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    Celulose bacteriana como penso curativo

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    Tese de doutoramento em Biomedical EngineeringWounds, in particular traumatic (e.g. burns) and chronic ones, are a major cause of morbidity, impaired life quality and high health care costs. They often result in long hospitalization stays, taking up substantial health resources in developed countries. Conventional treatments are often painful, expensive and may increase the infection risk, compromising the treatments‚Äô time and success. In recent years, there have been efforts to develop new advanced methodologies to heal chronic wounds, including the topic use of growth factors or cell-based therapies. However, in many cases, the therapeutic efficacy is low, the therapies are expensive and require application in a clinical facility. Therefore, development of new therapeutics is absolutely necessary and important to satisfy these unmet clinical needs. So, this work comprised the development of a safe, easy-to-use and non expensive novel dressing, aimed at efficiently addressing these issues, by attaining faster and proper wound healing. The use of bacterial nanocellulose (BNC) has already demonstrated positive results in the treatment of different kinds of wounds. Additionally, BNC is considered a promising drug delivery system. In this work, BNC was conceived as a protective barrier against exogenous agents (particles, microorganisms) that can impair wound healing, and as a drug carrier for the controlled release of hydrophobic drugs, namely of vitamin D3 (Vit D3 ), an inducer of the endogenous expression of antimicrobial peptide (AMP) LL37, known for accelerating the wound healing process. In a first part of this project, the optimization of the static BNC production was performed, aiming at making it viable and economic at large scale. First, an experimental design, based on response surface methodology (RSM) - central composite design (CCD) - was used to optimize the culture medium for BNC production by Komagataeibacter xylinus BPR 2001, using a simple culture medium composition based on byproducts from the food industry. The optimal conditions for BNC production were (% (m/v)): molasses 5.38; CSL 1.91; ammonium sulphate 0.63; disodium phosphate 0.270; citric acid 0.115 and ethanol 1.38 % (v/v). The experimental and predicted maximum BNC production yields were 7.5 ¬Ī0.54 g/L and 6.64 ¬Ī0.079 g/L, respectively, after 9 days at 30 ¬ļC. Furthermore, the effect of the surface area and culture medium depth on the BNC production yield and productivity were evaluated. BNC dry mass production increased with the surface area and with the medium volume (depth) and fermentation time. Also, as long as nutrients were still available in the culture media, the BNC mass productivity was maintained overtime. The pre-inoculum preparation (PIP) step was also optimized with regards to the (a) identification of an inexpensive culture medium for pre-inoculum leading to a high cell density; (b) analysis of the effect of the initial cellular concentration on the static production of BNC and (c) kinetics of cell growth throughout the different steps of pre-inoculum preparation, including static and stirred - laboratorial and pilot-scale ‚Äď fermentations. The best composition for PIP medium was (% (m/v)): Glucose and Fructose syrup 1.5- 2.0; Corn Step Liquor (protein basis) 0.7; citric acid 0.115; Na2HPO4 0.27. The analysis of the cell growth kinetics in the different steps of PIP showed that a careful control on the culture time in each stage is advisable. The time required to reach the exponential phase was very different in each stage of PIP, reducing significantly from the static culture to the stirred culture and for large scale stirred culture, in a 75 L Bioreactor. In a second part of this work, the use of BNC as a drug carrier was addressed. Since Vit D3 is poorly water soluble, and thus not easily incorporated in the highly hydrophilic environment of the BNC membrane, Vit D3 was encapsulated in a self-assembled hyaluronic acid (HA)-based amphiphilic nanogel and then incorporated in the BNC membrane. The carrier was obtained by grafting hexadecylamine (Hexa) into the HA backbone (HA-Hexa). Vit D3 was successfully loaded into the nanogel (HA-Vit D3 ) with an encapsulation efficiency between 60-91 %. The loaded system- HA-Vit D3 - was embedded into BNC, conceived as a transdermal delivery system. The release of Vit D3 was monitored over time using a Franz cell device. Around 70 % of the initial Vit D3 available was released from BNC membranes in the first 48 h. Most importantly, we observed that the released Vit D3 still remained within the HA-Hexa nanogel carrier. Vit D3 is known to stimulate the endogenous production of human cathelicidin (LL37), which is known to accelerate wound healing. Thus, formulations of HA-Vit D3 and HA-LLKKK18 (an analogue of LL37) were tested in vivo, using excision and chronic wound in dexamethasone treated C57BL/6 and db+/db+ mice models, as to evaluate and compare their efficiency in wound repairing. However, the results did not confirm any wound healing improvement.As feridas cr√≥nicas e traum√°ticas (e.g. queimaduras) apresentam uma elevada morbilidade, afetando severamente a qualidade de vida dos pacientes. Os tratamentos convencionais implicam longos per√≠odos de interna√ß√£o hospitalar, com