Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-20T15:04:22Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Luiza Lima_Tese de Doutorado_2015.pdf: 5654099 bytes, checksum: da2a7cb303b6562f1bc8af5fc0c76f13 (MD5) / Made available in DSpace on 2016-07-20T15:04:22Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Luiza Lima_Tese de Doutorado_2015.pdf: 5654099 bytes, checksum: da2a7cb303b6562f1bc8af5fc0c76f13 (MD5) Previous issue date: 2015-10-29 / CAPEs / Com o objetivo de criar um modelo biológico reprodutível e eficiente que possa mimetizar a interação proteína-carboidrato, nós reportamos o uso do ensaio quimiluminescente com lectinas conjugadas a éster de acridina (EA) para investigar a complexação da lectina ao carboidrato e para avaliação quantitativa da expressão de carboidratos em tumores cutâneos. Polissacarídeos de N-acetil-D-glicosamina (quitosana), glicose (phytagel) e galactose (carragenana) foram utilizados para sintetizar membranas (0,0314 – 0,6358 cm2). Concanavalina A-EA (Con A), Wheat germ aglutinina (WGA), Peanut aglutinina (PNA), Ulex europeaus aglutinina-I (UEA-I) e Maackia amurensis aglutinina (MAA) foram conjugadas a EA. A atividade hemaglutinante das lectinas-EA foi avaliada e a quimiluminescência quantificada e expressa em Unidades Relativas de Luz (URL). Biópsias de pele normal e de tumores cutâneos, tais como ceratose actínica (AK), ceratoacantoma (KA), carcinoma espinocelular (CEC) e basocelular (CBC), e as membranas foram incubadas com o conjugado lectina-EA (100 μL 100 μg mL-1) por 2h a 4°C. A emissão de fótons foi quantificada e correlacionada com a marcação de tecidos normais, transformados e das membranas. Inibições com carboidratos específicos foram realizadas. AK, KA , CBC e CEC apresentaram menor expressão de α -D- glucose/manose e resíduos de α-L-fucose, em comparação com o tecido normal. Os tumores cutâneos apresentaram maior expressão resíduos de β Gal-(1-3)-GalNAc que o tecido normal . AK e SCC exibiram maior expressão de resíduos Neu5Ac-α(2,3)Gal que a epiderme normal. KA e BCC apresentaram valores de URL equivalentes, em comparação com o tecido normal. Os valores de URL diminuíram quando as lectinas foram incubadas com seus carboidratos específicos. Os valores das constantes e os sítios de ligação foram calculados para cada lectina utilizando a equação obtida a partir das curvas hiperbólicas, 2,4 x 10-7 M-1 ± 0,8 x 10-7 M-1and 1,3 x 10-3 mol . mg-1 ± 0,3 x 10-3 mol . mg-1 (Con A); 0,9 x 10-6 M-1  0,4 x 10-6 M-1 and 0,021 x 10-3 mol . mg-1 ± 0,003 x 10-3 mol . mg-1 (WGA) and 2,0 x 10-6 M-1  0,9 x 10-6 M-1 and 0,069 x 10-3 mol . mg-1 ± 0,010 x 10-3 mol . mg-1 (PNA). O método quimiluminescente permitiu a avaliação quantitativa direta da expressão de carboidratos em neoplasias de pele e a investigação a complexação da lectina ao carboidrato através do modelo de Langmuir, combinando a especificidade desta interação e a sensibilidade dos ensaios quimiluminescentes. / Aiming to create a reproducible and efficient biological model that mimic the protein-carbohydrate interaction, we report the use of chemiluminescent assay with lectins labeled to acridinium ester (AE) to investigate the complexation of lectin to carbohydrate and for the quantitative evaluation of carbohydrates expression in cutaneous tumors. Polysaccharides made N-acetyl-D-glucosamine (chitosan), glucose (phytagel) and galactose (carrageenan) were used to synthetize membranes (0,0314 – 0,6358 cm2). Concanavalin A-AE (Con A), Wheat germ agglutinin (WGA), Peanut agglutinin (PNA), Ulex europeaus agglutinin-I (UEA-I) e Maackia amurensis agglutinin (MAA) were labeled to AE. The hemmagglutinating activity of lectins-AE was evaluated and the chemiluminescence was quantified and expressed in Relative Light Unit (RLU). Biopsies of normal skin and cutaneous tumors such as, actinic keratosis (AK), keratoacantoma (KA), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), and membranes were incubated with lectins-AE (100 μL 100 μg mL-1) for 2h at 4°C. Photon emission was measured and correlated to the labeling of the normal, transformed tissues and membranes. Inhibitions with specific carbohydrate were carried out. AK, KA, SCC and BCC showed lower expression of -D-glucose/mannose and -L-fucose residues compared to normal tissue. Cutaneous tumors displayed higher expression of Gal-(1-3)-GalNAc residues than normal tissue. AK and SCC exhibited