Logic-Based Delivery of Site-Specifically-Modified Proteins from Gels through Engineered Biomacromolecular Architecture

Thesis (Master's)--University of Washington, 2016-08

Obtenção de um hidrogel proveniente de proteínas da corvina (micropogonias furnieri) e solubilização das proteínas fibrosas residuais

Com o aumento na captura de pescado e da poluição do meio ambiente, esta-se à margem de exceder a estimativa do limite da sustentabilidade, e obviamente isto faz com se utilize os recursos marítimos com mais inteligência e precaução. Aplicando tecnologia enzimática ou química é possível recuperar as proteínas do processamento do pescado, produzindo hidrolisados e isolados protéicos. Uma grande quantidade de proteínas insolúveis está disponível em escamas, peles e ossos, subprodutos do processamento do pescado, que podem ser solubilizadas através de fungos e bactérias. Utilizando isolados protéicos é possível obter biopolímeros, estes têm chamado a atenção nos últimos anos, pois são biodegradáveis, não-tóxicos e geralmente biocompatíveis. Os hidrogéis protéicos são polímeros que podem absorver uma quantidade de água a partir de 10 até centenas de vezes o seu peso seco. O objetivo deste trabalho foi desenvolver um hidrogel protéico, com propriedades superabsorventes, a partir das proteínas solúveis e insolúveis da corvina (Micropogonias furnieri). Para a produção dos hidrolisados a partir das proteínas solúveis foi utilizado processo enzimático (Alcalase e Flavourzyme) e químico (solubilização ácida e alcalina). Nos processos de solubilização das proteínas insolúveis foram utilizados microrganismos (bactérias e fungos). Tanto as bactérias como os fungos avaliados apresentaram capacidade de solubilizar as proteínas insolúveis presentes nos resíduos (escamas, ossos, cartilagens e outros). A bactéria que atingiu a maior atividade proteolítica foi a Bacillus velesensis (47,56 U mL-1) e o fungo foi o Penicillium sp. (E20) (31,20 U mL-1). Para a produção dos hidrogéis, foram utilizados isolados protéicos provenientes de solubilização ácida ou alcalina, produzidos a partir de resíduos da industrialização de pescado, modificados quimicamente com dianidrido etilenodiamino tetraacético (EDTAD) e adicionados de agente de ligação cruzada (glutaraldeído). Algumas proteínas modificadas ainda foram submetidas a tratamento com etanol. Foram realizadas análise estrutural das proteínas modificadas e estudo da capacidade de retenção de água dos hidrogéis assim obtidos. Os hidrogéis produzidos apresentaram alta capacidade de retenção de água. A máxima absorção de água foi alcançada pelo hidrogel ácido sem o tratamento com etanol foi de 103,25 gágua/ggel seco, enquanto que a mesma amostra tratada com etanol alcançou 216,05 gágua/ggel seco. Os hidrogéis produzidos podem ser utilizados em diversas indústrias, tais como, farmacêutica, alimentícia, médica, agroindústria, entre outras, que necessitem de hidrogéis com alta capacidade de retenção de água.

Development of polymeric drug delivery vehicles for targeted cancer therapy

The aim of my Ph. D. thesis is to generalize a method for targeted anti-cancer drug delivery. Hydrophilic polymer-drug conjugates involve complicated synthesis; drug-encapsulated polymeric nanoparticles limit the loading capability of payloads. This thesis introduces the concept of nanoconjugates to overcome difficulties in synthesis and formulation. Drugs with hydroxyl group are able to initiate polyester synthesis in a regio- and chemo- selective way, with the mediation of ligand-tunable Zinc catalyst. Herein, three anti-cancer drugs are presented to demonstrate the high efficiency and selectivity in the method (Chapter 2-4). The obtained particles are stable in salt solution, releasing drugs over weeks in controlled manner. With the conjugation of aptamer, particles are capable to target prostate cancer cells in vitro. These results open the gateway to evaluate the in vivo efficacy of nanoconjugates for target cancer therapy (Chapter 5). Mechanism study of the polymerization leads to the discovery of chemosite selective synthesis of prodrugs with acrylate functional groups. Functional copolymer-drug conjugates will expand the scope of nanoconjugates (Chapter 6). Liposome-aptamer targeting drug delivery vehicle is well studied to achieve reversible cell-specific delivery of non-hydoxyl drugs e.g. cisplatin (Chapter 7). New monomers and polymerization mechanisms are explored for polyester in order to synthesize nanoconjugates with variety on properties (Chapter 8). Initial efforts to apply this type of prodrugs will be focused on the preparation of hydrogels for stem cell research (Chapter 9).

