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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
111

Quantitative analysis of surfactant deposits on human skin by liquid chromatography electrospray ionisation tandem mass spectrometry.

Massey, Karen A., Snelling, Anna M., Nicolaou, Anna January 2010 (has links)
No / Surfactants are commonly used as cleansing agents and yet there are concerns they may also have a role in skin irritation. Presently, the lack of suitable methods for quantitative and qualitative analysis of surfactant deposition on skin has hindered the in-depth investigation of such effects. Here, we report the application of reverse phase liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS/MS) assays for two surfactants commonly used in consumer products, namely sodium lauryl ether sulphate (SLES) and laurylamidopropyl betaine (LAPB), to a baseline study aiming to assess deposition levels on human skin. The linearity of the assays was established at 3-20 ng, with coefficient of variation below 5%. Detection limits were 100 pg for LAPB and 1 ng for SLES; quantitation limits were 500 pg for LAPB and 2.5 ng for SLES. The baseline study was conducted using a panel of 40 healthy volunteers. Skin extract samples were taken in triplicate from forearms, using ethanol. SLES was detected on most volunteers, with 75% of them having SLES deposits in the range of 100-600 ng/cm2. LAPB was detected on the skin of all volunteers with 85% of them having deposit levels within the concentration range of 1-100 ng/cm2. These results demonstrate the extent to which commonly used surfactants remain on the skin during the day. The analytical methods reported here can be applied to the investigation of surfactants in relation to general skin condition and the development and optimisation of new consumer wash products. / EPSRC
112

Desenvolvimento de pele humana reconstruída contendo equivalente dérmico glicado na avaliação da eficácia e toxicidade de compostos anti-glicação / Development of reconstructed human skin containing glycated dermal equivalent to toxicity and efficacy tests of anti-glycation compounds

Pennacchi, Paula Comune 03 February 2016 (has links)
A glicação não enzimática das proteínas é um fator comum para a fisiopatologia de uma série de transtornos relacionados ao envelhecimento e a doenças como o diabetes mellitus (DM). O geração dos produtos de glicação, os AGEs (do inglês: Advanced Glycation End Products) se dá através de reações de glicação da mariz extracelular (MEC) na derme e têm sido apontado como um dos fatores responsáveis pela perda de elasticidade e deficiência de cicatrização da pele. A permeação cutânea de compostos anti-AGE é uma limitação importante para eficiência terapêutica de compostos que devem atingir camadas mais profundas da pele. Modelos de pele reconstruída contendo equivalente dérmico glicado são estruturas tridimensionais geradas in vitro que mimetizam a pele humana e representam um eficiente modelo para o estudo de células e modificações provocadas na MEC no processo de envelhecimento e DM. O modelo 3D de pele reconstruída tem características metabólicas, de permeabilidade e atividade semelhantes à da pele original, potencializando seu papel nas investigações sobre permeabilidade de drogas, toxicidade, irritação, eficácia e segurança de compostos e diferenciação de queratinócitos. Uma série de compostos naturais ou sintéticos inibidores de AGEs têm sido descobertos e apresentados recentemente e podem representar inovação terapêutica no tratamento de modificações causadas pela a formação e acúmulo destes AGEs também na pele. Este estudo avaliou o desenvolvimento da pele reconstruída glicada e posteriormente, a avaliação da eficácia e toxicidade de compostos anti-glicação como aminoguanidina e carnosina em modelo de pele reconstruída glicada. Em perspectiva, este estudo contribuiu para o desenvolvimento de uma nova tecnologia in vitro, a pele reconstruída glicada, que auxiliará a compreensão da biologia da interação célula-MEC mimetizando processos fisiopatológicos importantes como o envelhecimento e o DM. / The Advanced Glycation End Products (AGEs) of proteins is a common factor to the pathophysiology of a number of disorders related to aging and diseases such as diabetes mellitus (DM). The generation of the AGEs products on skin occurs mainly through non-enzymatic glycation reactions of the dermal extracellular matrix and has been touted as one of the factors responsible for loss of elasticity and disability of skin healing. The skin permeation of compounds is an important limitation for therapeutic/cosmetic efficacy of anti-AGE compounds, which must reach the deepest layers of the skin. Reconstructed skin model containing dermal equivalent modified by in vitro glycation is able to mimic the elderly human skin and represent an efficient model for the study of cells interactions and changes in extracellular matrix induced by aging and diabetes. The 3D reconstructed skin model has metabolic characteristics, permeability and activity similar to the original skin, reinforcing its role in drug permeability of investigations toxicity, irritation, safety and efficacy evaluation of compounds and differentiation of keratinocytes. A number of natural or synthetic AGEs inhibitor compounds have been recently discovered and displayed and can represent therapeutic innovation for the treatment of changes caused by the aging of the skin. In this study we performed the development of reconstructed glycated skin model and evaluated the efficacy and toxicity of anti-glycation compounds such as aminoguanidine and carnosine. In perspective, this study has contributed to the development of a new technology in vitro, and for the understanding cell-extracellular matrix interaction during the aging of skin.
113