significativo consumo de recursos dos sistemas de sa√ļde nos pa√≠ses desenvolvidos. Al√©m disso, s√£o dolorosos, caros e podem aumentar o risco de infe√ß√£o, comprometendo a dura√ß√£o e o sucesso dos tratamentos. Recentemente, t√™m sido desenvolvidos esfor√ßos para o desenvolvimento de novas metodologias avan√ßadas para o tratamento de feridas cr√≥nicas, incluindo a aplica√ß√£o t√≥pica de fatores de crescimento ou terapias baseadas em c√©lulas. Em muitos casos, estas novas abordagens s√£o caras, devendo ser realizadas numa unidade hospitalar, e a sua efic√°cia terap√™utica √© baixa. Assim, o desenvolvimento de novas solu√ß√Ķes para satisfazer esta necessidade cl√≠nica ainda n√£o satisfeita √© absolutamente necess√°rio. Com este trabalho pretende se desenvolver um penso curativo eficiente, inovador, f√°cil de usar e n√£o dispendioso, atrav√©s de uma abordagem segura, visando uma cicatriza√ß√£o mais r√°pida e adequada da ferida. A nanocelulose bacteriana (BNC) demonstrou j√° resultados positivos no tratamento de diferentes tipos de feridas, assim como foi j√° demonstrado tamb√©m o seu potencial como sistema de entrega de f√°rmacos. Neste trabalho, a BNC foi utilizada como ve√≠culo para a liberta√ß√£o controlada de mol√©culas hidrof√≥bicas, nomeadamente a vitamina D3 (Vit D3 ), que √© um indutor da express√£o end√≥gena do pept√≠do antimicrobiano LL37, conhecido por acelerar o processo de cicatriza√ß√£o de feridas. Al√©m disso, a BNC funciona como uma barreira protetora contra agentes ex√≥genos (poeiras, microorganismos) que podem prejudicar a cicatriza√ß√£o de feridas. Numa primeira parte, foram desenvolvidos trabalhos visando tornar a produ√ß√£o em grande escala de BNC em cultura est√°tica econ√≥mica e vi√°vel. Nesse sentido, foi usado um desenho experimental, baseado na metodologia de superf√≠cie de resposta (RSM) - planeamento composto central (CCD) - para otimizar o meio de cultura, usando subprodutos da ind√ļstria alimentar. Foi utilizada a estirpe Komagataeibacter xylinus BPR 2001, a 30 ¬ļC. Foram identificadas as seguintes condi√ß√Ķes √≥timas para a produ√ß√£o de BNC (% (m/v)): mela√ßo 5,38, xarope de milho (CSL) 1,91; sulfato de am√≥nio 0,63; fosfato diss√≥dico 0,270; √°cido c√≠trico 0,115 e etanol 1,38 % (v/v). Os rendimentos m√°ximos experimentais e previstos de produ√ß√£o de BNC foram 7,5 ¬Ī0,54 g/L e 6,64 ¬Ī0,079 g/L, respetivamente, ap√≥s 9 dias. Adicionalmente, foram avaliados o efeito da √°rea superficial e da profundidade/altura do meio de cultura no rendimento e produtividade em BNC. Verificou-se que a produ√ß√£o de BNC aumenta com a √°rea superficial, com o volume de meio de cultura (profundidade) e com o tempo de fermenta√ß√£o. Al√©m disso, observou-se que a produtividade de BNC se mant√©m constante at√© se esgotarem os nutrientes no meio de cultura. Para a etapa de prepara√ß√£o pr√©-in√≥culo (PIP), a otimiza√ß√£o consistiu em diferentes estudos, especificamente: (a) otimiza√ß√£o dum meio de cultura de custos reduzidos, que permita a obten√ß√£o de uma elevada densidade celular; (b) avalia√ß√£o do efeito da concentra√ß√£o celular inicial na produ√ß√£o est√°tica de BNC e (c) estudo da cin√©tica de crescimento celular ao longo das diferentes etapas de PIP. A melhor composi√ß√£o para o PIP foi (% (m/v)): xarope de glucose e frutose 1,5- 2,0; CSL 0,7; √°cido c√≠trico 0,115 e Na2HPO4 0,27. Os estudos de cin√©tica de crescimento celular para as diferentes etapas do PIP evidenciam a necessidade dum controle cuidadoso do tempo de cultura em cada etapa do PIP. O tempo necess√°rio para atingir a fase exponencial foi muito diferente em cada fase do PIP, reduzindo significativamente da cultura est√°tica, para a cultura agitada, e para cultura agitada em larga escala num bioreator de 75 L. A segunda parte do trabalho relaciona-se com o desenvolvimento da BNC como sistema de entrega de f√°rmacos. A Vit D3 √© pouco sol√ļvel em √°gua e, portanto, n√£o √© facilmente incorporada no ambiente altamente hidrof√≠lico como o da membrana de BNC. Para esse efeito foi usado um nanogel anfif√≠lico auto-organizado obtido pela liga√ß√£o de hexadecilamina (Hexa) na cadeia do √°cido hialur√≥nico (HA). A Vit D3 foi ent√£o encapsulada no nanogel de (HA-Hexa) e em seguida impregnada na membrana de BNC, com uma efici√™ncia de encapsula√ß√£o entre 60-91 %. A liberta√ß√£o da Vit D3 foi monitorizada ao longo do tempo, usando uma c√©lula de Franz e realizando estudos de permea√ß√£o. Observou-se a liberta√ß√£o de cerca de 70 % da Vit D3 , ainda dentro do nanogel de HA-Hexa, das membranas de BNC em 48h. Finalmente, foi testada a utiliza√ß√£o de HA-Vit D3 e de HA-LLKKK18 (um p√©ptido an√°logo √† LL37) em modelos de feridas de excis√£o e cr√≥nicas em ratinhos tratados com dexametasona e diab√©ticos tipo II (db + / db +) C57BL/6. No entanto, os resultados n√£o revelaram uma maior efici√™ncia na cicatriza√ß√£o de feridas na presen√ßa das referidas formula√ß√Ķes.Ao Projeto BioTecNorte (NORTE-01-0145-FEDER-000004), n¬ļ 003435: ‚ÄúBUILD ‚Äď Bacterial cellulose Leather‚ÄĚ, financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) atrav√©s do Programa Operacional do Regional do Norte (NORTE 2020) e ao projeto SkinChip: Disruptive cellulose-based microfluidic device for 3D skin modelling, PTDC/BBB-BIO/1889/2014 e ainda √† Funda√ß√£o para a Ci√™ncia e Tecnologia no √Ęmbito do financiamento estrat√©gico da unidade UID / BIO / 04469/2019 e pela atribui√ß√£o da bolsa de doutoramento SFRH/BD/89547/2012