Dynamic Coculture of a Prevascularized Engineered Bone Construct

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of tissue engineered bone constructs can succeed, in vitro fabrication needs to address limitations in large-scale tissue development, including controlled osteogenesis and an inadequate vasculature network to prevent necrosis of large constructs. The tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cell (hMSC)-seeded scaffolds while they are developed in vitro. To further enhance this process, we developed a novel osteogenic growth factors delivery system for dynamically cultured hMSCs using microparticles encapsulated in three-dimensional alginate scaffolds. In light of this increased differentiation, we characterized the endogenous cytokine distribution throughout the TPS bioreactor. An advantageous effect in the ‘outlet’ portion of the uniaxial growth chamber was discovered due to the system’s downstream circulation and the unique modular aspect of the scaffolds. This unique trait allowed us to carefully tune the differentiation behavior of specific cell populations. We applied the knowledge gained from the growth profile of the TPS bioreactor to culture a high-volume bone composite in a 3D-printed femur mold. This resulted in a tissue engineered bone construct with a volume of 200cm3, a 20-fold increase over previously reported sizes. We demonstrated high viability of the cultured cells throughout the culture period as well as early signs of osteogenic differentiation. Taking one step closer toward a viable implant and minimize tissue necrosis after implantation, we designed a composite construct by coculturing endothelial cells (ECs) and differentiating hMSCs, encouraging prevascularization and anastomosis of the graft with the host vasculature. We discovered the necessity of cell to cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. Notably, the results suggested increased osteogenic and angiogenic potential of the encapsulated cells when dynamically cultured in the TPS bioreactor, suggesting a synergistic effect between coculture and applied shear stress. This work highlights the feasibility of fabricating a high-volume, prevascularized tissue engineered bone construct for the regeneration of a critical size defect.

Dyskusja programowa na łamach czasopism Międzyzakładowego Komitetu Założycielskiego i Zarządu Regionu Wielkopolska NSZZ "Solidarność" w latach 1980-1981

This article analyses the programme discussion in the trade union’s press supported by the trade union’s regional structures: the Inter-Enterprise Founding Committee, and subsequently the Wielkopolska Regional Board of the Independent Self-Governing Trade Union “Solidarność”. The press of „Solidarność” in the Wielkopolska region is distinguished by two groups of articles: reprints from other independent papers and first-time published articles. This article only interprets the latter group of publications. Those articles mainly focused on dominant topics such as discussions on the trade union’s current programme and its organization, the economic reform, the preparation to local self-government elections and the trade union’s global strategy.

Bogusław Śliwa (1944-1989) - działacz opozycji demokratycznej i "Solidarności" w Kaliszu