Auswirkungen der Bestrahlung mit UVB, UVA-1 und PUVA-1 auf das Funktionsverhalten humaner dermaler Mastzellen nach Stimulation mit anti-IgE und Substanz P

Strathmann, Marc 16 September 2004 (has links)
Die in dieser Arbeit isolierten und hochaufgereinigten, humanen dermalen Mastzellen wurden mit UVB, UVA-1 und PUVA-1 bestrahlt und anschließend entweder mit Substanz P oder anti-IgE stimuliert. Dadurch sollten Einblicke in die unterschiedlichen Wirkmechanismen der bei zahlreichen mastzellassoziierten Erkrankungen eingesetzten UV Licht Therapie gewonnen werden. Ein Schwerpunkt dieser Arbeit lag in der Erforschung der Histamin-, Tryptase- und Zytokinfreisetzung von menschlichen Mastzellen. Zusätzlich wurde die Expression von Lysosomen-assoziierten Membran Proteinen (LAMPs) auf der Oberfläche dieser Zellen untersucht. Im Gegensatz zu der durch UV Licht induzierbaren spontanen Histaminfreisetzung zeigte sich die anti-IgE stimulierte Histaminausschüttung durch UVB, UVA-1 und PUVA-1 signifikant und dosisabhängig inhibierbar. Auch die durch Substanz P stimulierten Mastzellen gaben nach UVA-1 Bestrahlung deutlich weniger Histamin ab. Demgegenüber blieb die sezernierte Menge des Mediators nach UVB und PUVA-1 konstant. Analog zu den erhobenen Histaminergebnissen konnte eine signifikante Inhibition der anti-IgE induzierten Tryptasefreisetzung nachgewiesen werden. Ebenso zeigte sich eine signifikante Verringerung, der innerhalb von vierundzwanzig Stunden basal freigesetzten Menge an Interleukin-6 und Interleukin-8, nach UV Bestrahlung. Die spontane Ausschüttung vom Tumornekrosefaktor-a verblieb in dem Untersuchungszeitraum auf sehr geringem Niveau, so dass eine relevante Beeinflussung durch UV Licht nicht stattfand. Des weiteren konnte eine vermehrte basale Expression von CD107a und CD63 auf der Mastzellmembran durch UV Bestrahlung demonstriert werden. Auch die anti-IgE induzierte Mehrexpression der LAMPs konnte bei den drei Bestrahlungsarten nachweisbar supprimiert werden. Die in dieser Arbeit gewonnenen Erkenntnisse zeigen, dass die Auswirkungen der UV Licht Therapie auf Mastzellen sehr komplex sind. Zu beachten ist, dass unter physiologischen Bedingungen nicht nur isolierte Mastzellen, sondern auch weitere bestrahlte Zellverbände eine Veränderung erfahren. Erst durch das Zusammenspiel aller Zellen lassen sich Rückschlüsse auf Krankheitsbilder und die anzuwendende Therapien ziehen. Letztendlich soll diese Arbeit dazu beitragen, dass das Verständnis der unterschiedlichen Wirkungen von UV Licht begriffen und so ein sinnvolles und gezieltes Einsetzen im klinischen Alltag ermöglicht wird. / For all experiments highly purified dermal mast cells were used. The aim of the current study was to systematically investigate the effects of UVB, UVA-1 and PUVA-1 on skin derived human mast cells. Baseline and stimulated release of histamine, tryptase and of the proinflammatory cytokines IL-6, IL-8 and TNF-a were examined. Furthermore, the CD 107a and CD 63 surface levels in UV treated cells were investigated representing two members of lysosome associated membrane proteins which were found to appear on mast cell surface during degranulation. Prior treatment with UV resulted in a striking suppression of the anti-IgE induced release of preformed mediators such as histamine and tryptase in a dose dependent manner. This inhibition was accompanied by a diminished, anti-IgE mediated increase in CD 107a and CD 63 surface expression. In sharp contrast, UV-light slightly preactivates mast cells as indicated by a marginal but statistically significant direct UV-caused histamine release. Theses findings matched the observation of slightly enhanced CD 107a and CD 63 surface levels in UV treated but unstimulated cells. After UVA-1 treatment the histamine release of substance P stimulated mast cells is statistically significant reduced which contrasts to the unchanged liberation of this preformed marker after UVB and PUVA-1 irradiation. Furthermore the release of mediators that are not or only partially preformed was examined. In the present study all types of UV-irradiation inhibits baseline IL-6 and IL-8 secretion from mast cells. The very low baseline level of TNF-a remained unaffected. Taken together, the present findings identify cutaneous human mast cells as important targets of UV-induced immunomodulation. They help explain both the dose-dependent adverse effects of UV-light and the beneficial and desired antiinflammatory effects during therapy.
114

Avaliação fotoquimiopreventiva do extrato de maçã e da rutina em modelos de pele in vitro e in vivo / Photochemoprotective evaluation of apple extract and rutin in in vitro and in vivo skin models

