O objetivo deste trabalho foi avaliar se as enzimas secretadas pelo fungo de decomposição parda Gloeophyllum trabeum podem atuar como auxiliares às celulases comerciais na hidrólise de substratos celulósicos oxidados. Para isto foi realizada a oxidação de celulose microcristalina Avicel mediante reação de Fenton (Fe+2, H2O2) e tratamento com CuSO4. O substrato oxidado foi caracterizado e posteriormente foi determinado o efeito desta modificação sobre a eficiência hidrolítica de enzimas comerciais (Celluclast/Novozym) e sobre misturas destas enzimas comerciais suplementadas com extrato enzimático obtido de G. trabeum. As reações realizadas com Avicel indicaram que ambos tratamentos químicos foram efetivos para modificar grupos funcionais presentes na celulose. A caracterização fisicoquímica dos substratos oxidados permitiu concluir que o tratamento Fenton gerou transformações mais intensas na celulose do que o tratamento com CuSO4. O análise dos hidrolisados ácidos das amostras oxidadas por Fenton mediante espectrometria de massas (GC-MS) confirmou que a oxidação ocorre principalmente no C1. As hidrólises enzimáticas utilizando preparações com diferentes cargas das enzimas comerciais apresentaram uma diminuição significativa da conversão de celulose quando as amostras são tratadas com o reagente Fenton. Por outro lado, a celulose oxidada gerada pelo tratamento com CuSO4 ainda é eficientemente hidrolisada pelas enzimas. As duas amostras de celulose Avicel oxidadas também foi hidrolisada utilizando misturas de enzimas comerciais e extratos enzimáticos de G. trabeum. Em todas as condições ensaiadas a suplementação da mistura reacional com extratos enzimáticos do fungo de decomposição parda G. trabeum diminuíram a eficiência de hidrólise enzimática da celulose oxidada. O emprego do dobro de carga enzimática de enzimas comerciais permitiu recuperar os níveis de hidrólise observados na celulose in natura. / The goal of this work was to evaluate whether enzymes produced by the brown-rot fungus Gleophyllum trabeum would be able to act as auxiliary enzymes of commercial cellulases during the enzymatic hydrolysis of oxidized cellulose. To reach this goal an oxidation of microcrystalline-cellulose (Avicel) was performed by two differente reaction systems: Fenton reaction (Fe2+, H2O2) or CuSO4 treatments. The oxidized subtracts were characterized and the effect of cellulose modification on the hydrolytic efficiency of commercial enzymes, supplemented or not with the enzymes recovered from a G. trabeum culture, was determined. The Avicel treatment by the oxidative systems resulted in cellulose structural changes as evidenced by infrared spectroscopy, viscosity measurements and chemical characterization by HPLC and GC/MS chromatography of the monomers recovered after acid hydrolysis. The physicochemical characterization of the oxidized celluloses allowed to conclude that the transformation of the cellulose was more intense when Fenton treatment was used as compared with the CuSO4 treatment. The analysis of the acid hydrolysates by GC-MS indicated that the oxidation by Fenton reaction occurred mainly at the C1 of the cellulose chain, resulting in gluconic acid in the acid hydrolysates. A meaningful decrease of the cellulose conversion was observed when Fetnon-treated samples were submitted to enzymatic hydrolysis using different amounts of commercial enzymes. On the other hand, the CuSO4-oxidized cellulose was still efficiently hydrolyzed by high enzyme dosages. The oxidized Avicel substrates were also hydrolyzed using a mixture of the commercial enzymes with enzymes recovered from G. trabeum cultures. At all tested conditions, the supplementation of the reaction mixture with enzymatic extracts from G. trabeum decreased the enzymatic hydrolysis efficiency. Nevertheless, the normal levels of enzymatic hydrolysis were recovered when the commercial enzymes dosage two-fold increased.