Bogusław Śliwa was born in Lvov on 6 October 1944. He graduated in law studies at Adam Mickiewicz University in Poznań in 1969. Following the completion of his prosecutor’s apprenticeship he worked, among others, in Wolsztyn, Świebodzin and – from 1975 – in Kalisz. On 22 August 1978 Śliwa was fired from the public prosecutor’s office because he had attempted to detect a person who murdered during the robbery committed by a Civic Militia officer. That time he established and maintained close contacts with activists of the Workers’ Defence Committee (KOR), among others: Jacek Kuroń, Mirosław Chojecki, Adam Michnik, Bronisław Geremek, Jan Lityński, Zofia Romaszewska and Zbigniew Romaszewski. In 1978 he began to cooperate with the Kalisz group of the Movement for the Defence of Human and Civil Rights (ROPCiO). In the early 1979 this group started to publish “Wolne Słowo” in which Śliwa was a co-editor. On 28 June 1979 in Poznań he was involved in founding the Social Self-Defence Club of the Wielkopolska and Kujawy Region. In September 1980, during strikes at the FWR “Runotex” and KZKS “Winiary” in Kalisz Śliwa was an expert representing the workers. On 29 September of that year, he arranged in Kalisz a meeting of representatives and delegates of enterprises in Kalisz aimed at appointing the Board of the Inter-Enterprise Founding Committee of the Independent Self-Governing Trade Union. He became the secretary. Bogusław Śliwa also engaged in setting up and developing an information team. He was informally responsible for developing an information and printing base. Bogusław Śliwa set up “NSZZ Solidarność” magazine where he published his own articles. He also founded the “Solidarność” Workers’ Community Centre in Kalisz. it is noteworthy that it was the only community centre in Poland established by „Solidarność”. In December the Nationwide Liaison Commission of „Solidarność” appointed him to the Committee for the Defence of Prisoners of Conscience established on 10 December of that year. He participated in the information meeting of the Independent Self-Governing Trade Union of Independent Farmers “Solidarność Wiejska” held in Staw, in Szczytniki commune. During that meeting “Solidarność Wiejska” led by Mieczysław Walczykiewicz requested the authorities to liquidate the “Świt” Agricultural Production Cooperative in Cieszyków, in Szczytniki commune. Bogusław Śliwa was involved in this successful event. It was the first liquidation of cooperative in Poland. On 11 January 1981 Śliwa co-organized the 1st Regional Convention of „Solidarność” Wiejska in Kalisz. Following the so-called Bydgoszcz events of 19 March 1981 he advocated the general strike. Due to his attitude, Śliwa was listed as one of 146 „Solidarność” activists executed by the 3rd “A” Department of the Ministry of Internal Affairs. According to the authorities those activists presented radical views. On 30 June 1981 at the 1st General Delegates Convention of the Kaliskie province, Śliwa, as a delegate of the Kaliskie province, was appointed to the Regional Board of „Solidarność” – Southern Wielkopolska. In July Śliwa set up in Kalisz the underground branch of the Polish Democratic Party. In 1981 Śliwa was a delegate to the 1st National Delegates Convention of „Solidarność” and co-edited with Jan Lityński the document entitled: “Message to the Working People of Eastern Europe” originated by Henryk Siciński and adopted by the 1st National Delegates Convention. On 22 November he participated in the Warsaw-held meeting founding the Self-Governing Republic Clubs “Liberty – Equality– Independence” and signed the founding declaration. On 28 of that month he co-organized with Antoni Pietkiewicz a founding meeting of the Club in Kalisz. When martial law was declared he began to hide in Kalisz. Śliwa was arrested on 25 February 1982 and interned in Ostrów Wielkopolski and then in Gębarzew and Kwidzyn. After being released on 25 November 1982, he was immediately involved in the activity of the underground movement of „Solidarność”. He edited the first two issues of “Nasza Solidarność” magazine published in Kalisz. Śliwa co-invented and co-organized the 1st May march that was independent from the authorities’ one held in Kalisz in 1983. Consequently, he was temporarily arrested and detained in Ostrów Wielkopolski. On 7 June 1983 he was released from custody. The amnesty declared on 21 July 1983 caused that the investigation against him was discontinued. In July of the same year he co-founded the Inter-Regional Coordination Commission of the Independent Self-Governing Trade Union “Solidarność” Kalisz-Konin-Sieradz. As he could not find any work and he and his family were exposed to psychological harassment, he emigrated to Sweden on 30 December 1983. He worked, among other positions, as bookbinder. He was the board secretary of the Congress of Poles in Sweden. In 1984 he commented the death of priest Jerzy Popiełuszko in “Dagens Nyheter” daily. He was also interviewed by Radio Liberty. Śliwa commenced cooperating with representatives of the „Solidarność” Coordination Office in Paris, Brussels and Stockholm. On 18 April 1985 the Military Garrison Prosecutor’s Office in Wrocław initiated investigations against Śliwa, charging him with activities detrimental to political interests of the People’s Republic of Poland. Subsequently, on 10 July 1985 this public prosecutor’s office decided to issue an arrest warrant for him. On the same day the public prosecutor suspended criminal proceedings against him. In December 1985, after the courageous escape of two brothers, Adam and Krzysztof Zieliński, from Poland to Sweden, he helped them prevent their deportation and stay in their new homeland. He expressed his opinion on this issue on Swedish television and in “ Dagens Nyheter” daily. His intervention helped them legally stay in Sweden. In 1989 he arrived in Poland. During this short visit he met and talked with his colleagues from the so-called first „Solidarność”. Bogusław Śliwa died in Stockholm on 23 November 1989. He was buried there on 7 December 1989. On 18 October 2006 he was posthumously honoured by Lech Kaczynski, President of Poland, with the Order of Polonis Restitution. On 15 June 2007 Bogusław Śliwa was posthumously granted the title of an Honorary Citizen of Kalisz by the Town Council of Kalisz.