    Aplica√ß√Ķes biom√©dicas de materiais fotoativos √† base de amido

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    The centrality that light presents in the human being‚Äôs life since antiquity to its use in industrial and biomedical applications makes it a topic of intense research. The knowledge of its properties and the way it affects the environment and living beings allows us to find new applications, namely biomedical. Photodynamic therapy is known since the end of the 1970‚Äôs as a promising approach in the treatment of neoplasia. This therapeutic approach takes advantage of light‚Äôs interaction with photosensitive compounds, named photosensitizers, which in the presence of molecular oxygen produce reactive oxygen species capable of leading tumoral cells to cellular death. From tumoral cells to microbial cells was only a step, and this photodynamic approach has proved very successful, more specifically using porphyrins as photosensitizers, in inactivating microorganisms, including microbial strains multi-resistant to conventional antimicrobial agents (e.g., antibiotics, antifungals). With the rise of microbial resistance to conventional antimicrobials and increasing emergence of chronical conditions such as diabetes and its complications, it becomes imperative to find new therapies which allow to treat and help meliorate patients‚Äô quality of life. The goal of this study was to evaluate the possibility of using cost-friendly materials, such as starch, to support porphyrinic derivatives and in this way to produce photoactive materials with the ability to act as photosensitizers and inactivate common skin infections such as the ones incited by methicillin resistant Staphylococcus aureus and Pseudomonas aeruginosa, and which arise in diabetic ulcers, and furthermore to evaluate its ability to promote tissue regeneration. The porphyrins selected to incorporate in starch were cationic porphyrin 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TPP5F) and neutral porphyrin 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TMPyP) which were prepared and later immobilized in the starch-based materials. The photoactive films prepared by incorporation of porphyrin in starch were then tested and their antimicrobial properties evaluated in in vitro and ex vivo studies. The TMPyP incorporated starch-based film revealed promising antimicrobial activity as it was able to inactivate S.aureus (MRSA), both in vitro and ex vivo, after white light exposure with an irradiance of 50 mW cm-2 for 60 min and 24h, respectively. Using porcine skin as skin infection model, this TMPyP film revealed potential in preventing the onset of bacterial infections. To evaluate the biocompatibility and possible stimulating effects in tissue regeneration of the starch/ porphyrin films, in vitro studies were conducted in cell lines of fibroblasts (HDF) and endothelial cells (HMEC), two important components of tissue regeneration and wound healing. Four different parameters were tested: cellular viability, cellular migration, ROS formation and cellular adhesion. For that, the starch/porphyrin-based materials were applied and irradiated with a red light from a LED system with an irradiance of 5 mW cm-2 for 15 min. The films of 7.06 mm2 revealed the best results in viability and at these conditions the cells present high levels of cellular ROS. The application of light seems to have influenced all parameters except ROS formation. Low dose red-light seems to positively influence viability and wound healing in endothelial cells, revealing itself as a potential promoter of vascularization. The results obtained with the starch/porphyrin films allow for a perspective of their potential application in the treatment of diabetics‚Äô wounds/ulcers.A centralidade que a luz apresenta na vida do ser Humano desde a antiguidade at√© √† sua utiliza√ß√£o em aplica√ß√Ķes industriais