Os modelos de reconstrução do microambiente são úteis para investigar as propriedades biológicas dos melanócitos humanos com a matriz e como plataforma para testes de novos fármacos. Existe uma demanda crescente para a utilização de pele e derme reconstruídas em laboratório, em ensaios in vitro de citotoxicidade, viabilidade celular, crescimento celular, irritabilidade e avaliação dos constituintes da matriz extracelular. Caracterizamos, em equivalente dérmico, alguns mecanismos de viabilidade e invasão de melanoma metastático humano quando na presença da matriz, a fim de ampliar o conhecimento a respeito de novas terapias contra o melanoma e entender os mecanismos que favorecem seu potencial invasivo, mimetizando com mais fidelidade o que ocorre in vivo. Através da validação deste modelo, constatamos que ele é essencial para resgatar a fisiopatologia do tumor, pois o melanoma, na presença de equivalente dérmico, torna-se capaz de evadir mecanismos de morte e aumentar a secreção de metaproteinases e citocinas que favorecerão a evolução tumoral. Avaliamos também as propriedades antineoplásicas do ácido clorogênico, que já foram relatadas em um grande número de tumores, porém os mecanismos que levam à sua ação antitumoral ainda não foram bem elucidados. Diante dos trabalhos enfatizando o efeito quimioprotetor dos polifenóis, referentes à sua ação antioxidante em células neoplásicas e com potencial metastático, não há na literatura estudos que comprovem a eficácia do ácido clorogênico sobre células de melanoma metastático humano. Portanto, este estudo visou recriar a estrutura dérmica in vitro e, a partir disso, comparar a resposta de fármacos em células de melanoma cultivada em equivalente dérmico, a fim de avaliar se há modulação diferencial nesse substrato, sugerindo, portanto, um protocolo mais eficaz para a prospecção de fármacos antimelanoma. / The microenvironment reconstruction models are useful for investigating the biological properties of human melanocytes onto the matrix and as platform for testing new drugs. There is a growing demand for the use of reconstructed skin and dermis in the laboratory for in vitro assays of cytotoxicity, viability, cell growth, irritability, and evaluation of the extracellular matrix. We characterize in dermal equivalent some mechanisms for cell viability and invasion of human metastatic melanoma in the presence of matrix, the order to increase knowledge about new therapies against melanoma and to understand the mechanisms that favor its invasive potential, to more closely mimic what occurs in vivo. By means of this model, we find that it is essential to rescue the tumor physiopathology as melanoma, in the presence of dermal equivalent, it is able to evade death mechanisms and increase the expression of metalloproteinases and cytokines which favor the tumor evolution. We also evaluated the antineoplastic properties of chlorogenic acid, that have been reported in a large number of tumors, but the mechanisms that lead to its antitumor action has not been well elucidated. Before the work emphasizing the chemoprotective effect of polyphenols related to its antioxidant action in neoplastic cells and with metastatic potential, there is no literature studies confirming the effectiveness of chlorogenic acid on human metastatic melanoma cells. Therefore, this study aims to rebuild the dermal structure in vitro, and from this, to compare the response to drugs in melanoma cells dermal equivalent cultured in order to evaluate whether modulation differential on the substrate, thus suggesting a protocol more effective for prospecting antimelanoma drugs.