Siqueira, Silvia de 24 October 2014 (has links)
Diversos estudos têm demonstrado que os danos causados pela exposição da pele à radiação ultravioleta (RUV) são relacionados aos fotodanos ao DNA, geração de espécies reativas de oxigênio e ativação de mediadores do processo inflamatório. Há, portanto, um crescente interesse pelo uso de antioxidantes com potencial fotoquimiopreventivo, como o extrato de maçã e a rutina. O modelo mais utilizado para avaliação de agentes fotoquimiovreventivos é a exposição de camundongos sem pelos à RUV. Porém, os esforços para diminuir ou mesmo evitar a utilização de animais em ensaios científicos tem levado a busca por métodos alternativos à experimentação animal. Na primeira etapa desse trabalho visou-se a otimização de parâmetros relativos ao processo de extração do pó da maçã, bem como a caracterização do extrato obtido e da rutina. Assim, as condições de extração da maçã otimizadas foram tempo de extração de 22 h, teor de etanol no solvente de 60 % (p/p) e proporção solvente:planta de 18 (p/p). A concentração dos ativos presentes na maçã levou ao extrato enriquecido com polifenóis da maçã (EEPM), que apresentou elevada atividade antioxidante in vitro e teor de rutina de 6,1 ± 0.3 ?g/g de extrato. Ambos os ativos apresentaram baixa ou nenhuma toxicidade contra os fibroblastos MRC5, bem como protegeram os fibroblatos contra a morte induzida pela RUV e inibiram a formação de peróxidos lipídicos gerados pelas células irradiadas no tratamento com 4000 e 100 ?g/mL de EEPM e rutina, respectivamente. Na segunda etapa desse trabalho visou-se a avaliação do potencial fotoquimiopreventivo do EEPM e da rutina adicionados a uma formulação tópica em modelos de biópsia de pele humana e pele humana reconstruída in vitro e de camundongo sem pelos in vivo. O EEPM (1,25 %) e a rutina (0,75 %) em formulação foram avaliados quanto à retenção cutânea in vitro utilizando célula de difusão vertical de Franz e, embora não tenha sido possível detectar compostos do EEPM, foi demonstrado que 2,04 ± 0,19 ?g/cm2 da rutina ficou retida na biópsia de pele humana. Na avaliação da eficácia fotoquimiopreventiva em modelos de pele humana in vitro o EEPM e a rutina adicionados em formulação foram capazes de evitar/diminuir a formação de sunburn cells, a indução de caspase-3, dímeros de ciclobutanodipirimidina, metaloproteinases e peroxidação lipídica em pele exposta à RUV. Quanto a atividade funcional in vivo, o extrato enriquecido com polifenóis da maçã apresentou leve efeito inibidor do infiltrado inflamatório induzido pela RUV, enquanto que tanto a rutina como o extrato inibiram a depleção dos níveis de GSH endógeno, o que sugere uma potente atividade fotoquimiopreventiva para estes princípios ativos. Estes resultados são promissores e apontam para o uso do EEPM e da rutina na prevenção/tratamento dos danos induzidos pela RUV na pele. / Several studies have shown that the ultraviolet radiation (UVR) - induced skin damage are related to DNA photolesions, generation of reactive oxygen species and activation of inflammatory mediators. Therefore, there is an increasing interest in the use of antioxidants with photochemoprotective potential, such as apple extract and rutin. The most used model for evaluation of photochemoprotective agents is the exposure of hairless mice to UVR. However, efforts to reduce or even avoid the use of animals in scientific trials has pursued for alternative methods to replace/reduce animal testing. The first step of this work aimed to optimize parameters for the extraction of apple powder and the characterization of the obtained extract and rutin. Thus, the optimized apple extraction conditions were extraction time of 22 h, ethanol content of 60% (w/w) and plant:solvent ratio of 18 (w/w). The apple extract concentration led to an enriched apple polyphenols extract (EEPM), which showed strong in vitro antioxidant activity and rutin content of 6.1 ± 0.3 ?g / g. The actives showed low or no toxicity against MRC5 fibroblasts, protected these cells against UVR - induced death and inhibited lipid peroxidation in irradiated cells (treatment with 4000 and 100 ?g/mL of EEPM and rutin, respectively). The second step of this study aimed to evaluate the photochemoprotective potential of EEPM and rutin added in a topical formulation and assayed in in vitro models of human skin biopsy and human reconstructed skin and in vivo hairless mouse. The EEPM (1.25%) and rutin (0.75%) formulations were evaluated for skin retention in vitro using the Franz diffusion cell and, although it was not possible to detect compounds of EEPM, rutin was retained in the human skin biopsy (2.04 ± 0.19 mg/cm2). As regard the The photochemoprotective efficacy in in vitro models of human skin, EEPM and rutin formulations were able to prevent/reduce the formation of sunburn cells, induction of caspase-3, cyclobutane pyrimidine dimers, matrix metalloproteinases and lipid peroxidation in skin exposed to UVR. As for in vivo functional activity, EEPM showed a slight inhibitory effect of UVR-induced inflammatory infiltrate, while both EEPM and rutin completely inhibited endogenous GSH levels depletion. These results are promising and suggest the use of EEPM and rutin in the prevention/treatment of ultraviolet radiationinduced damage to the skin.
115

