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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

    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

    Management of valvular heart disease in patients with cancer: Multidisciplinary team, cancer-therapy related cardiotoxicity, diagnosis, transcatheter intervention, and cardiac surgery. Expert opinion of the Association on Valvular Heart Disease, Association of Cardiovascular Interventions, and Working Group on Cardiac Surgery of the Polish Cardiac Society

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    The Association on Valvular Heart Disease, Association of Cardiovascular Interventions, and the Working Group on CardiacSurgery of the Polish Cardiac Society have released a position statement on risk factors, diagnosis, and management of patients with cancer and valvular heart disease (VHD). VHD can occur in patients with cancer in several ways, for example, it can exist or be diagnosed before cancer treatment, after cancer treatment, be an incidental finding during imaging tests, endocarditis related to immunosuppression, prolonged intravenous catheter use, or combination treatment, and nonbacterial thrombotic endocarditis. It is recommended to employ close cardiac surveillance for patients at high risk of complications during and after cancer treatment and for cancer treatments that may be cardiotoxic to be discussed by a multidisciplinary team. Patients with cancer and pre-existing severe VHD should be managed according to the 2021 European Society of Cardiology (ESC) and European Association for Cardio-Thoracic Surgery (EACTS) guidelines for VHD management, taking into consideration cancer prognosis and patient preferences

    Centella asiatica (L.) Urb: A comprehensive bibliometric analysis of published studies between 1857 and 2022

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    Centella asiatica (L.) Urban (C. asiatica) is a medicinal plant that generates terpenes, phenols, vitamins, minerals, polyacetylene and fatty acids among other phyto-constituents. This review focuses on the bibliometric analysis of 1164 documents on C. asiatica that were retrieved from the Scopus database. Harzing Publish or Perish and VOSviewer were used for citation and network analyses. According to the bibliometric analysis, publications are on the rise, particularly in the fields of pharmacology, toxicology and pharmaceutics, medicine, biochemistry, genetics and molecular biology, agricultural and biological sciences, and chemistry. "Phytoremediation", "secondary metabolites", "Andrographis paniculata", and "cognitive impairment" are emerging areas for C. asiatica research. However, currently there is a lack of international collaboration in C. asiatica research among contributing countries. Researchers can utilise the findings cited in this reiew to locate potential collaborators, top authors, countries and documents

    Solutions in Breast Reconstruction

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    Breast reconstruction, after cancer surgery, is not only a reconstructive surgery but also an esthetic surgery. No woman should be expected to give up the breast tissue, which is the symbol of female identity, easily. The reconstruction stage after breast cancer is difficult enough in the early and late stages. It is generally not possible to cover the defect and to equalize the two breasts in a single step. General surgery and plastic surgery should work together. Recently, innovative solutions have been offered in breast reconstruction. Starting from skin grafts and local flaps, various flap options, dermal equivalents, fat transfer, and tissue expansion operations are among the options. Breast reconstruction is difficult enough in breasts that have undergone radiotherapy, and reconstruction with autologous tissue is preferred

    Gene expression changes in therapeutic ultrasound-treated venous leg ulcers

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    IntroductionLow-frequency, low-intensity ultrasound has been previously shown to promote healing of chronic wounds in humans, but mechanisms behind these effects are poorly understood. The purpose of this study was to evaluate gene expression differences in debrided human venous ulcer tissue from patients treated with low-frequency (20 kHz), low-intensity (100 mW/cm2) ultrasound compared to a sham treatment in an effort to better understand the potential biological mechanisms.MethodsDebrided venous ulcer tissue was collected from 32 subjects one week after sham treatment or low-frequency, low-intensity ultrasound treatment. Of these samples, 7 samples (3 ultrasound treated and 4 sham treated) yielded sufficient quality total RNA for analysis by ultra-high multiplexed PCR (Ampliseq) and expression of more than 24,000 genes was analyzed. 477 genes were found to be significantly differentially expressed between the ultrasound and sham groups using cut-off values of p‚ÄČ<‚ÄČ0.05 and fold change of 2.Results and DiscussionThe top differentially expressed genes included those involved in regulation of cell metabolism, proliferation, and immune cell signaling. Gene set enrichment analysis identified 20 significantly enriched gene sets from upregulated genes and 4 significantly enriched gene sets from downregulated genes. Most of the enriched gene sets from upregulated genes were related to cell-cell signaling pathways. The most significantly enriched gene set from downregulated genes was the inflammatory response gene set. These findings show that therapeutic ultrasound influences cellular behavior in chronic wounds as early as 1 week after application. Considering the well-known role of chronic inflammation in impairing wound healing in chronic wounds, these results suggest that a downregulation of inflammatory genes is a possible biological mechanism of ultrasound-mediated venous chronic wound healing. Such increased understanding may ultimately lead to the enhancement of ultrasound devices to accelerate chronic wound healing and increase patient quality of life

    Central extracorporeal circulatory life support (cECLS) in selected patients with critical cardiogenic shock