higher expression ofNeu5Ac-(2,3)Gal residues than normal epidermis. KA and BCC showed equivalent RLU values compared to normal tissue. The RLU values decreased when the lectins were incubated with their specific carbohydrate. The constant values and maximum binding sites on the membranes were calculated for each lectin using the equation obtained from hyperbolic curves, 2.4 x 10-7 M-1 ± 0.8 x 10-7 M-1and 1.3 x 10-3 mol . mg-1 ± 0.3 x 10-3 mol . mg-1 (Con A); 0.9 x 10-6 M-1  0.4 x 10-6 M-1 and 0.021 x 10-3 mol . mg-1 ± 0.003 x 10-3 mol . mg-1 (WGA) and 2.0 x 10-6 M-1  0.9 x 10-6 M-1 and 0.069 x 10-3 mol . mg-1 ± 0.010 x 10-3 mol . mg-1 (PNA). The chemiluminescence method allowed quantitative assessment of the carbohydrate expression in cutaneous tissues and was an analytical technique efficient to investigate the complexation of lectin to carbohydrate through Langmuir model, combining the specifity this interaction and sensibility of chemiluminescent assays.

Anfíbios possuem uma microbiota cutânea que os protege contra patógenos. Essa proteção se dá pela produção de moléculas antimicrobianas e pela competição por espaço e nutrientes contra patógenos. Alterações na composição da microbiota, causadas por fatores bióticos e abióticos do ambiente e por fatores ecofisiológicos do hospedeiro, podem afetar a resistência dos anfíbios à doenças. Assim, é possível que ambientes artificiais, por conter condições ambientais diferentes dos naturais e por alterarem aspectos ecofisiológicos dos indivíduos, devem modular a microbiota cutânea de animais mantidos e nascidos em cativeiro. Nós avaliamos diferenças inter e intra-populacionais no perfil da comunidade bacteriana de Scinax alcatraz entre três grupos: indivíduos selvagens; indivíduos nascido em cativeiro; e indivíduos mantidos em cativeiro por dois anos. Também verificamos o efeito temporal de ambientes artificiais no perfil da microbiota cutânea entre e dentre indivíduos selvagens mantidos em cativeiro ao longo de 312 dias. Os parâmetros microbiológicos utilizados foram riqueza de morfotipos bacterianos e abundância de colônias bacterianas. As diferenças encontradas entre populações apontam para o ambiente como um importante modulador da microbiota cutânea. No entanto, as diferenças encontradas entre indivíduos de uma mesma população apontam para a importância de aspectos fisiológicos do hospedeiro na modulação. Por fim, a avaliação temporal foi importante para mostrar que tanto aspectos ambientais quanto aspectos ecofisiológicos atuam juntos na modulação da comunidade bacteriana cutânea de anfíbios mantidos em cativeiro / Amphibians harbor a skin microbiota that provides protection against pathogens. This protection happens by production of antimicrobial substances and by competition for space and nutrients against pathogens. Changes in the microbiota composition, caused by biotic and abiotic factors of the environment and ecophysiology factors of the host, may affect disease resistance of amphibians. As artificial environments contain different environmental conditions compared to natural ones and may alter physiological aspects of individuals, it may modulate the cutaneous microbiota of captive animals. Our study evaluated inter- and intra-population differences in the profile of bacterial community of Scinax alcatraz from three distinct groups: wild individuals; individuals born in captivity and individuals kept in captivity for two years. We also investigated the temporal effects of artificial environments on the cutaneous microbiota profile between and within wild individuals kept in captivity over 312 days. Microbiological parameters analyzed were richness of bacterial morphotypes and abundance of bacterial colonies. The differences found between populations show that the environment may be an important modulator of the microbial community. However the differences between individuals within a population demonstrate the importance of physiological aspects of the host for the composition of the microbiota. Finally, the temporal evaluation performed was important to show that both environmental and ecophysiological aspects act together in modulating the cutaneous microbiota community of amphibians kept in captivity