Das Renin-Angiotensin-System in menschlicher Haut

Wollschläger, Tanja 04 May 2006 (has links)
In der vorliegenden Arbeit wurde die Expression von Angiotensinogen, Renin, Angiotensin-Converting-Enzym (ACE) und von den Agiotensin-Rezeptoren AT1 und AT2 in humaner Haut untersucht, um zu sehen, ob humane Haut ein lokales Gewebe Renin-Angiotensin-System (RAS) besitzt und fähig ist, Angiotensin II (Ang II) zu synthetisieren sowie welche physiologische Rolle Ang II in humaner Haut haben könnte. Außerdem wurde das Expressionsmuster von Angiotensinogen, Renin und ACE in gesunder humaner Haut mit dem in Psoriasis, Basaliom und Spinaliom (SCC) verglichen, um einen Einblick in pathophysiologische Funktionen des RAS zu gewinnen. Mit Hilfe von RT-PCR konnten alle Komponenten des RAS in vitro auf mRNA Ebene in kultivierten primären Keratinozyten, Melanozyten, dermalen Fibroblasten und dermalen mikrovaskulären Endothelzellen (MVEC´s) nachgewiesen werden, mit einer Ausnahme: Melanozyten scheinen keine AT2-Rezeptoren zu exprimieren. Immunhistochemische Untersuchungen zeigten die Expression aller Komponenten auf Proteinebene in Epidermis und dermalen Gefäßwänden in Gewebeschnitten humaner Haut. Zusätzlich erfolgte der Nachweis von Ang II in kultivierten Keratinozyten mittels enzymatischen immunometrischen Assays. Während Angiotensinogen, Renin und ACE bei immunhistochemischen Untersuchungen an Gewebeschnitten gesunder menschlicher Haut in allen Epidermalschichten gleichmäßig verteilt waren, zeigte sich bei der Psoriasis eine deutliche Betonung der unteren Epidermalschichten. Immunhistochemische Untersuchungen von Basaliomen erbrachten eine verminderte Expression von Angiotensinogen und Renin innerhalb der Tumornester. ACE wurde in den Tumorzellen noch weniger exprimiert. In immunhistochemischen Untersuchungen von Spinaliomen färbten sich die Tumorzellen deutlich homogen an. Die Experimente haben gezeigt, dass alle Komponenten des RAS in enger Lokalisation in menschlicher Haut vorkommen und dass folglich ein lokales Gewebe RAS in humaner Haut existiert sowie dass humane Haut fähig ist, Ang II ohne Zufuhr weiterer Komponenten und ohne regulatorische Einflüsse aus der Zirkulation zu synthetisieren. Eine mögliche physiologische Rolle von Ang II könnte die Regulation von Keratinozyten-Proliferation und –Differenzierung über seine Rezeptoren sein. Bezüglich der pathophysiologischen Rolle haben die Untersuchungen eine Fehlregulation des kutanen RAS in Epidermis psoriatisch veränderter Haut gezeigt, welches ein Hinweis auf eine pathogenetische Rolle des RAS bei der gestörten Keratinozyten-Proliferation und –Differenzierung sein könnte. Das Expressionsmuster in den untersuchten Tumoren war uneinheitlich, weshalb eine Interpretation der Rolle des RAS in kutanen Tumoren ohne weitere Untersuchungen kaum möglich erscheint. 1 / The present study was designed to elucidate whether a local tissue renin-angiotensin system (RAS) is expressed in human skin, whether cutaneous cells are able to autonomously synthesise angiotensin II (Ang II), and to get a first insight into a putative physiological role of Ang II in this location. For this purpose, the expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE) and of the angiotensin receptors AT1 and AT2 was examined in human skin samples and in diverse cutaneous cells in primary culture on mRNA- and protein-level. Furthermore, the study compared the expression pattern of angiotensinogen, renin and ACE in healthy human skin with that in psoriasis, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) to look for possible differences between healthy and diseased skin. Using mRNA derived from cultured primary keratinocytes, melanocytes, dermal fibroblasts and dermal microvascular endothelial cells (MVECs), all components of the RAS could be demonstrated by RT-PCR except for AT2 receptors in melanocytes. Immunohistochemical stainings of cryostat sections of human skin revealed the expression of all components at protein level within the epidermis and in dermal vessel walls. In addition, the presence of Ang II in cultured keratinocytes and their supernatants could be proven by enzyme immunometric assay giving strong evidence for the ability of keratinocytes to autonomously synthesise Ang II. Regarding the comparison of RAS expression in healthy versus diseased skin, expression of angiotensinogen, renin and ACE was altered in all dermatoses examined. While in normal skin, RAS components were distributed equally and homogenously throughout all layers of the epidermis, in psoriatic skin their expression was more intense in the basal epidermal layers and less intense in the upper layers. In BCC sections, expression of angiotensinogen and renin was down-regulated, and tumour cells stained negatively for ACE. In SCC cryostat sections, tumour cells stained positively for all RAS components with an intensity comparable to normal skin. Taken together, the experiments revealed that a local tissue RAS exists in human skin, and that human skin is able to autonomously synthesise Ang II without any supply of components from the circulation. The physiological role of Ang II in normal skin may comprise the regulation of keratinocyte proliferation and differentiation. Concerning a putative pathophysiological role of Ang II in skin, this study provides evidence for a deregulation of the RAS in psoriatic skin and in BCC pointing to an involvement of the RAS in the pathomechanisms of these dermatoses. 1
116