Os fatores bioquímicos de cronicidade de úlceras venosas ainda não estão totalmente esclarecidos, porém, sabe-se que há desequilíbrio entre produção de metaloproteinases de matriz (MMPs) e fatores de inibição tissular de metaloproteinases (TIMPs), metabolismo anormal de colágeno, senescência de fibroblastos e alterações na ação de fatores de crescimentos, tais como fator transformador de crescimento beta (TGF-?). Diante das falhas no processo de reparo tecidual, várias terapias têm sido desenvolvidas com destaque para celulose oxidada regenerada (oxidized regenerated cellulose - ORC)/colágeno e ORC/colágeno/prata cuja função é alterar o microambiente diminuindo MMPs/TIMPs presente no leito da ferida. Nesse estudo, objetivamos avaliar o comportamento in vitro de fibroblastos oriundos de leito de úlcera venosa (VUF) na presença de ORC/colágeno, associada ou não à prata, quanto aspectos morfofuncionais celulares e síntese de MMPs, TIMPs, TGF beta, colágeno tipo I e colágeno tipo III. Após padronização do tempo de exposição e concentração de ORC/colágeno e ORC/colágeno/prata, foi realizada avaliação dos sinalizadores de interação celular utilizando-se imunoensaio Multiplex bead-based nos sobrenadantes da cultura de VUFs e imunofluorescência indireta nas células cultivadas. Como resultado, os estímulos ORC2 (correspondente à 2.8 mg/mL) e 4.2 mg/mL (ORC3+Ag) foram os que apresentaram maior homogeneidade proliferativa. A ORC/colágeno interferiu positivamente na síntese de MMP1, já a presença de prata interferiu negativamente na síntese de MMP2, MMP10, MMP13, TIMP1, TIMP 2 e TIMP3 e positivamente no colágeno tipo III, as sínteses de TGFbeta e colágeno tipo I não sofreram interferência dos estímulos. Tais achados sugerem que a presença de celulose oxidada regenerada pode modular a formação de fibrose no leito da ferida. Quando há presença de prata, nota-se tendência anti-inflamatória sobre a célula com maior síntese de colágeno tipo III / The biochemical factors of chronicity of venous ulcers are not yet fully understood, however, it is known that there is imbalance between producing matrix metalloproteinases (MMPs) and tissue inhibition factors of metalloproteinases (TIMPs), abnormal collagen metabolism, fibroblastic senescence and changes in growth factors action, such as transforming growth factor beta (TGF-?). In the face of failures in the tissue repair process, many therapies have been developed. The use of oxidized regenerated cellulose (ORC)/collagen and ORC/collagen/silver, to modify the microenvironment by decreasing MMPs/TIMPs present in the wound bed, has been emphasized. In the present study, we aimed to evaluate the in vitro behavior of fibroblasts derived from venous ulcer (venous ulcer fibroblasts - VUF) in the presence of ORC/collagen associated or not to silver with regard to cellular morph functional aspects and synthesis of MMPs, TIMPs, TGFbeta, type I and type III collagen. After standardization of the exposure time and concentration of ORC/collagen and ORC/collagen/silver stimulation, an evaluation of cell interaction signaling was performed by using a bead-based multiplex immunoassay in cells supernatants and indirect immunofluorescence in cultured cells. We demonstrated that the ORC2 (corresponding to 2.8 mg/mL) and 4.2 mg/mL (ORC3+Ag) stimuli had the highest proliferative homogeneity. The ORC/collagen interfered positively in the synthesis of MMP1, since the presence of silver interfered negatively in the synthesis of MMP2, MMP10, MMP13, TIMP1, TIMP2 and TIMP3 and positively in type III collagen, the synthesis of TGFbeta and type I collagen did not suffer interference. Such findings suggest that the presence of regenerated oxidized cellulose may modulate the formation of fibrosis in the wound bed. When silver is associated antiinflammatory tendency is observed on fibroblasts with increased type III collagen synthesis