Class of 2015 Abstract / Objectives: The aim of this study was to develop a computational process capable of hypothesizing potential chemotherapeutic agents for the treatment of skin cutaneous melanoma given an annotated chemotherapy molecular target database and patient-specific genetic tumor profiles. Methods: Aberrational profiles for a total of 246 melanoma patients indexed by the Cancer Genome Atlas (TCGA) for whom complete somatic mutational, mRNA expression, and protein expression data was available were queried against an annotated targeted therapy database using Visual Basic for Applications and Python in conjunction with Microsoft Excel. Identities of positively and negatively associated therapy-profile matches were collected and ranked. Results: Subjects included in the analysis were predominantly Caucasian (93%), non-Hispanic (95.9%), female (59%), and characterized as having stage III clinical disease (37.4%). The most frequently occurring positive and negative therapy associations were determined to be 17-AAG (tanespimycin; 42.3%) and sorafenib (41.9%), respectively. Mean total therapy hypotheses per patient did not differ significantly with regard to either positive or negative associations (p=0.1951 and 0.4739 by one-way ANOVA, respectively) when stratified by clinical melanoma stage. Conclusions: The developed process does not appear to offer discernably different therapy hypotheses amongst clinical stages of cutaneous melanoma based upon genetic data alone. The therapy-matching algorithm may be useful in quickly retrieving potential therapy hypotheses based upon the genetic characteristics of one or many subjects specified by the user.

Le mélanome cutané est le cancer de la peau le plus agressif. Il représente moins de 5% des cancers de la peau mais sa forme métastatique est responsable de 60 à 80% des décès. La médiane de survie des patients atteints d’un mélanome métastatique n’est que de 6 à 9 mois avec les chimiothérapies classiques ou ciblées. L’immunothérapie a été un grand progrès thérapeutique, néanmoins la prise en charge du mélanome métastatique souffre encore de trois points négatifs, tous les patients ne répondent pas aux différents traitements, l’efficacité des chimiothérapies ciblées est limitée par l’apparition rapide de résistances, les cliniciens manquent de marqueurs prédictifs de l’évolution dès les stades précoces pour la prise en charge. Dans ce contexte, notre groupe s’intéresse à la méthylation de l’ADN associées à l’agressivité du mélanome. La méthylation est catalysée par les méthyltransférases de l’ADN (DNMTs). Lorsque celle-ci est présente au niveau d’îlots CpGs localisés dans les promoteurs des gènes, elle empêche la machinerie transcriptionnelle de se mettre en place et inhibe l’expression du gène correspondant. Le profil de méthylation globale de l’ADN étant altéré dans le cancer, elle a donc un rôle fonctionnel dans le processus tumoral mais peut aussi être utilisée en tant que biomarqueur, chaque cancer ayant un profil de méthylation différent. De plus, au sein d’un même cancer, ce profil évolue avec la progression de la tumeur. Au cours de cette thèse, j’ai identifié des modifications du profil de méthylation de l’ADN associées à l’agressivité du mélanome et j'ai sélectionné 9 loci hyperméthylés candidats biomarqueurs. J’ai d’abord comparé les profils de méthylation au niveau du génome entier de lignées cellulaires de mélanome correspondant à des stades d’agressivité différents. À partir de ces informations, des loci candidats ont été choisis par une analyse de la répartition de ces loci hyperméthylés sur le génome couplée à une analyse bioinformatique des fonctions et interactions des gènes associés. Ensuite, leur statut de méthylation a été validé par une technique différente (collaboration avec le Dr. J. Tost, CNG, Evry). Une fois leur hyperméthylation confirmée, j’ai entrepris l’analyse de la méthylation de l’ADN au niveau de ces gènes dans des échantillons de tumeurs primaires issues de patients montrant des survies différentes (Collaboration : L. Lamant, N. Meyer, IUCT, Toulouse, France; L. Lanfrancone IFOM, Milan, Italie). Notre étude confirme le statut hyperméthylé des gènes retenus dans les échantillons métastatiques par rapport aux échantillons de tumeurs primaires. De plus il apparaît un lien entre le niveau de méthylation de la tumeur primaire et le délai d’apparition de métastases ainsi que la survie globale des patients. Parmi les loci sélectionnés, j’ai déterminé si le statut hyperméthylé de ces loci est corrélé à leur sous- expression dans le but