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    BackgroundPercutaneous extracorporeal life support (pECLS) is increasingly applied in cardiogenic shock (CS) despite a lack of evidence from randomized trials. The in-hospital mortality rate of pECLS still reaches up to 60%, while vascular access site complications remain a shortcoming. Surgical approaches with central cannulation for ECLS (cELCS) have emerged as a bail-out option. To date, no systematic approach exists that allows a definition of inclusion or exclusion criteria for cECLS.Methods and resultsThis single-center, retrospective, case-control study includes all patients fulfilling criteria for CS at the West German Heart and Vascular Center Essen/Germany between 2015 and 2020 who underwent cECLS (n‚ÄČ=‚ÄČ58), excluding post-cardiotomy patients. Seventeen patients received cECLS (29.3%) as a first-line treatment strategy and 41 patients as a second-line strategy (70.7%). The main complications leading to the use of cECLS as a second-line strategy were limb ischemia (32.8%) and ongoing insufficient hemodynamic support (27.6%). The first-line cECLS cohort showed a 30-day mortality rate of 53.3% that was constant during follow-up. The 30-day mortality rate of secondary cECLS candidates was 69.8% and the rate at 3 and 6 months was 79.1%. Younger patients (<55 years) were more likely to exhibit survival benefit with cECLS (p‚ÄČ=‚ÄČ0.043).ConclusionSurgical cECLS in CS is a feasible therapy for highly selected patients with hemodynamic instability, vascular complications, or peripheral access site limitations as complementary strategy in experienced centers

    Macrophages from naked mole-rat possess distinct immunometabolic signatures upon polarization

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    The naked mole-rat (NMR) is a unique long-lived rodent which is highly resistant to age-associated disorders and cancer. The immune system of NMR possesses a distinct cellular composition with the prevalence of myeloid cells. Thus, the detailed phenotypical and functional assessment of NMR myeloid cell compartment may uncover novel mechanisms of immunoregulation and healthy aging. In this study gene expression signatures, reactive nitrogen species and cytokine production, as well as metabolic activity of classically (M1) and alternatively (M2) activated NMR bone marrow-derived macrophages (BMDM) were examined. Polarization of NMR macrophages under pro-inflammatory conditions led to expected M1 phenotype characterized by increased pro-inflammatory gene expression, cytokine production and aerobic glycolysis, but paralleled by reduced production of nitric oxide (NO). Under systemic LPS-induced inflammatory conditions NO production also was not detected in NMR blood monocytes. Altogether, our results indicate that NMR macrophages are capable of transcriptional and metabolic reprogramming under polarizing stimuli, however, NMR M1 possesses species-specific signatures as compared to murine M1, implicating distinct adaptations in NMR immune system

    In vitro investigation of the effect of disulfiram on hypoxia induced NFőļB, epithelial to mesenchymal transition and cancer stem cells in glioblastoma cell lines

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    A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.Glioblastoma multiforme (GBM) is one of the most aggressive and lethal cancers with a poor prognosis. Advances in the treatment of GBM are limited due to several resistance mechanisms and limited drug delivery into the central nervous system (CNS) compartment by the blood-brain barrier (BBB) and by actions of the normal brain to counteract tumour-targeting medications. Hypoxia is common in malignant brain tumours such as GBM and plays a significant role in tumour pathobiology. It is widely accepted that hypoxia is a major driver of GBM malignancy. Although it has been confirmed that hypoxia induces GBM stem-like-cells (GSCs), which are highly invasive and resistant to all chemotherapeutic agents, the detailed molecular pathways linking hypoxia, GSC traits and chemoresistance remain obscure. Evidence shows that hypoxia induces cancer stem cell phenotypes via epithelial-to-mesenchymal transition (EMT), promoting therapeutic resistance in most cancers, including GBM. This study demonstrated that spheroid cultured GBM cells consist of a large population of hypoxic cells with CSC and EMT characteristics. GSCs are chemo-resistant and displayed increased levels of HIFs and NFőļB activity. Similarly, the hypoxia cultured GBM cells manifested GSC traits, chemoresistance and invasiveness. These results suggest that hypoxia is responsible for GBM stemness, chemoresistance and invasiveness. GBM cells transfected with nuclear factor kappa B-p65 (NFőļB-p65) subunit exhibited CSC and EMT markers indicating the essential role of NFőļB in maintaining GSC phenotypes. The study also highlighted the significance of NFőļB in driving chemoresistance, invasiveness, and the potential role of NFőļB as the central regulator of hypoxia-induced stemness in GBM cells. GSC population has the ability of self-renewal, cancer initiation and development of secondary heterogeneous cancer. The very poor prognosis of GBM could largely be attributed to the existence of GSCs, which promote tumour propagation, maintenance, radio- and chemoresistance and local infiltration. In this study, we used Disulfiram (DS), a drug used for more than 65 years in alcoholism clinics, in combination with copper (Cu) to target the NFőļB pathway, reverse chemoresistance and block invasion in GSCs. The obtained results showed that DS/Cu is highly cytotoxic to GBM cells and completely eradicated the resistant CSC population at low dose levels in vitro. DS/Cu inhibited the migration and invasion of hypoxia-induced CSC and EMT like GBM cells at low nanomolar concentrations. DS is an FDA approved drug with low toxicity to normal tissues and can pass through the BBB. Further research may lead to the quick translation of DS into cancer clinics and provide new therapeutic options to improve treatment outcomes in GBM patients
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