Desenvolvimento de nanopartículas inovadoras a partir de constituintes da biodiversidade brasileira destinadas à aplicação tópica de antioxidantes / Development of innovative nanoparticles using brazilian compounds intended for antoxidants topical application

Colomé, Letícia Marques January 2011 (has links)
Nanopartículas lipídicas têm sido desenvolvidas para aplicação tópica de fármacos e ativos cosméticos. Neste trabalho, foi proposta a primeira aplicação de um lipídeo natural não-refinado biodegradável e biocompatível - manteiga de cupuaçu (Theobroma grandiflorum) - para a preparação de nanopartículas lipídicas, as quais foram denominadas teosferas. As teosferas foram preparadas por emulsificação-evaporação do solvente (EES) e por homogeneização à alta pressão (HAP), apresentando tamanho nanométrico e distribuição granulométrica estreita quando preparadas por ambos os métodos. O trabalho teve continuidade com a preparação de teosferas pelo método de EES utilizando manteiga de cupuaçu ou sua mistura com óleo de castanha do Brasil (Bertholletia excelsa) - também derivado da biodiversidade Amazônica - visando a incorporação de antioxidantes. Idebenona (IDB) foi selecionada por sua conhecida ação antioxidante e pela sua utilização em formulações cosméticas antienvelhecimento. IDB foi incorporada nas teosferas com eficiência de encapsulação superior a 99%, sendo que os estudos de liberação in vitro mostraram que a liberação de IDB a partir das teosferas foi mais lenta em comparação à IDB livre. Estes experimentos foram capazes ainda de demonstrar as características elásticas das teosferas. Além disso, foi evidenciada in vitro a atividade antioxidante superior das teosferas contendo IDB em relação ao ativo livre. Visando possibilitar a aplicação tópica de teosferas contendo IDB, em um trabalho subseqüente, suspensões de teosferas preparadas por HAP foram incorporadas em géis hidrofílicos. As formulações apresentaram características pseudoplásticas e demonstraram efeito oclusivo in vitro, o qual foi dependente da composição dos colóides. Finalmente, os estudos de permeação in vitro utilizando pele humana demonstraram que teosferas e nanocápsulas de núcleo lipídico, utilizadas neste estudo de modo comparativo, modificaram a permeção da IDB, permitindo a acumulação do ativo nas camadas superficiais da pele. / Lipid nanoparticles have been developed for administration of active substances to the skin, both for pharmaceutical and cosmetic uses. In the present work, we proposed the first use of a none-refined natural biodegradable and biocompatible lipid – Cupuaçu seed butter (Theobroma grandiflorum) – for the preparation of lipid nanoparticles, which were called theospheres. Theospheres were prepared by emulsification-solvent evaporation (ESE) and by high pressure homogenization technique (HPH), presenting size in nanometrical range and narrow particle size distribution for both methods. Taking these results into account, the next step of this work was the preparation of theospheres by ESE method using Cupuaçu seed butter with or without Brazil nut (Bertholletia excelsa) seed oil - another ingredient derived from an Amazonian fruit - intending the encapsulation of an antioxidant. Idebenone (IDB) has been selected due to its known antioxidant action and because it has been used in antiaging cosmetic formulations. IDB was incorporated in the theospheres presenting encapsulation efficiency higher than 99%. The in vitro release evaluation demonstrated that the release of IDB from theospheres was lower than that of free drug. Besides, the in vitro release study highlighted the elastic characteristics of theospheres. Additionally, IDB-loaded theospheres showed higher antioxidant activity compared to free IDB. Viewing the cutaneous administration, theosphere suspensions prepared by HPH technique were incorporated into hydrogels. The rheograms of the semi-solid formulations exhibited a non-Newtonian behavior presenting pseudoplastic characteristics. In vitro occlusion study highlighted the dependence of the occlusive effect on the lipidic composition of the theospheres. Finally, in vitro human skin permeation studies showed that theospheres and lipid-core nanocapsules, used in this study in a comparative way, changed the permeation of IDB, increasing the accumulative amount of IDB in the upper skin layer.
117