O objetivo deste trabalho foi avaliar se as enzimas secretadas pelo fungo de decomposição parda Gloeophyllum trabeum podem atuar como auxiliares às celulases comerciais na hidrólise de substratos celulósicos oxidados. Para isto foi realizada a oxidação de celulose microcristalina Avicel mediante reação de Fenton (Fe+2, H2O2) e tratamento com CuSO4. O substrato oxidado foi caracterizado e posteriormente foi determinado o efeito desta modificação sobre a eficiência hidrolítica de enzimas comerciais (Celluclast/Novozym) e sobre misturas destas enzimas comerciais suplementadas com extrato enzimático obtido de G. trabeum. As reações realizadas com Avicel indicaram que ambos tratamentos químicos foram efetivos para modificar grupos funcionais presentes na celulose. A caracterização fisicoquímica dos substratos oxidados permitiu concluir que o tratamento Fenton gerou transformações mais intensas na celulose do que o tratamento com CuSO4. O análise dos hidrolisados ácidos das amostras oxidadas por Fenton mediante espectrometria de massas (GC-MS) confirmou que a oxidação ocorre principalmente no C1. As hidrólises enzimáticas utilizando preparações com diferentes cargas das enzimas comerciais apresentaram uma diminuição significativa da conversão de celulose quando as amostras são tratadas com o reagente Fenton. Por outro lado, a celulose oxidada gerada pelo tratamento com CuSO4 ainda é eficientemente hidrolisada pelas enzimas. As duas amostras de celulose Avicel oxidadas também foi hidrolisada utilizando misturas de enzimas comerciais e extratos enzimáticos de G. trabeum. Em todas as condições ensaiadas a suplementação da mistura reacional com extratos enzimáticos do fungo de decomposição parda G. trabeum diminuíram a eficiência de hidrólise enzimática da celulose oxidada. O emprego do dobro de carga enzimática de enzimas comerciais permitiu recuperar os níveis de hidrólise observados na celulose in natura. / The goal of this work was to evaluate whether enzymes produced by the brown-rot fungus Gleophyllum trabeum would be able to act as auxiliary enzymes of commercial cellulases during the enzymatic hydrolysis of oxidized cellulose. To reach this goal an oxidation of microcrystalline-cellulose (Avicel) was performed by two differente reaction systems: Fenton reaction (Fe2+, H2O2) or CuSO4 treatments. The oxidized subtracts were characterized and the effect of cellulose modification on the hydrolytic efficiency of commercial enzymes, supplemented or not with the enzymes recovered from a G. trabeum culture, was determined. The Avicel treatment by the oxidative systems resulted in cellulose structural changes as evidenced by infrared spectroscopy, viscosity measurements and chemical characterization by HPLC and GC/MS chromatography of the monomers recovered after acid hydrolysis. The physicochemical characterization of the oxidized celluloses allowed to conclude that the transformation of the cellulose was more intense when Fenton treatment was used as compared with the CuSO4 treatment. The analysis of the acid hydrolysates by GC-MS indicated that the oxidation by Fenton reaction occurred mainly at the C1 of the cellulose chain, resulting in gluconic acid in the acid hydrolysates. A meaningful decrease of the cellulose conversion was observed when Fetnon-treated samples were submitted to enzymatic hydrolysis using different amounts of commercial enzymes. On the other hand, the CuSO4-oxidized cellulose was still efficiently hydrolyzed by high enzyme dosages. The oxidized Avicel substrates were also hydrolyzed using a mixture of the commercial enzymes with enzymes recovered from G. trabeum cultures. At all tested conditions, the supplementation of the reaction mixture with enzymatic extracts from G. trabeum decreased the enzymatic hydrolysis efficiency. Nevertheless, the normal levels of enzymatic hydrolysis were recovered when the commercial enzymes dosage two-fold increased.

