vivo as nanodispersões aumentaram a penetração cutânea da PpIX e ClAlPc no estrato córneo em, respectivamente, 13,7 e 7,0 vezes, quando comparadas ao controle, sendo que na epiderme mais derme sem estrato córneo somente as nanodispersões foram capazes de promover a penetração cutânea destes fotossensibilizadores nestas camadas da pele. A visualização da penetração cutânea dos fotosensibilizadores por microscopia de florescência confirmou os resultados obtidos nos experimentos in vivo de que as nanodispersões foram superiores ao controle em aumentar a penetração cutânea da PpIX e ClAlPc em camadas mais profundas da pele. Os resultados obtidos mostraram que as nanodispersões desenvolvidas são sistemas de liberação promissores para a PpIX e ClAlPc no tratamento do câncer de pele na TFD uma vez que aumentaram in vitro e in vivo a penetração cutânea destes na epiderme. / Photodynamic therapy (PDT) is a relatively new and promising cancer treatment modality that involves the administration of a photosensitizing drug and its subsequent activation by light to produce activated oxygen species that selectively destroy target cells. Recently, PDT to treat skin cancer is focused on the development of drug delivery systems and penetration enhancers that aim to optimize the topical release of photosensitizers. Drug delivery systems based on monoolein, a biocompatible polar lipid, and water are able to enhance the cutaneous penetration of drugs and control their release. Liquid crystal nanodispersions were developed and characterized in this project as delivery systems for the photosensitizers Protoporphyrin IX (PpIX) and Zinc and Chloroaluminum phthalocyanines (ZnPc and ClAlPc), aiming to increase their topical penetration in the epidermis. The physical stability of the nanodispersions, their encapsulation efficiency and the chemical stability of the photosensitizers incorporated were tested. In vitro and in vivo skin penetration tests were performed to verify the efficacy of the nanodispersions in enhancing the topical delivery of the photosensitizers. In vitro pre-treatment tests were conducted to determine if the nanodispersions are able to increase the skin penetration of the photosensitizers by a penetration enhancing effect. Stability and encapsulation efficiency tests showed that nanodispersions are an adequate topical delivery system for photosensitizers. In vitro experiments showed increased PpIX and ClAlPc penetration in the stratum corneum, respectively, of 6.5- and 9.7-fold for the nanodispersions compared to the control. In the epidermis with dermis, without stratum corneum, the increase promoted by the nanodispersions for PpIX and ClAlPc were, respectively, of 6.5- and 9.7-fold. Experimental retention in vivo confirmed that when the nanodispersions were used as carrier, PpIX and ClAlPc concentrations in the stratum corneum were about 13.7- and 7.0-fold higher, respectively, than control. In the epidermis with dermis, without stratum corneum, only the nanodispersions were able to promote the skin penetration of these photosensitizers. Visualization of PpIX and ClAlPc skin penetration by fluorescence microscopy confirmed that the nanodispersions increased the skin penetration of these photosensitizers in deeper skin layers. The results showed that the nanodispersions are promising topical delivery systems in the PDT of skin cancer once they increased in vitro and in vivo the topical penetration of PpIX and ClAlPc in the epidermis.