An examination of the bioactive lipids involved in skin cell inflammation and in response to ultraviolet radiation : effect of n-3 polyunsaturated fatty acid supplementation on red blood cell and human dermal fatty acid and production of eicosanoids by HaCaT keratinocytes and 46BR.1N fibroblasts following exposure to UVR

Al-Aasswad, Naser M. I. January 2013 (has links)
Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.
118

Desenvolvimento de nanopartículas inovadoras a partir de constituintes da biodiversidade brasileira destinadas à aplicação tópica de antioxidantes / Development of innovative nanoparticles using brazilian compounds intended for antoxidants topical application

Colomé, Letícia Marques January 2011 (has links)
Nanopartículas lipídicas têm sido desenvolvidas para aplicação tópica de fármacos e ativos cosméticos. Neste trabalho, foi proposta a primeira aplicação de um lipídeo natural não-refinado biodegradável e biocompatível - manteiga de cupuaçu (Theobroma grandiflorum) - para a preparação de nanopartículas lipídicas, as quais foram denominadas teosferas. As teosferas foram preparadas por emulsificação-evaporação do solvente (EES) e por homogeneização à alta pressão (HAP), apresentando tamanho nanométrico e distribuição granulométrica estreita quando preparadas por ambos os métodos. O trabalho teve continuidade com a preparação de teosferas pelo método de EES utilizando manteiga de cupuaçu ou sua mistura com óleo de castanha do Brasil (Bertholletia excelsa) - também derivado da biodiversidade Amazônica - visando a incorporação de antioxidantes. Idebenona (IDB) foi selecionada por sua conhecida ação antioxidante e pela sua utilização em formulações cosméticas antienvelhecimento. IDB foi incorporada nas teosferas com eficiência de encapsulação superior a 99%, sendo que os estudos de liberação in vitro mostraram que a liberação de IDB a partir das teosferas foi mais lenta em comparação à IDB livre. Estes experimentos foram capazes ainda de demonstrar as características elásticas das teosferas. Além disso, foi evidenciada in vitro a atividade antioxidante superior das teosferas contendo IDB em relação ao ativo livre. Visando possibilitar a aplicação tópica de teosferas contendo IDB, em um trabalho subseqüente, suspensões de teosferas preparadas por HAP foram incorporadas em géis hidrofílicos. As formulações apresentaram características pseudoplásticas e demonstraram efeito oclusivo in vitro, o qual foi dependente da composição dos colóides. Finalmente, os estudos de permeação in vitro utilizando pele humana demonstraram que teosferas e nanocápsulas de núcleo lipídico, utilizadas neste estudo de modo comparativo, modificaram a permeção da IDB, permitindo a acumulação do ativo nas camadas superficiais da pele. / Lipid nanoparticles have been developed for administration of active substances to the skin, both for pharmaceutical and cosmetic uses. In the present work, we proposed the first use of a none-refined natural biodegradable and biocompatible lipid – Cupuaçu seed butter (Theobroma grandiflorum) – for the preparation of lipid nanoparticles, which were called theospheres. Theospheres were prepared by emulsification-solvent evaporation (ESE) and by high pressure homogenization technique (HPH), presenting size in nanometrical range and narrow particle size distribution for both methods. Taking these results into account, the next step of this work was the preparation of theospheres by ESE method using Cupuaçu seed butter with or without Brazil nut (Bertholletia excelsa) seed oil - another ingredient derived from an Amazonian fruit - intending the encapsulation of an antioxidant. Idebenone (IDB) has been selected due to its known antioxidant action and because it has been used in antiaging cosmetic formulations. IDB was incorporated in the theospheres presenting encapsulation efficiency higher than 99%. The in vitro release evaluation demonstrated that the release of IDB from theospheres was lower than that of free drug. Besides, the in vitro release study highlighted the elastic characteristics of theospheres. Additionally, IDB-loaded theospheres showed higher antioxidant activity compared to free IDB. Viewing the cutaneous administration, theosphere suspensions prepared by HPH technique were incorporated into hydrogels. The rheograms of the semi-solid formulations exhibited a non-Newtonian behavior presenting pseudoplastic characteristics. In vitro occlusion study highlighted the dependence of the occlusive effect on the lipidic composition of the theospheres. Finally, in vitro human skin permeation studies showed that theospheres and lipid-core nanocapsules, used in this study in a comparative way, changed the permeation of IDB, increasing the accumulative amount of IDB in the upper skin layer.
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Desenvolvimento de nanopartículas inovadoras a partir de constituintes da biodiversidade brasileira destinadas à aplicação tópica de antioxidantes / Development of innovative nanoparticles using brazilian compounds intended for antoxidants topical application