d’étudier leur rôle dans l’agressivité du mélanome, et si ces loci peuvent être déméthylés et ré-exprimés par un inhibiteur de DNMTs. Cela a amené à l’identification d’un gène et d'un miR peu décrits dans la littérature, dont les expressions sont corrélées au statut de méthylation et modulées par un traitement déméthylant l'ADN. J’ai entrepris de comprendre leur rôle dans l’agressivité du mélanome et évaluer leur intérêt potentiel en tant que cibles anti-tumorales. Pour cela, j’ai utilisé des tests fonctionnels pour étudier les conséquences de la surexpression dans la lignée métastatique ou de l’inhibition dans la lignée primaire. Les résultats suggèrent une régulation épigénétique fine de ce miR et de ce gène pendant la progression du mélanome, retrouvée indépendamment par notre analyse des tumeurs de patients de la banque TCGA. / Cutaneous melanoma is the most aggressive skin cancer and represents less than 5% of skin cancers but its metastatic form is responsible for 60-80% of the deaths. The median survival for patients with metastatic melanoma is only 6 to 9 months with conventional chemotherapy or targeted therapy. Despite recent therapeutic advances with the immune-therapies, the treatment of metastatic melanoma still suffers from three negatives drawbacks: 1) All patients do not respond to the different treatments. 2) The effectiveness of the targeted chemotherapy is limited by the rapid emergence of resistance. 3) Predictive biomarkers of the evolution of the disease are lacking for the clinicians. In this context, we studied the epigenetic regulations associated with the aggressiveness of melanoma, particularly in DNA methylation. DNA methylation is catalyzed by DNA methyltransferases (DNMTs). When CpGs islands are methylated and located in the promoters of genes, expression of the corresponding gene is inhibited. Commonly, DNA methylation profile is altered in cancer and plays a role in tumorigenesis and tumor maintenance. In addition, the alterations of the DNA methylation profile can be used as biomarkers for prognostic and diagnostic. Here, I identified changes in the DNA methylation profile associated with the aggressiveness of melanoma and selected 9 candidates loci that are hypermethylated in the most aggressive forms of melanoma. First, I compared the methylation patterns genome-wide (450K BeadChip methylation) of melanoma cell lines bearing different aggressiveness. The loci biomarker candidates were selected by combining an analysis of the distribution of these hypermethylated loci on the genome and a bioinformatic analysis of the functions and interactions of the associated genes. Their methylation status was validated by a different technique in collaboration with Dr. J. Tost, CNG, Evry. Once confirmed their hypermethylation, I started to analyze the DNA methylation of the selected genes in the pairs of cell lines of different aggressiveness, such as the primary tumor compared to the metastasis from the same patient, and in patients samples of primary tumors (Collaboration L. Lamant, N. Meyer, iUCT, Toulouse, France; L. Lanfrancone IFOM, Milan, Italy). A total of twenty samples were analyzed. Our study confirms the status of selected hypermethylated genes in metastatic samples compared to primary tumors. Moreover there is a link between the level of methylation of the primary tumor and the overall survival. A patent is being filed in and new samples are collected in order to extend the patient cohort to validate the biomarkers in prognosis of the evolution of the disease. Among the selected loci, I determined whether their hypermethylated status is correlated with their under-expression. For this, I measured their level of expression in a couple of cell lines WM115 / WM266-4 by RT-PCR and RT-qPCR. Then, I chose the loci for which I observed a correlation between methylation and expression. In addition, I studied whether these loci can be demethylated and re-expressed by a DNMTs inhibitor. This led to the identification of a microRNA and gene not fully described in the literature, of which the expression is correlated to DNA methylation status and are reactivated upon treatment with demethylating agents. I explored the role of the microRNA and the gene in the aggressive features of the metastatic melanoma suggesting a tumor suppressive function. These data were further comforted by the data analysis of patient samples.