e biom√©dicas faz com que esta continue a ser objeto de intensa investiga√ß√£o. O conhecimento das suas propriedades e da forma como afeta o ambiente e os seres vivos permite encontrar novas aplica√ß√Ķes nomeadamente biom√©dicas. A terapia fotodin√Ęmica √© reconhecida desde finais da d√©cada de 70 como uma abordagem promissora no tratamento de neoplasias. Esta abordagem terap√™utica tira partido da intera√ß√£o da luz com compostos fotossens√≠veis designados de fotosensibilizadores que na presen√ßa de oxig√©nio molecular produzem esp√©cies reativas de oxig√©nio capaz de levar √† morte celular de c√©lulas tumorais. Das c√©lulas tumorais √†s c√©lulas microbianas foi um passo, e esta abordagem fotodin√Ęmica tem sido muito bem sucedida nomeadamente utilizando porfirinas como fotosensibilizadores, na inativa√ß√£o de microorganismos incluindo estirpes microbianas multi-resistentes aos agentes antimicrobianos convencionais (eg. antibi√≥ticos, antif√ļngicos, etc). Com o aumento da resist√™ncia microbiana aos antimicrobianos convencionais e o surgimento crescente de condi√ß√Ķes cr√≥nicas como diabetes e as suas complica√ß√Ķes, torna-se imperativo encontrar novas terapias que permitam tratar e melhorar a qualidade de vida dos pacientes. Este estudo teve como objetivo avaliar a possibilidade de utilizar materiais baratos, como seja o amido, para suportar derivados porfir√≠nicos e desta forma produzir materiais fotoactivos com capacidade de atuar como fotosensibilizadores e inactivarem comuns infe√ß√Ķes de pele como sejam as provocadas por Staphylococcus aureus resistente √† meticilina (MRSA) e Pseudomonas aeruginosa e que surgem em √ļlceras de diab√©ticos, e mais do que isso, avaliar a sua capacidade de promoverem a regenera√ß√£o de tecidos. As porfirinas selecionadas para incorporar no amido foram a porfirina cati√≥nica 5,10,15,20-tetraquis(1- metilpiridinium-4-il)porfirina (TMPyP) e porfirina neutra 5,10,15,20- tetraquis(pentafluorofenil)porfirina (TPP5F) que foram preparadas e posteriormente imobilizadas nos materiais √† base de amido. Os filmes fotoactivos preparados por incorpora√ß√£o de porfirina em amido, foram ent√£o testados e as suas propriedades antimicrobianas em estudos in vitro e ex vivo avaliadas. O filme √† base de amido com a TMPyP incorporada revelou promissora atividade antimicrobiana sendo que conseguiu inativar a S. aureus (MRSA), tanto in vitro como ex vivo, ap√≥s exposi√ß√£o a luz branca com uma irradi√Ęncia de 50 mW cm-2 por 60 min e 24h, respetivamente. Utilizando pele de porco como um modelo de infe√ß√£o de pele, este filme contendo TMPyP revelou potencial em prevenir a instala√ß√£o de infe√ß√Ķes bacterianas. Para avaliar a biocompatibilidade dos filmes de amido-porfirina preparados e poss√≠veis efeitos estimulantes na regenera√ß√£o de tecidos, foram realizados estudos in vitro em linhas celulares de fibroblastos (HDF) e c√©lulas endoteliais (HMEC), dois componentes importantes da regenera√ß√£o de tecido e cura de feridas. Quatro par√Ęmetros diferentes foram testados: viabilidade celular, migra√ß√£o celular, forma√ß√£o de ROS e ades√£o celular. Para tal os filmes √† base de amido-porfirinas foram aplicados e irradiados com luz vermelha proveniente de um sistema LED com uma irradi√Ęncia de 5 mW cm-2 por 15 min. Os filmes de dimens√Ķes 7.06 mm2 revelaram os melhores resultados nos ensaios de viabilidade celular, e as c√©lulas nestas condi√ß√Ķes apresentam elevada quantidade de ROS celular. A aplica√ß√£o de luz parece ter influenciado todos os par√Ęmetros exceto a forma√ß√£o de ROS. A luz vermelha em baixa dose parece influenciar positivamente a viabilidade e regenera√ß√£o em c√©lulas endoteliais, revelando-se um potencial promotor de vasculariza√ß√£o. Os resultados obtidos com os filmes amido-porfirina permitem perspetivar a sua potencial aplica√ß√£o no tratamento de feridas/√ļlceras de diab√©ticos.Mestrado em Biomedicina Molecula