Correct diagnosis of disease is essential in the effort to save and improve lives. Point of care (POC) diagnostics are in-vitro tests that assist in patient diagnosis and can be used at the location of patient care. POC diagnostics are easy to use and provide near-instant readouts allowing medical providers and patients to make rapid decisions about treatment. Increased access to POC testing is especially beneficial to low-income and low resource areas that cannot afford expensive lab testing. The World Health Organization (WHO) has outlined at least 113 diseases for which POC diagnostics are needed. Because of this, developing effective, efficient, and economical methods for creating new POC tests is essential. Work in section one of this thesis describes strategies by which new POC bio-diagnostics can be created. The use of oxidized cellulose as a vector for antibody immobilization was explored in several cellulose-based materials to provide quick, economical tests while still obtaining effective limits of detection when used to detect the pregnancy hormone Human Chorionic Gonadotropin (HCG) in a proof of concept study. The majority of these tests could detect as low as 100 ng/mL of HCG well below the clinical level necessary for detection at 2400 ng/mL. The use of a hand-powered syringe-based POC named the fast flow immunoassay (FFI) was tested for its ability to increase observable signal in a sandwich immunoassay by passing the sample through the test filter multiple times. 10 passes through the filter resulted in a signal approximately 17x more intense than a 1-hour dot-blot sandwich immunoassay. Both oxidized cotton and FFI systems can be used to develop new POC assays quickly and economically. Future use of these POC systems could help expand the availability of diagnostic testing to disadvantaged areas. Gold-based drugs have been used and investigated as medications multiple times throughout history to treat various diseases such as Rheumatoid arthritis, parasitic infections, and cancer. In the last few decades, gold nanoparticles have been used as drug delivery agents and catalysts for various reactions. Recently catalytic gold nanocrystals have been characterized for their ability to treat neurodegenerative diseases. Although these results were promising, much is still unknown about their mechanism of action. Section two of this thesis investigates potential molecular pathways that gold nanocrystals could be affecting, specifically the IL-6/Jak/STAT3 inflammation pathway and the Nrf2 antioxidant pathway. The gold nanocrystals we tested did not affect these pathways at physiologically obtainable concentrations. Additional work was done to characterize protein interactome or protein corona of gold nanocrystals. Preliminary proteomic characterization of this protein corona in fetal bovine serum (FBS) identified 118 potential interactors and classified those based on function and structure. Future work will need to be done to follow up on these identifications and to determine what mechanistic implications they may have.

Předkládaná disertační práce se zabývá přípravou a charakterizací nových biokompatibilních nanovláken s potenciální aplikací v medicíně. V této práci byl výběr jednotlivých složek pro přípravu nanovlákenného materiálu zvolen tak, aby vyhovoval nárokům tkáňového inženýrství. Literární rešerše shrnuje poznatky o elektrostatickém zvlákňování a o jeho parametrech. Dále se věnuje možnostem elektrostatického zvlákňování proteinů kolagenu a želatiny a jejich směsmi se syntetickými polymery a biopolymery a anorganickými plnivy. Teoretická část řeší také různé postupy síťování nanovláken vedoucí ke zlepšení jejich hydrolytické stability a mechanických vlastností. Poslední část je zaměřena na anorganické nanotrubky halloysitu (HNT), které získaly svou pozornost díky svým vynikajícím fyzikálním a biologickým vlastnostem. V experimentální části byly zpracovány dvě případové studie, z nichž každá se zabývá přípravou nanovlákenných biomateriálů s potenciální aplikací v medicíně. První studie je zaměřena na přípravu a charakterizaci nových hydrolyticky stabilních antibakteriálních želatinových nanovláken modifikovaných pomocí oxidované celulózy. Unikátní inhibiční účinky nanovláken byly testovány na kmenu bakterie Escherichia coli pomocí metody chemické bioluminiscence. Kultivované buňky lidského papilárního adenokacinomu plic prokázaly dobrou adhezi a proliferaci k povrchu nanovláken. Druhá část popisuje vliv zdroje a množství anorganických halloysitových nanotrubek na strukturu a vlastnosti amfifilních nanovláken ze směsi želatiny a syntetického polykaprolaktonu. Přídavek HNT zlepšil tepelnou stabilitu, mechanické vlastnosti (jak tuhost, tak prodloužení) a snížil krystalinitu nanovláken. HNT z různých zdrojů neměl vliv na chování buněk, ale mírně ovlivnil proliferaci a životaschopnost buněk na povrchu nanovláken.