A administração tópica do ácido 5-aminolevulínico (5-ALA) tem se destacado na Terapia Fotodinâmica (TFD) do câncer de pele pela eficiência no tratamento de tumores e pelos reduzidos efeitos colaterais fototóxicos. Entretanto, esta eficácia é limitada devido à baixa penetração do 5-ALA na pele. Uma proposta para aumentar a penetração do 5-ALA na pele é a utilização de promotores de absorção cutânea, que visam alterar a barreira cutânea para várias moléculas bioativas. O objetivo deste trabalho foi o desenvolvimento farmacotécnico de formulações contendo ácido oléico (AO) como promotor de absorção cutânea, visando o aumento da penetração cutânea do 5-ALA para a TFD do câncer de pele. O fluxo in vitro de 5-ALA das formulações contendo AO (10%) foi significativamente aumentado após 12 horas de experimento, em relação às formulações sem AO, principalmente para os grupos contendo 5% e 10% de 5-ALA. A retenção no estrato córneo (EC) e na[epiderme + derme] sem EC também aumentou significativamente, mostrando um efeito promotor do AO. Foram também realizados experimentos in vivo em camundongos, com o intuito de analisar o efeito da formulação sobre a produção e o acúmulo in vivo de protoporfirina IX (PpIX) na pele. Observou-se que a presença do promotor de absorção aumentou significativamente a quantidade de PpIX extraída da pele comparado às formulações sem este aditivo. Para visualizar a presença de PpIX no tecido, realizou-se microscopia confocal das peles de animais tratados com as formulações. As imagens foram condizentes com os experimentos in vivo, observando-se um aumento da intensidade de fluorescência nas amostras de pele que foram tratadas com as formulações contendo AO. Os resultados in vitro e in vivo mostraram a potencialidade das formulações contendo AO como promotor de absorção cutânea na liberação do 5-ALA na TFD do câncer de pele. / The topical administration of 5-ALA has been distinguished on skin cancer photodynamic therapy (PDT) because of its efficiency in the treatment of tumors and the reduced phototoxic collateral effects. However, this effectiveness is limited by its low penetration in the skin. A proposal to optimize the 5-ALA penetration in the skin is the use of cutaneous penetration enhancers, which seek to alter the cutaneous barrier for many bioactive molecules. The aim of this work was the pharmaceutical development of formulations containing oleic acid (OA) as a cutaneous penetration enhancer, seeking the increase of the cutaneous penetration of 5-ALA for skin cancer PDT. The in vitro flux of 5-ALA from formulations containing OA (10% w/w) was significantly increased after 12 hours of experiment, in comparison with formulations without OA, mainly for the groups containing 5% and 10% of 5-ALA. The stratum corneum (SC) and [epidermis + dermis] without SC retention were also significantly increased, showing an enhancer effect of the fatty acid. In addition, in vivo experiments were accomplished in mice, with the purpose of analyzing the formulation effect on the PpIX production and accumulation in the skin. It was observed that the penetration enhancer\'s presence significantly increased the amount of PpIX extracted from the skin, in contrast with formulations without this addition. To visualize the PpIX presence in the tissue, confocal microscopy of the treated animal skins was made. The images were suitable with the in vivo experiments, with increased intensity of fluorescence in the tissue samples that were treated with formulations containing OA. Both in vitro and in vivo results showed the potentiality of the formulations containing OA as a cutaneous penetration enhancer for 5-ALA delivery on the skin cancer PDT.

A terapia fotodinâmica (TFD) associada à administração tópica de agentes fotossensibilizantes é uma terapia promissora para o tratamento tópico do câncer de pele. A protoporfirina IX (PpIX) é uma substância fotodinamicamente ativa usada na TFD, entretanto, devido a sua alta lipofilia ela forma agregados em meio aquoso, o que diminui sua atividade fotodinâmica e dificulta sua administração na pele. Assim, sistemas de liberação nanoparticulados vêm sendo investigados para melhorar a distribuição da PpIX na pele e facilitar sua penetração até as células tumorais. Os dendrímeros de poliamidoamina (PAMAM) representam uma nova geração de nanosistemas que tem despertado grande interesse nos últimos anos. Eles são uma classe especial de polímeros que apresentam estrutura muito ramificada e regular e que interagem com a PpIX formando complexos (nanopartículas dendriméricas de PpIX-PAMAM). A aplicação de uma corrente elétrica de baixa intensidade, conhecida como iontoforese, pode influenciar na penetração cutânea dessas nanopartículas, direcionando-as para o interior das células. Desta forma, o objetivo deste trabalho foi avaliar a influência da iontoforese e de nanopartículas de PAMAM de geração 4 hidroxilado (PAMAM G4-OH) com a PpIX na localização subcelular e penetração deste agente fotossensibilizante em tumores cutâneos. Assim foram preparados complexos de