    Phytochemical prospection and larvicidal bioactivity of the janaguba (Himatanthus drasticus) Mart. Plumel (Apocynaceae) latex against Aedes aegypti L. (Diptera: Culicidae)

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    Abstract The aim of this study was to carry out phytochemical prospecting and evaluate the larvicidal activity of Himatanthus drasticus latex extracts against Aedes aegypti. The extracts were obtained by maceration from 5 g of latex powder concentrated separately in 100 mL of methanol, ethyl acetate, and hexane solvents. The concentrations of 100, 200, 300, 400, and 500 ppm of each extract were tested in triplicate with a solution of pyriproxyfen as the positive control and distilled water and dimethylsulfoxide as the negative control. The phytochemical prospection of the methanolic extract showed the presence of phenolic compounds, such as anthocyanins, anthocyanidins, catechins, chalcones, aurones, leucoanthocyanidins, and condensed tannins. The insecticidal bioactivity was most significant for the methanolic extract. The methanolic extract lethal concentrations (LC) of 50 and 90% were 190.76 and 464.74 ppm, respectively. After 48 hours of exposure, the extracts using methanol, ethyl acetate, and hexane at their highest concentrations (500 ppm) caused larval mortality of 100, 73.33, and 66.67%, respectively. These extracts also promoted changes in the external morphology of the larvae, such as damage to the anal papillae, darkening of the body, and reduction in the number of bristles. The methanolic extract showed greater expressivity for morphological changes. The latex of H. drasticus has larvicidal activity against third-stade larvae of A. aegypti and it is more significant when obtained through maceration in methanol. The methanolic extract of H. drasticus latex contains phenolic compounds with insecticidal activity against A. aegypti larvae