Colomé, Letícia Marques January 2011 (has links)
Nanopartículas lipídicas têm sido desenvolvidas para aplicação tópica de fármacos e ativos cosméticos. Neste trabalho, foi proposta a primeira aplicação de um lipídeo natural não-refinado biodegradável e biocompatível - manteiga de cupuaçu (Theobroma grandiflorum) - para a preparação de nanopartículas lipídicas, as quais foram denominadas teosferas. As teosferas foram preparadas por emulsificação-evaporação do solvente (EES) e por homogeneização à alta pressão (HAP), apresentando tamanho nanométrico e distribuição granulométrica estreita quando preparadas por ambos os métodos. O trabalho teve continuidade com a preparação de teosferas pelo método de EES utilizando manteiga de cupuaçu ou sua mistura com óleo de castanha do Brasil (Bertholletia excelsa) - também derivado da biodiversidade Amazônica - visando a incorporação de antioxidantes. Idebenona (IDB) foi selecionada por sua conhecida ação antioxidante e pela sua utilização em formulações cosméticas antienvelhecimento. IDB foi incorporada nas teosferas com eficiência de encapsulação superior a 99%, sendo que os estudos de liberação in vitro mostraram que a liberação de IDB a partir das teosferas foi mais lenta em comparação à IDB livre. Estes experimentos foram capazes ainda de demonstrar as características elásticas das teosferas. Além disso, foi evidenciada in vitro a atividade antioxidante superior das teosferas contendo IDB em relação ao ativo livre. Visando possibilitar a aplicação tópica de teosferas contendo IDB, em um trabalho subseqüente, suspensões de teosferas preparadas por HAP foram incorporadas em géis hidrofílicos. As formulações apresentaram características pseudoplásticas e demonstraram efeito oclusivo in vitro, o qual foi dependente da composição dos colóides. Finalmente, os estudos de permeação in vitro utilizando pele humana demonstraram que teosferas e nanocápsulas de núcleo lipídico, utilizadas neste estudo de modo comparativo, modificaram a permeção da IDB, permitindo a acumulação do ativo nas camadas superficiais da pele. / Lipid nanoparticles have been developed for administration of active substances to the skin, both for pharmaceutical and cosmetic uses. In the present work, we proposed the first use of a none-refined natural biodegradable and biocompatible lipid – Cupuaçu seed butter (Theobroma grandiflorum) – for the preparation of lipid nanoparticles, which were called theospheres. Theospheres were prepared by emulsification-solvent evaporation (ESE) and by high pressure homogenization technique (HPH), presenting size in nanometrical range and narrow particle size distribution for both methods. Taking these results into account, the next step of this work was the preparation of theospheres by ESE method using Cupuaçu seed butter with or without Brazil nut (Bertholletia excelsa) seed oil - another ingredient derived from an Amazonian fruit - intending the encapsulation of an antioxidant. Idebenone (IDB) has been selected due to its known antioxidant action and because it has been used in antiaging cosmetic formulations. IDB was incorporated in the theospheres presenting encapsulation efficiency higher than 99%. The in vitro release evaluation demonstrated that the release of IDB from theospheres was lower than that of free drug. Besides, the in vitro release study highlighted the elastic characteristics of theospheres. Additionally, IDB-loaded theospheres showed higher antioxidant activity compared to free IDB. Viewing the cutaneous administration, theosphere suspensions prepared by HPH technique were incorporated into hydrogels. The rheograms of the semi-solid formulations exhibited a non-Newtonian behavior presenting pseudoplastic characteristics. In vitro occlusion study highlighted the dependence of the occlusive effect on the lipidic composition of the theospheres. Finally, in vitro human skin permeation studies showed that theospheres and lipid-core nanocapsules, used in this study in a comparative way, changed the permeation of IDB, increasing the accumulative amount of IDB in the upper skin layer.
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Estratégias computacionais como métodos alternativos para avaliação da sensibilização cutânea / Computational strategies as alternative methods to chemical prediction of skin sensitization