PpIX-PAMAM, os quais foram caracterizados em função do tamanho de partículas e potencial zeta. A localização subcelular da PpIX a partir dos complexos foi investigada em carcinoma de células escamosas. A influência dos complexos na geração de oxigênio singleto quando a PpIX sofre irradiação também foi avaliada. Por fim, a penetração da PpIX a partir dos complexos PpIX-PAMAM foi avaliada, in vivo, em pele sadia e em tumores induzidos em camundongos Nude BalbC, com e sem aplicação da iontoforese. O tamanho médio das nanopartículas dendriméricas contendo a PpIX dispersas em meio aquoso foi de aproximadamente 220 nm. Quando avaliadas em função do tempo, este tamanho sofreu um aumento de apenas 5% depois de 24 h, permanecendo constante por 7 dias. O potencial zeta das dispersões foi de 10 mV, em pH 7, e de 30 mV, em pH 5,5, possibilitando a contribuição da eletromigração durante a iontoforese. Nos estudos em cultura de células tumorais observou-se que a complexação com o PAMAM aumentou 30 vezes a localização da PpIX na mitocôndria quando comparada a PpIX livre. Além disso, a quantidade de oxigênio singleto gerada foi semelhante para a PpIX livre não agregada e complexada, 4,3 x 10-3 e 4,6 x 10-3 , respectivamente, sugerindo que o PAMAM manteve a atividade fotodinâmica da PpIX. Nos experimentos in vivo, em pele sadia, verificou-se que a PpIX administrada a partir do complexo com o PAMAM se distribuiu homogeneamente pela pele, enquanto que a PpIX livre apresentou uma fluorescência localizada em apenas algumas área da superfície da pele. A iontoforese facilitou a penetração da PpIX para as camadas mais profundas da pele. Finalmente, no tratamento dos tumores cutâneos, a administração tópica dos complexos por apenas 30 min possibilitou a penetração da PpIX até os tumores localizados abaixo da pele, em concentrações semelhantes para a aplicação passiva e iontoforética. Portanto, a complexação da PpIX com o PAMAM é um sistema de liberação nanoparticulado promissor para o tratamento tópico de tumores cutâneos por TFD. / Photodynamic therapy (PDT) associated with topical administration of photosensitizer agents is a promising therapy for topical treatment of skin cancer. Protoporphyrin IX (PpIX) is a photosensitizer commonly used in PDT; however, due to its high lipophilicity it aggregates in aqueous medium, which decreases its photodynamic activity and hinders its penetration through the skin. In this way, nanoparticles have been designed to improve the distribution of PpIX in the skin and enhance its tumor cell penetration. The polyamidoamine dendrimers (PAMAM) represent a new generation of nanosystems that has aroused great interest in recent years. They are hyberbranched polymers capable to form complexes with PpIX (PpIX-PAMAM), increasing PpIX aqueous solubility. The application of a low intensity electrical current, known as iontophoresis, may influence the nanoparticles skin penetration, directing them to the tumor cells. Therefore, the aim of this study was to evaluate the influence of iontophoresis and PpIX-PAMAM G4-OH complexes in PpIX subcellular localization and penetration into skin tumors. The complexes were prepared and characterized as a function of particle size and zeta potential. The subcellular localization of PpIX from the complexes was investigated in squamous cell carcinoma. The influence of PpIX-PAMAM on the generation of singlet oxygen after irradiation was also evaluated. Finally, the penetration of PpIX from the PpIX-PAMAM complexes was evaluated in vivo in healthy skin and in tumors induced in BalbC nude mice with and without application of iontophoresis. The average size of PpIX-PAMAM nanoparticles dispersed in aqueous medium was approximately 220 nm. When evaluated as a function of time, this size was increased only 5% after 24 h and remained constant for 7 days. The zeta potential of the dispersions was 10 mV at pH 7 and 30 mV at pH 5.5, allowing the contribution of electromigration during iontophoresis. In studies in culture tumor cells it was observed that complexation with PAMAM increased 30 times the localization of PpIX in the mitochondria compared to free PpIX. Furthermore, the amount of singlet oxygen generated when PpIX-PAMAM was irradiated was similar to that generated by the irradiation of the non-aggregated free PpIX, 4.6 x 10-3 and 4.3 x 10-3, respectively, suggesting that PAMAM did not modify the photodynamic activity of PpIX. In vivo experiments on healthy skin have shown that PpIX from the PpIX-PAMAM was homogeneously distributed throughout the skin, whereas free PpIX fluorescence was visualized only in some restricted areas of the skin surface. Iontophoresis facilitated PpIX diffusion to deep layers of the skin. Finally, the treatment of skin tumors have shown that the topical administration of the PpIX-PAMAM for only 30 min, passively or by iontophoresis, allowed the penetration of PpIX into the tumors located below the skin. Therefore, the PpIX complexation with PAMAM is a promising nanoparticle delivery system for the topical treatment of skin tumors by PDT.