Separation and identification fresh water leeches of waters resources in Kordistan Province

In the primary studying, known leeches have included into free living and parasitic which the parasitic group, besides of direct impacts like: growth detraction, anemia, making wound in the connecting part with the skin, with making plat for secondary bacterial and molding infections in the toll place, is able to cause to transfer blood flagellate and virus to the fish too. Therefore, by accusing information related to fauna leeches of each area a risk factor from the viewpoint of the possibility of being or accusing transferred diseases by these leeches, one can predict and forehand about them. Freshwater leeches of Iran to present accurate recognition (morphological, molecular) have not been, and there are some limited reports from different parts of the country about them. One of the areas that its leeches have not been identified yet is Kurdistan, By having five latrines and big permanent rivers and 32000 springs and a lot of deep and semi-deep wells and this province is a convenient bed for growing aquatics in the country. Therefore, identifying risk factors for development of aquaculture on water resources is one important factor to access achieving development goals. For recognizing leeches of this province, some samples from 10 stands were token. Samples from under stones, sticking to the fish, turtles, plants and solid substances in the water were separated and after recording their physical characteristics, calming with 10% ethylic alcohol with 10% formalin become fixed and after painting with Carmen acetic acid by standard keys for 7 species of Helobdella stagnalis, Placobdella costata, Hemiclepsis marginata, Erpobdella octoculata, Hirudo medicinalis, Dina lineate lineata have been identified and described. Which Helobdella stagnalis has the highest distribution in the province and the minimum one is Hirudo medicinalis. However, that the data obtained in leeches in Kurdistan is a relatively complete collection in this research, recognizing fauna of these areas needs more studying. The Placobdella costata and Hemiclepsis marginata sticking to the fish were separated among identified species which showed that these are parasites for the fish. The sticking area of those leeches to the skin was accompanied with scales cast, damage to mucous membranes beneath the parasite and bleeding Was associated with Histopathology studying effect includes observing break and disconnection in the leech connecting place to the epithelial layer of epidermis in the skin, destroyed nucleus in skin Epithelial cells with observing necrosis in ulcerative place become of the leech and the sub acute inflammated penetration until acute necrosis with opening in Dermis layer is observable. Kidney of this fish have changes such as: proliferation, like proliferative kidney disease with increasing proliferative glomerular cells and increasing in membranous cells in Capillary corpuscle, observing necrotic cells in haematopoietic tissue of kidney along with increasing in infiltration of leukocyte's cells generally mono nucluars such as lymphocytes and less poly morpho nucluars such as neutrophiles that are symptoms of disorders causing anemia become of nourishing and sucking blood by the leech and creating a chronic kidney infection that originally root is in another place like the skin. Also Hemorrhagic anemia causes losing RBC's is because of using the host blood by the leech. (In this situation, one can see immature RBC red cells in Peripheral blood. To identify potential carriers of the leech to the viruses, after finding them in recorded stands and putting them in 75% ethanol for viruses cause IPN, VHS, IHN, they were tested by PCR that the conclusion of these experiments approved IPN virus in Hemiclepsis marginata and Hirudo medicinalis. This kind of leeches can act like a mechanical carrier and causing spreading the agent of this disease. It is worth mentioning that studying the pathogenicity of this virus for aquaculture sources, mentioned before needs more research. During the study of infected fish with leeches that was done after preparing bloody slides and staining them, no case blood parasites was observed. During a research about infecting fish experimentally to known leeches it become clear that 5 days after being in aquarium including leeches, samples of sticking Hirudo medicinalis leech to the golden carp with scales cast were observed. Including leeches to the fish started with molting the scales in the sticking area in the fish and fish become too uneasy and by rubbing themselves to the malls and things inside the aquarium, tried to separate them. Finally, after around 30 hours, leeches penetrate the skin, feeding from blood and tissue liquids and cause mortality the fish and then they become separated from them. If the corpse of these fish stayed in the aquarium, the Helobdella stagnalis and Erpobdella octoculata would start feeding them.