    Pathogenesis and treatment of chronic rhinosinusitis from the perspective of sinonasal epithelial dysfunction

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    BackgroundChronic rhinosinusitis (CRS) is a clinical syndrome primarily characterized by long-term mucosal inflammation of the nasal cavity and sinuses. The pathogenesis of CRS is still unclear due to its high heterogeneity. A number of studies have recently focused on the sinonasal epithelium. Thus, there has been a quantum leap in awareness of the role of the sinonasal epithelium, which is now understood as an active functional organ rather than simply an inert mechanical barrier. Undoubtedly, epithelial dysfunction plays a vital role in the onset and development of CRS.ObjectiveIn this article, we discuss the potential contribution of sinonasal epithelium dysfunction to CRS pathogenesis and explore a few current and developing therapeutic options targeting the sinonasal epithelium.ResultsImpaired mucociliary clearance (MCC) and an abnormal sinonasal epithelial barrier are usually considered to be the main causative factors in CRS. Epithelial-derived bioactive substances, such as cytokines, exosomes, and complements, play a vital role in the regulation of innate and adaptive immunity and contribute to the pathophysiological alterations of CRS. The phenomena of epithelial‚Äďmesenchymal transition (EMT), mucosal remodeling, and autophagy observed in CRS offer some novel insights into the pathogenesis of this disease. In addition, existing treatment options targeting disorder of sinonasal epithelium can help to relieve the main symptoms associated with CRS to some extent.ConclusionThe presence of a normal epithelium is fundamental for maintaining homeostasis in the nasal and paranasal sinuses. Here, we describe various aspects of the sinonasal epithelium and highlight the contributions of epithelial dysfunction to CRS pathogenesis. Our review provides sound evidence of the need for in-depth study of the pathophysiological alterations of this disease and for the development of novel epithelium-targeting alternative treatments

    IDO1 and inflammatory neovascularization: bringing new blood to tumor-promoting inflammation

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    In parallel with the genetic and epigenetic changes that accumulate in tumor cells, chronic tumor-promoting inflammation establishes a local microenvironment that fosters the development of malignancy. While knowledge of the specific factors that distinguish tumor-promoting from non-tumor-promoting inflammation remains inchoate, nevertheless, as highlighted in this series on the ‚ÄėHallmarks of Cancer‚Äô, it is clear that tumor-promoting inflammation is essential to neoplasia and metastatic progression making identification of specific factors critical. Studies of immunometabolism and inflamometabolism have revealed a role for the tryptophan catabolizing enzyme IDO1 as a core element in tumor-promoting inflammation. At one level, IDO1 expression promotes immune tolerance to tumor antigens, thereby helping tumors evade adaptive immune control. Additionally, recent findings indicate that IDO1 also promotes tumor neovascularization by subverting local innate immunity. This newly recognized function for IDO1 is mediated by a unique myeloid cell population termed IDVCs (IDO1-dependent vascularizing cells). Initially identified in metastatic lesions, IDVCs may exert broader effects on pathologic neovascularization in various disease settings. Mechanistically, induction of IDO1 expression in IDVCs by the inflammatory cytokine IFNő≥ blocks the antagonistic effect of IFNő≥ on neovascularization by stimulating the expression of IL6, a powerful pro-angiogenic cytokine. By contributing to vascular access, this newly ascribed function for IDO1 aligns with its involvement in other cancer hallmark functionalities, (tumor-promoting inflammation, immune escape, altered cellular metabolism, metastasis), which may stem from an underlying role in normal physiological functions such as wound healing and pregnancy. Understanding the nuances of how IDO1 involvement in these cancer hallmark functionalities varies between different tumor settings will be crucial to the future development of successful IDO1-directed therapies