Alves, Vinícius de Medeiros 12 May 2017 (has links)
Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-12T15:19:44Z No. of bitstreams: 2 Dissertação - Vinicius de Medeiros Alves - 2014.pdf: 3082084 bytes, checksum: da4838d5fe24841429f43de84204d98a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-06-12T15:21:40Z (GMT) No. of bitstreams: 2 Dissertação - Vinicius de Medeiros Alves - 2014.pdf: 3082084 bytes, checksum: da4838d5fe24841429f43de84204d98a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-12T15:21:40Z (GMT). No. of bitstreams: 2 Dissertação - Vinicius de Medeiros Alves - 2014.pdf: 3082084 bytes, checksum: da4838d5fe24841429f43de84204d98a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-05-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: Skin sensitization is a major environmental and human health hazard. Although many chemicals have been evaluated in humans, there have been no efforts to model these data to date. Skin sensitization is commonly evaluated using structural alerts. However, there has been a growing concern that alerts disproportionally flag too many chemicals as toxic, which questions their reliability as toxicity markers. The main goal of this thesis was to develop and apply new cheminformatics methods to predict skin sensitization of chemical compounds that lack experimental data. Methodology: It has been compiled, curated, analyzed, and compared the available human data and the murine (performed in mice) animal model data, named LLNA (local lymph node assay). Using these data, it was developed reliable computational models and applied them for virtual screening of chemical libraries to identify putative skin sensitizers. It was developed a freely accessible web-based application for the identification of potential skin sensitizers. In addition, it was demonstrated that contrary to the common perception of QSAR models as “black boxes” they can be used to identify statistically significant chemical substructures (QSAR-based alerts) that influence toxicity. Results and discussion: The overall concordance between murine LLNA and human skin sensitization responses for a set of 135 unique chemicals was low (R = 28-43%), although several chemical classes had high concordance. We have succeeded to develop predictive QSAR models of all available human data with the external correct classification rate of 71%. A consensus model integrating concordant QSAR predictions and LLNA results afforded a higher correct classification rate of 82% but at the expense of the reduced external dataset coverage (52 %). We used the developed QSAR models for virtual screening of CosIng database and identified 1,061 putative skin sensitizers; for seventeen of these compounds, we found published evidence of their skin sensitization effects. The developed Pred-Skin web app (http://www.labmol.com.br/predskin/) is based on binary QSAR models of human (109 compounds) and LLNA (515 compounds) data with good external correct classification rate (70-81% and 72-84%, respectively). It is also included a multiclass potency model based on LLNA data (accuracy ranging between 73-76%). Conclusions: Models reported herein provide more accurate alternative to LLNA testing for human skin sensitization assessment across diverse chemical data. In addition, they can also be used to guide the structural optimization of toxic compounds to reduce their skin sensitization potential. The Pred-Skin web app is a fast, reliable, and user-friendly tool for early assessment of chemically-induced skin sensitization. A new approach that synergistically integrates structural alerts and rigorously validated QSAR models for a more transparent and accurate safety assessment of new chemicals was also proposed. / Introdução: A sensibilização cutânea é um importante parâmetro de avaliação de toxicidade humana e ambiental. Embora muitos compostos tenham sido avaliados em seres humanos, não foi reportado até o momento modelos de QSAR (do inglês, quantitative structure-activity relationships) gerados com esses dados. Comumente, a sensibilização cutânea é avaliada computacionalmente usando-se alertas estruturais. No entanto, tem havido uma preocupação crescente de que alertas sinalizam a maioria dos compostos como tóxicos, o que questiona sua confiabilidade como marcadores de toxicidade. O objetivo geral do presente trabalho foi desenvolver e aplicar novos métodos de quimioinformática para predizer a sensibilização cutânea de compostos químicos que carecem de dados experimentais. Metodologia: Foram compilados, preparados, analisados e comparados os dados de sensibilização cutânea de pele humana e do modelo animal murino (realizado em camundongos), denominado LLNA (local lymph node assay). Modelos de QSAR foram desenvolvidos utilizando esses dados e aplicados para a triagem de quimiotecas virtuais para identificar potenciais sensibilizadores. Foi desenvolvido um aplicativo gratuito para a identificação de potenciais sensibilizadores cutâneos. Além disso, foi demonstrado que modelos de QSAR podem ser usados para identificar subestruturas químicas estatisticamente significativas (alertas estruturais baseados em QSAR) que influenciam a toxicidade. Resultados e discussão: A concordância global (R) entre respostas de sensibilização cutânea humana e murina para um conjunto de 135 substâncias químicas únicas foi baixa (R = 28-43%), embora várias classes químicas apresentassem alta concordância. Foi possível desenvolver modelos de QSAR preditivos com taxa de classificação correta externa de 71%. Um modelo de consenso que integrava predições concordantes de QSAR e dados de LLNA proporcionaram uma acurácia 82%. Utilizou-se os modelos de QSAR desenvolvidos para a triagem virtual da base de dados CosIng e foram identificados 1061 potenciais sensibilizadores cutâneos. Para dezessete desses compostos, encontrou-se evidências publicadas de seus efeitos de sensibilização cutânea em seres humanos. O aplicativo desenvolvido, Pred-Skin (http://www.labmol.com.br/predskin/), baseia-se em modelos de QSAR classificatórios de dados humanos (109 compostos) e murinos (515 compostos) com boa taxa de classificação correta externa (70-81% e 72-84%, respectivamente). Esse aplicativo também possui um modelo de multiclassificatório desenvolvido com dados de LLNA (precisão que varia entre 73-76%). Conclusões: Os modelos de QSAR desenvolvidos forneceram uma alternativa mais precisa do que o modelo animal para avaliação da sensibilização cutânea humana. Além disso, a interpretação dos modelos de QSAR permitem orientar a otimização estrutural de compostos tóxicos para reduzir o potencial de toxicidade. O aplicativo Pred-Skin é uma ferramenta rápida, confiável e de fácil utilização para a avaliação da sensibilização cutânea de compostos químicos. Foi também proposta uma nova abordagem que integra sinergicamente alertas estruturais e modelos de QSAR rigorosamente validados para uma avaliação de toxicidade mais transparente e precisa de novos produtos químicos.

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