Interest within the healthcare profession in transdermal delivery of pharmaceuticals through passive, mechanical (phonophoresis) or electromotive (iontophoresis) forces has increased significantly throughout the past decade. The current review will examine the histology and cellular biology of the integument system as related to regulation of transcutaneous delivery of pharmaceutics, and examine currently accepted mechanism(s) of iontophoretic delivery. Additionally, a survey of current iontophoretic devices and electrodes available within the U.S. market, and the limitations of current technology will be presented. Experimental research supporting the use of iontophoresis for local delivery of pharmaceuticals will also be presented in conjunction with the outcomes of clinical investigations where iontophoresis was utilized for the local delivery of these pharmaceuticals. Topic areas to be covered within this section include iontophoresis of antibiotics into integument wounds, local anesthetics, and steroidal and nonsteroidal anti- inflammatory drugs. Finally, an examination of the benefits of combining various forces to enhance transcutaneous drug delivery and future direction(s) of research within this field will be discussed. The purpose of the present review is to provide both researchers and clinical practitioners with an objective basis for the current use of iontophoresis in rehabilitation medicine.

Background and Purpose. Iontophoresis is a process that uses bipolar electric fields to propel molecules across intact skin and into underlying tissue. The purpose of this study was to describe and experimentally examine an iontophoresis drug delivery model. Subjects and Methods. A mechanistic model describing delivery was studied in vitro using agarose gels and was further tested in vivo by evaluation of cutaneous vasoconstriction following iontophoresis in human volunteers. Results. In vitro cathodic iontophoresis at 4 mA and 0.1 mA each delivered dexamethasone/dexamethasone phosphate (DEX/DEX-P) from a 4-mg/mL donor solution to a depth of 12 mm following a 40 mA·minute stimulation dosage. Delivery of DEX/DEX-P to at least the depths of the vasculature in humans was confirmed by observation of cutaneous vasoconstriction. This cutaneous vasoconstriction was longer lasting and greater in magnitude when using low-current, long-duration (∼0.1 mA) iontophoresis compared with equivalent dosages delivered by higher-current, shorter-duration (1.5-4.0 mA) iontophoresis. Discussion and Conclusion. From data gathered with the gel model, the authors developed a model of a potential mechanism of drug depot formation following iontophoresis. The authors believe this drug depot formation to be due to exchange of drug ions for chloride ions as the ionic current carriers. Furthermore, diffusion, not magnitude of current, appears to govern the depth of drug penetration. Although the authors did not address the efficacy of the drug delivered, the results of human experiments suggest that current magnitude and duration should be considered as factors in treating musculoskeletal dysfunctions with iontophoresis using DEX/DEX-P at a concentration of 4 mg/mL. [Anderson CR, Morris RL, Boeh SD, et al. Effects of iontophoresis current magnitude and duration on dexamethasone deposition and localized drug retention.