Hidrógeis de Poli(N-Vinil 2-Pirrolidona) com nano argilas laponite RD sinterizados por irradiação gama

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Historiografia dziejów najnowszych po 1989 roku wobec innych nauk społecznych (na przykładzie psychologii). Refleksja metodologiczna i postulaty badawcze

Celem artykułu jest krytyczna rekonstrukcja stanu refleksji metodologicznej w kwestii interdyscyplinarnej współpracy między naukami historycznymi i psychoiogicznymi.

Wood-derived biomaterials for biomedical applications

Driven by the global trend in the sustainable economy development and environmental concerns, the exploring of plant-derived biomaterials or biocomposites for potential biomedical and/or pharmaceutical applications has received tremendous attention. Therefore, the work of this thesis is dedicated to high-value and high-efficiency utilization of plant-derived materials, with the focus on cellulose and hemicelluloses in the field of biomedical applications in a novel biorefinery concept. The residual cellulose of wood processing waste, sawdust, was converted into cellulose nanofibrils (CNFs) with tunable surface charge density and geometric size through 2,2,6,6-tetramethylpiperidinyloxy (TEMPO)-mediated oxidation and mechanical defibrillation. The sawdust-based CNFs and its resultant free-standing films showed comparable or even better mechanical properties than those from a commercial bleached kraft pulp at the same condition, demonstrating the feasibility of producing CNFs and films thereof with outstanding mechanical properties from birch sawdust by a process incorporated into a novel biorefinery platform recovering also polymeric hemicelluloses for other applications. Thus, it is providing an efficient route to upgrade sawdust waste to valuable products. The surface charge density and geometric size of the CNFs were found to play key roles in the stability of the CNF suspension, as well as the gelling properties, swelling behavior, mechanical stiffness, morphology and microscopic structural properties, and biocompatibility of CNF-based materials (i.e. films, hydrogels, and aerogels). The CNFs with tunable surface chemistry and geometric size was found promising applications as transparent and tough barrier materials or as reinforcing additive for production of biocomposites. The CNFs was also applied as structural matrices for the preparation of biocomposites possessing electrical conductivity and antimicrobial activity by in situ polymerization and coating of polypyrrole, and incorporation of silver nanoparticles, which make the material possible for potential wound healing application. The CNF-based matrices (films, hydrogels, and aerogels) with tunable structural and mechanical properties and biocompatibility were further prepared towards an application as 3D scaffolds in tissue engineering. The structural and mechanical strength of the CNF matrices could be tuned by controlling the charge density of the nanocellulose, as well as the pH and temperature values of the hydrogel formation conditions. Biological tests revealed that the CNF scaffolds could promote the survival and proliferation of tumor cells, and enhance the transfection of exogenous DNA into the cells, suggesting the usefulness of the CNF-based 3D matrices in supporting crucial cellular processes during cell growth and proliferation. The CNFs was applied as host materials to incorporate biomolecules for further biomedical application. For example, to investigate how the biocompatibility of a scaffold is influenced by its mechanical and structural properties, these properties of CNF-based composite matrices were controlled by incorporation of different hemicelluloses (O-acetyl galactoglucomanan (GGM), xyloglucan (XG), and xylan) into CNF hydrogel networks in different ratios and using two different approaches. The charge density of the CNFs, the incorporated hemicellulose type and amount, and the swelling time of the hydrogels were found to affect the pore structure, the mechanical strength, and thus the cells growth in the composite hydrogel scaffolds. The mechanical properties of the composite hydrogels were found to have an influence on the cell viability during the wound healing relevant 3T3 fibroblast cell culture. The thusprepared CNF composite hydrogels may work as promising scaffolds in wound healing application to provide supporting networks and to promote cells adhesion, growth, and proliferation.