    Identifizierung prädiktiver und prognostischer Biomarker in unterschiedlichen Tumorkompartimenten des ösophagealen Adenokarzinoms

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    Das √∂sophageale Adenokarzinom zeigt eine global steigende Inzidenz und hat mit einer 5-Jahres-√úberlebensrate von weniger als 25% eine schlechte Prognose. Personalisierte Therapieans√§tze sind selten und prognostische/pr√§diktive Biomarker des Tumormikromilieus sind unzureichend charakterisiert. Die kumulative Promotion n√§hert sich dieser Problematik in drei unterschiedlichen Schwerpunkten. 1. Zur Identifizierung Kompartiment-spezifischer Biomarker wurde eine Methode entwickelt, welche als kosteng√ľnstige Alternative zum sc-Seq Expressionsprofile individueller Zelltypen generiert. Dabei erfolgt die Extraktion der RNA nicht aus Einzelzellen, sondern aus flowzytometrisch-getrennten Zellkompartimenten. Die Separation der Proben in Epithelzellen, Immunzellen und Fibroblasten wurde durch verschiedene Verfahren validiert und eine suffiziente Ausbeute an RNA auch f√ľr kleine Gewebemengen gezeigt. 2. Biomarker des Immunzellkompartiments als therapeutische Angriffspunkte wurden in einem Patientenkollektiv von bis zu 551 Patienten auf ihre Bedeutung beim EAC √ľberpr√ľft. Es zeigte sich eine Expression der Immuncheckpoints LAG3, VISTA und IDO auf TILs durch IHC und RNA-Sonden basierte Verfahren in einem relevanten Anteil (LAG3: 11,4%, VISTA: 29%, IDO: 52,6%). Es konnte eine prognostisch g√ľnstige Bedeutung der VISTA, LAG3 und IDO Expression gezeigt werden. Durch den Vergleich von Genexpressionsprofilen aus therapienaiven und vorbehandelten Tumoren konnte zudem ein immunsuppressiver Effekt von neoadjuvanten Therapiekonzepten auf das Tumormikromilieu des EACs gezeigt werden. Dabei kam es zur verminderten Expression von Checkpoints und Anzahl TILs nach (Radio-) Chemotherapie. 3. Im Tumorzellkompartiment wurde die Rolle von Amplifikationen in ErbB-Rezeptor abh√§ngigen Signalwegen durch FISH-Technik und Immunhistochemie evaluiert. Es fanden sich KRAS Amplifikationen in 17,1%, PIK3CA Amplifikationen in 5% sowie eine HER2/neu-√úberexpression in 14,9% der untersuchten Tumore

    Preparation, modification, and clinical application of porous tantalum scaffolds

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    Porous tantalum (Ta) implants have been developed and clinically applied as high-quality implant biomaterials in the orthopedics field because of their excellent corrosion resistance, biocompatibility, osteointegration, and bone conductivity. Porous Ta allows fine bone ingrowth and new bone formation through the inner space because of its high porosity and interconnected pore structure. It contributes to rapid bone integration and long-term stability of osseointegrated implants. Porous Ta has excellent wetting properties and high surface energy, which facilitate the adhesion, proliferation, and mineralization of osteoblasts. Moreover, porous Ta is superior to classical metallic materials in avoiding the stress shielding effect, minimizing the loss of marginal bone, and improving primary stability because of its low elastic modulus and high friction coefficient. Accordingly, the excellent biological and mechanical properties of porous Ta are primarily responsible for its rising clinical translation trend. Over the past 2 decades, advanced fabrication strategies such as emerging manufacturing technologies, surface modification techniques, and patient-oriented designs have remarkably influenced the microstructural characteristic, bioactive performance, and clinical indications of porous Ta scaffolds. The present review offers an overview of the fabrication methods, modification techniques, and orthopedic applications of porous Ta implants

    Investigation of suture surgery with ant by Hakim Mohammad the Iranian surgeon of Safavid Era (1501 to 1736)