Orientador: Rosemary Adriana Chiérici Marcantonio / Banca: Joni Augusto Cirelli / Banca: Elizabeth Pimentel Rosetti / Banca: Washington Rodrigues Camargo / Banca: Giuseppe Alexandre Romito / Resumo: O propósito deste estudo foi avaliar histológica e histometricamente o resultado da utilização das matrizes de colágeno de Pericárdio Bovino (MPB) e Dérmica Acelular (MAD) no tratamento de deiscências ósseas criadas cirurgicamente em cães. Foram criados defeitos periodontais do tipo deiscência óssea na superfície vestibular dos 2os, 3os e 4os pré-molares inferiores de seis cães. Estes dentes foram divididos em 3 grupos: grupo I: MAD; grupo II: controle e grupo III: MPB, ambos com reposição coronária do retalho. Após 70 dias os animais foram sacrificados, os dentes retirados em bloco e processados histologicamente. Realizou-se análise histológica descritiva e histométrica, medindo-se a extensão do tecido epitelial, neoformação óssea e cementária e nível gengival para os grupos I, II e III. Os resultados demonstraram valores estatisticamente iguais para as medianas (em mm) dos valores de epitélio: 1,49, 1,91 e 1,82; cemento: 3,18, 3,09 e 3,31 respectivamente para os grupos I, II e III. Foi encontrada diferença estatisticamente significante em relação à neoformação óssea, com medianas (em mm) de 1,23, 1,82 e 1,62, respectivamente para os grupos I, II e III. Foi observado escasso infiltrado inflamatório. Com isso, pudemos concluir que as matrizes de MPB e MAD são materiais que permitem a reparação dos tecidos periodontais. / Abstract: The goal of this investigation was to compare, histologically and histometrically, the healing process of dehiscence-type defects treated by acellular dermal matrix (AD) and bovine pericardium matrix (PB). Six mongrel dogs were used. Buccal osseous deiscences were surgically created on mesial roots of the mandibular second, third and fourth premolars. The defects were randomly assigned to one of the treatments: AD, control and PB. After 70 days of healing, the dogs were sacrificed and the blocks were processed. The histometric parameters evaluated included: gingival level, epithelial length, new cementum and new bone. No statistically significant differences were found between PB, AD and control groups for epithelial length and new cementum parameters (p<0,05). Statistically significant differences were found between PB, AD and control groups for new bone (minor for AD). The presence of a slight inflammation was observed. Withim the limits of this study, it can be concluded that PB and AD showed good biocompatibility, but did not bring a complete periodontal tissue regeneration. / Doutor

The sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A2 (cPLA2a) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA2a with an ablated C1P interaction site (KI) to examine the cPLA2a/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was dramatically enhanced by loss of the C1P/cPLA2α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii) increased wound tensile strength; and iii) higher Type I/Type III collagen ratios. These findings were recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed significantly increased collagen deposition and migration velocity compared to WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of reduced pro-inflammatory prostaglandins (e.g., PGE2) and increased levels of specific HETE species (e.g., 5-HETE). Elevated 5-HETE levels promoted increased dermal fibroblast migration and collagen deposition. This “gain of function” role for the mutant cPLA2a was also linked to differential cellular localization of cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of key in vivo biological mechanisms by a defined protein:lipid interaction and provide new insights into cPLA2a function.

acase@tulane.edu

acase@tulane.edu

Background: Respiratory conditions are prevalent and cause an enormous burden on society. In recent decades, there has been a global increase in asthma in children and adults, yet the diagnosis of asthma must be made on clinical grounds as the diagnostic use of pharmacological reversibility of airway obstruction remains controversial. It is possible however that tools exist from different medical paradigms that may assist in the clinical diagnosis of asthma. Tools such as Traditional Chinese Medicine (TCM) enquiry, Breath Holding Time (BHT) or Electro-Dermal Screening Test (EDST) may provide useful clinical information, yet their use has not been widely explored or validated. Integrative medicine may be considered to represent a new frontier in medicine where each therapy and diagnostic method is seen to have its own advantages and limitations and where an integration of both diagnostic and therapeutic techniques from conventional and complementary medicine is seen to produce the best results. However, while there is a high community use of complementary therapies for conditions such as asthma, there is also a need to maintain accepted standards of medical and scientific principles and foster high quality research into complementary therapies. Objectives: The current study sought to determine: • If there is a correlation between conventional measures of lung function such as Forced Expiratory Volume in one second (FEV1) and less conventional measures such as airways expired NO level (eNO), electrodermal measures, TCM enquiry and BHT. • If any of the lung function measures are able to distinguish between asthmatic and healthy subjects. • If the use of a rye grass extract is better than placebo in improving requirements for bronchodilator medication, peak flow, forced expiratory volume in one second or quality of life in mild to moderate childhood asthma. Rational for carrying out two separate studies as one study; The author was interested on both the integration of diagnostic techniques as well as the effect of the herbal extract on asthma so it was decided to use the opportunity and put both in one study. Method: A range of conventional and non-conventional measures were conducted in healthy and asthmatic children including demographic details, quality of life data, spirometry measures, airway nitric oxide levels, electro-dermal measures, TCM history enquiries, breath holding time, and skin prick tests. The data were analysed to determine any significant correlations between these measures. A double blind randomized controlled pilot clinical trial was also performed to assess the effect of using rye grass extract in asthmatic children aged 8 to 16 years. Results: There were significant correlations between forced expiratory volume in one second and active quality of life, TCM spleen score and fraction of exhaled nitric oxide measurements. There were also statistically significant differences between asthmatic and healthy subjects in TCM history, breath holding time and exhaled nitric oxide. The use of rye grass extract did not produce any significant improvement over placebo in any of the asthma outcome measures. Conclusions: The use of inexpensive measures such as BHT and TCM enquiry may provide useful clinical information when assessing respiratory conditions such as asthma. However, further research is required in larger populations to confirm their use. The use of the rye grass extract at the dose given in this study did not provide any clinical benefit for the asthmatic children in this study.

Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors. One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin. The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications. Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV). In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.

Towards understanding regional meniscus characteristics important for tissue engineering efforts, meniscus cells were characterized biomechanically and an effective method for isolating these cells for tissue engineering was determined. It was found that the meniscus contains cells that are biomechanically distinct, with outer meniscus cells showing higher stiffness than inner cells. It was also determined that meniscus cells as a whole were more biomechanically similar to ligament cells than to articular chondrocytes, indicating that tissue properties may correlate with cellular mechanics. In addition to showing regionally distinct biomechanical properties, enzymatic isolation of meniscus cells was found to cause varying phenotypic changes in cells from the inner, middle, and outer regions. A comparison of isolation techniques also indicated that sequential digestion of meniscus tissue with pronase and collagenase was able to yield more cells with higher viability than other techniques tested, and those isolated cells created stiffer and more glycosaminoglycan (GAG) rich constructs when used in a tissue engineering modality than cells isolated using only collagenase. The identification of an effective mode of isolating meniscus cells is of great use to tissue engineering efforts, as they often require a large cell numbers. These findings illustrate that known regional variations in meniscus cell phenotype and biochemical composition are also evident in cellular mechanics, and phenotypic responses of these cells to isolation are varied and distinct. To be successful tissue replacements, tissue engineered meniscus constructs must not elicit an immune response and must have sufficient mechanical properties to survive when implanted. To determine if allogeneic or xenogeneic implantation of scaffold-free meniscus constructs could be feasible, the immunogenicity of bovine and leporine meniscus cells and articular chondrocytes were determined in an in vitro model system. It was found that neither bovine nor leporine meniscus cells or articular chondrocytes caused activation of leporine immune cells, suggesting that they may serve as allogeneic or xenogeneic cell sources for meniscus engineering. Additional analysis of the mechanical role of meniscus GAGs indicated that they are mechanically important in all regions of the meniscus, but especially in the inner region where the relatively high GAG content affects both compressive and tensile properties. Therefore, tissue engineering efforts should try to recapitulate GAG content and distribution to enhance the functionality of meniscus replacements. As a major obstacle for meniscus engineering is the identification of an abundant cell source, this thesis also investigated the use of skin cells as an alternative to primary cells for tissue engineering. Previously identified chondroinducible dermis cells were found to have multilineage differentiation capacity, and were subsequently termed dermis isolated adult stem cells (DIAS). DIAS cells were also able to be expanded in monolayer without losing chondroinductive capacity, and were able to create constructs with cartilaginous properties which could be varied with growth factor application. Given the ease of expansion and ability of DIAS cells to form fibrocartilaginous tissue, these cells present an abundant cell source for meniscus tissue engineering.