Study Design: A single-blind, 2-factor (4 treatments by 8 time points) repeated-measures study design. Objective: To analytically determine dexamethasone and dexamethasone phosphate concentrations in plasma derived from proximal effluent venous blood, following cathodic iontophoresis. Methods and Measures: Six volunteers received the following dexamethasone phosphate (2.5 ml, 4 mg/ml) treatments to their wrists on separate occasions: cathodic iontophoresis (4 mA, 10 minutes or 4 mA, 20 minutes), passive application (10 or 20 minutes). Plasma samples from the ipsilateral antecubital vein were obtained 10 minutes prior to and half way through the treatment (5 or 10 minutes), at the end of the treatment (10 or 20 minutes), and posttreatment (15, 30, 60, 90, and 120 minutes). The present investigation examined: (1) the sensitivity and linearity of extraction and analysis of dexamethasone and dexamethasone phosphate; (2) the necessity for determining both; and (3) the plasma levels from proximal effluent venous blood following cathodic iontophoresis. Results: The aggregate (n= 18) of the 6-point standard curves were linear for dexamethasone (r > 0.974) and dexamethasone phosphate (r > 0.829). In vitro dephosphorylation of dexamethasone phosphate to dexamethasone occurred in plasma at 37°C and during freeze-thaw. Measurable dexamethasone or dexamethasone phosphate concentrations were absent at all time points and under all conditions in the human subjects. Conclusions. These results demonstrate the sensitivity of the current assay and the need for evaluating both forms of the drug, as in vitro dephosphorylation results in the presence of dexamethasone and dexamethasone phosphate in samples. Absence of measurable dexamethasone or dexamethasone phosphate in the proximal effluent venous blood may require re-evaluation of the extent of drug delivery during the clinical iontophoresis of dexamethasone phosphate.

Interest within the healthcare profession in transdermal delivery of pharmaceuticals through passive, mechanical (phonophoresis) or electromotive (iontophoresis) forces has increased significantly throughout the past decade. The current review will examine the histology and cellular biology of the integument system as related to regulation of transcutaneous delivery of pharmaceutics, and examine currently accepted mechanism(s) of iontophoretic delivery. Additionally, a survey of current iontophoretic devices and electrodes available within the U.S. market, and the limitations of current technology will be presented. Experimental research supporting the use of iontophoresis for local delivery of pharmaceuticals will also be presented in conjunction with the outcomes of clinical investigations where iontophoresis was utilized for the local delivery of these pharmaceuticals. Topic areas to be covered within this section include iontophoresis of antibiotics into integument wounds, local anesthetics, and steroidal and nonsteroidal anti-inflammatory drugs. Finally, an examination of the benefits of combining various forces to enhance transcutaneous drug delivery and future direction(s) of research within this field will be discussed. The purpose of the present review is to provide both researchers and clinical practitioners with an objective basis for the current use of iontophoresis in rehabilitation medicine.

Current ACL reconstruction techniques are performed with little regard to cutaneous nerves despite the evidence that iatrogenic nerve injury often leads to painful neuromas, reflex sympathetic dystrophy, or anterior knee/kneeling pain which directly impacts patients whose profession requires kneeling (Cerulli et al. 2013; Ruffilli et al. 2016; Hurley et al. 2018). In this study, the femoral cutaneous and saphenous nerves from two embalmed lower extremities were dissected in exquisite detail to create topographic maps of the anterior thigh and knee, detailing the nerves' anatomical course and relationship to relevant landmarks. To further characterize the cutaneous innervation at the knee joint, ultrasound techniques to locate sensory nerves were also explored. In both specimens, all branches of the femoral cutaneous and saphenous nerves were located, dissected, and mapped. In addition, the ability to locate terminal branches of the anterior femoral cutaneous nerve in a cadaveric specimen on ultrasound was successfully demonstrated. However, due to the small sample size, further research implementing similar methods is necessary to ensure the results are representative of the population and can be applied to surgical innervation. These findings highlight the importance of considering cutaneous nerves in ACL reconstruction techniques to minimize the risk of iatrogenic nerve injury and associated complications.

No description available.