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    Hakim Mohammad, a military physician, and surgeon of the Safavid Era (1501 to 1736) and the author of Dhakhira-Yi-Kamilah book, served as a young man in the Ottoman Empire Officer as a surgeon physician. In this study, the method of suturing by Hakim Mohammad has been introduced. Suturing with ant was a wound healing method that was carried out by Hakim Muhammad to treat the wounds of certain areas of the body, by a special species of ants called fire ants, and by the lower jaw of these ants. Also, Hakim Mohammed suggested specific food and drug in order to take care of the wound and control infection and pain followed by this type of suture. Study of the treatment methods of predecessors shows us developments and the path to the progress of surgical affairs and can be used in the direction of further advances

    Investigation of the Antineoplastic Effects of 2-(4-Chlorophenyl)-13őĪ-Estrone Sulfamate against the HPV16-Positive Human Invasive Cervical Carcinoma Cell Line SiHa

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    Cervical carcinoma is one of the most frequent malignant gynecological cancers in women of reproductive age. Because of the poor tolerability of currently available chemotherapeutic agents, efforts have been focused on developing innovative molecules, including steroids, that exert antineoplastic effects with a better safety profile. In addition to their endocrine properties, certain estrogens exhibit additional biological activities, such as antiangiogenic and anticancer effects. Based on previous studies, the antineoplastic properties of 13őĪ-estrone sulfamate derivatives (13AES1-3) were investigated, and the mechanism of action for the most promising compound 13AES3 was explored. Based on their effects on the viability of different human adherent gynecological cancer cells, the SiHa cervical cell line was used for mechanistic experiments. The most active analog 13AES3 was shown to exert considerable proapoptotic effects, as evidenced by a colorimetric caspase-3 assay and fluorescent double staining. It also elicited antimigratory and anti-invasive effects in a concentration-dependent manner, as evidenced by wound healing and Boyden chamber assays, respectively. Regarding their mechanism of action, 13AES derivatives were shown to inhibit tubulin polymerization, and computer simulations provided a possible explanation for the importance of the presence of the chlorophenyl ring on the estrane skeleton. 13AES3 is considered to be the first 13őĪ-estrone derivative with a significant antineoplastic potency against SiHa cancer cells. Therefore, it might serve as a valuable lead molecule for the design of anticancer agents targeting cervical carcinomas

    Role of NLRP3 in the pathogenesis and treatment of gout arthritis

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    Gout arthritis (GA) is a common and curable type of inflammatory arthritis that has been attributed to a combination of genetic, environmental and metabolic factors. Chronic deposition of monosodium urate (MSU) crystals in articular and periarticular spaces as well as subsequent activation of innate immune system in the condition of persistent hyperuricemia are the core mechanisms of GA. As is well known, drugs for GA therapy primarily consists of rapidly acting anti-inflammatory agents and life-long uric acid lowering agents, and their therapeutic outcomes are far from satisfactory. Although MSU crystals in articular cartilage detected by arthrosonography or in synovial fluid found by polarization microscopy are conclusive proofs for GA, the exact molecular mechanism of NLRP3 inflammasome activation in the course of GA still remains mysterious, severely restricting the early diagnosis and therapy of GA. On the one hand, the activation of Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome requires nuclear factor kappa B (NF-őļB)-dependent transcriptional enhancement of NLRP3, precursor (pro)-caspase-1 and pro-IL-1ő≤, as well as the assembly of NLRP3 inflammasome complex and sustained release of inflammatory mediators and cytokines such as IL-1ő≤, IL-18 and caspase-1. On the other hand, NLRP3 inflammasome activated by MSU crystals is particularly relevant to the initiation and progression of GA, and thus may represent a prospective diagnostic biomarker and therapeutic target. As a result, pharmacological inhibition of the assembly and activation of NLRP3 inflammasome may also be a promising avenue for GA therapy. Herein, we first introduced the functional role of NLRP3 inflammasome activation and relevant biological mechanisms in GA based on currently available evidence. Then, we systematically reviewed therapeutic strategies for targeting NLRP3 by potentially effective agents such as natural products, novel compounds and noncoding RNAs (ncRNAs) in the treatment of MSU-induced GA mouse models. In conclusion, our present review may have significant implications for the pathogenesis, diagnosis and therapy of GA
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