p-Dichlorobenzene (p-DCB) and naphthalene are compounds classified as Group C carcinogens according to the USEPA. Sources of p-DCB and naphthalene include moth repellents and deodorizers typically used in closets, garment bags, and toilet bowls found in pure form. In this study, laboratory, closet, and garment bag experiments were used to determine emission rates of p-DCB and naphthalene from consumer products (closet air freshener, toilet bowl deodorizer, and moth repellent). Emission rates varied considerably between products that contain p-DCB, primarily due to product packaging, and were generally suppressed when the product was used in a closed closet or garments bag, relative to products placed in well-ventilated chambers. Experimental mass emission rates were used in conjunction with a well-mixed reactor model to predict indoor p-DCB and naphthalene concentrations for a range of reasonable residential scenarios. Results suggest that exposures under worst-case scenarios could lead to excess lifetime cancer risks of greater than 20,000 in a million (2%) for those who use consumer products that are pure p-DCB, a risk that dwarfs any reported environmental cancer risks over large segments of the US population. Since such products are typically used where clothing is kept, significant chemical adsorption onto clothing is possible following sublimation from the solid product. Chamber experiments were used to determine the amount of p-DCB and naphthalene mass that adsorbs onto selected clothing materials made of cotton, polyester, or wool. Cloth specimens were kept inside a chamber through which an air stream containing p-DCB or naphthalene was passed for one month. After this time, p-DCB or naphthalene were chemically extracted from the cloth specimens. Polyester was determined to be the most adsorbent material, while cotton was the least adsorbent for each chemical. Equilibrium partition coefficients of 0.01 m³/g for p-DCB and 0.02 m³/g for naphthalene were determined experimentally for wool. Desorption rates were determined in both laboratory chambers and a closet in a test house. Results suggest prolonged persistence of p-DCB and naphthalene on polyester and wool, e.g., half-lives of 12 to 20 days after a moth repellent is removed from the clothes storage environment. An exposure scenario was also carried out to compare the inhalation and dermal exposure risks associated with contaminated clothing. / text

Cobalt is a commonly known sensitiser in industrial settings and has been classified by the IARC as a possible group (2B) human carcinogen. Workers at a South African base metal refinery are potentially exposed to cobalt in the cobalt packaging area. The respiratory and dermal exposure to cobalt is a possible health risk. Quantifying the exposures levels assists in determining the degree of the risk as well as the management thereof. The objectives of this study were to assess dermal and respiratory exposure of workers at a cobalt packaging area and to assess their skin condition by measuring transepidermal water loss (TEWL) and skin hydration indices. The skin hydration index was measured on the back of the hand, forehead, wrist and palm at the start, during and end of the shift. The TEWL index was measured at the start and end of the shift on the same areas as the hydration index. Ghostwipes was chosen as preferred wipe sampling media to collect dermal and surface samples. Wipe samples were also taken on suspected contaminated workplace surfaces. Respiratory samples were taken by using the Institute of Occupational Medicine (IOM) inhalable aerosol sampler at a flow rate of 2 l/min. Wipes and respiratory samples were analysed for cobalt according to NIOSH method 9102 using inductively coupled plasma–atomic emission spectrometry (ICP–AES). The hydration indices indicated that worker’s skin are slightly dry to normal at the beginning of the shift. Hydration on the wrist increased significantly during the shift. TEWL indices increased significantly on the back of the hand, wrist and forehead during the shift. TEWL indices of the palm showed a low barrier function before the shift and deteriorated further to a very low barrier function at the end of the shift. Significant dermal cobalt loading occurred on the back of the hand, forehead, wrist and palm during the shift. The palm was the most exposed and the forehead least. The barrier function of the skin is most likely to be affected by exposure to cobalt. The skin condition of workers put them at greater risk to develop adverse health effects of cobalt. Workplace surfaces were contaminated with cobalt. Airborne cobalt was visible at different working stations. Cobalt exposure is due to numerous sources in the packaging area, thus contributing to the dermal exposure. Respiratory exposure exceeded the 8 hour occupational exposure limit for most of the workers. The guidance limit for short term exposure was exceeded by half of the workers. It is important to minimise the exposure to cobalt in packaging area. Manifestation of the adverse health effects are usually not visible in the short term, the necessary precautions have to be taken to protect the workers. / Thesis (M.Sc. (Occupational Hygiene))--North-West University, Potchefstroom Campus, 2011.