This master's thesis was focused on the study of interaction of some organic azo dyes (model diffusion probes) with cationic biopolymer chitosan. This interactions were realized via diffusion processes in hydrogel media based on thermoreversible agarose. The main aim was study of influence of pH of solution on the diffusion process. Interactions of used dyes are based on electrostatic character. The aminogroup of chitosan interacts with the functional group of chosen dyes (anionic sulfonic group) and thus affects the process of diffusion. The model diffusion compounds were chosen organic anionic dyes Chicago sky blue 6B (C.I. 24 410), Sirius red (C.I. 35 780) and Reactive blue 49 (C.I. 621 526). The important content of this diffusion method is a monitoring of the time progression of diffusion profile by UV-VIS spectrophotometry. The presented work follows the bachelor thesis and shows comprehensive view of the reactivity of chitosan and its behavior in different systems. Unsteady diffusion in cuvettes appears to be a universal method for the study of reactivity of biopolymers and for the study of transport processes in hydrogel media. The diffusion method is universal, easy and cheap.

The presented thesis on thermosensitive polymer gel is focused especially on a thermosensitive triblock copolymer, which is composed of hydrophobic polylactide, polyglycolid and hydrophilic polyethylene glycol (PLGA-PEG-PLGA). Thermosensitive copolymers are very attractive for their phase sol-gel transitions and gel-suspension transitions. The aqueous solution of this copolymer behaves like a sol at laboratory temperature and like a gel at body temperature. These systems are used as injectable carriers for targeted drug delivery with controlled release. However, the influence of the resulting polymer concentration and temperature on the thermosensitive hydrogel nanostructure was not yet fully studied. In the experimental part, the viscoelastic behavior of hydrogels was observed by dynamic rheological analysis at different polymer concentrations and temperature conditions. The average size and distribution of micelles of triblock copolymer in aqueous solution were measured using dynamic light scattering technique. Characterization of fibrous micelles was complemented by imaging technique, cryogenic transmission electron microscopy.

This diploma thesis deals with preparation and subsequent characterization of albumin (BSA) and mixed hyaluronan-albumin (HA+BSA) hydrogels. The experimental characterization was based on the rheological measurements of the influence of ionic strength, pH and molar mass of HA on the gelation process and the properties of formed gels, as well as the time stability of these properties. Following object of the study was swelling of hydrogels in water and buffers with different pH value. Characterization was extended by thermal analysis, morphology description using scanning electron microscopy (SEM) and study of transport properties of hydrogels for selected model substance. Due to increasing ionic strength, the gel point was shifted towards higher temperature values and the resulting gels exhibited higher values of complex moduli. In case when pH was lower than isoelectric point of BSA, attractive electrostatic interactions between albumin and hyaluronan took place and the faster gelation occurred, compared to the case when pH was higher than isoelectric point of BSA. On the contrary, values of complex moduli increased with increasing pH. The gel samples prepared in the presence of low molecular weight HA reached higher values of complex moduli, compared to samples with the high molecular weight HA. Rheological properties of BSA gels were stable over time, whereas the mixed HA+BSA were characterized by significant decrease of complex moduli during the first three days of storage. Due to heating up to 100 °C, more than 80 % of the weight of studied samples was lost, as a result of water evaporation. Further heating of hydrogels, up to 250 °C, did not cause other changes associated with weight loss, in contrast with crystalline powder BSA, in which another weight loss at 220 °C was observed, as a result of the beginning degradation. Swelling studies, as well as SEM images, indicated higher crosslink density of BSA gels, compared to HA+BSA samples, which exhibited higher values of swelling ratio. The lowest values of equilibrium swelling ratio were observed at pH 4, which is close to the isoelectric point of albumin. Calculated diffusion coefficients relative to diffusivity of methylene blue in water ranged between the order of 10E-2 for diffusion into physiological solution and the order of 10E-3 for the diffusion into water. Ongoing transport of model dye from gel to solution was significantly slower, compared to its diffusion in water, as a result of gel porosity and electrostatic immobilization of positively charged molecules of methylene blue on the negatively charged BSA and HA chains.

This diploma thesis was focused on the system of agarose hydrogels with micellar domains. Polysaccharide agarose was used for the preparation of hydrogel matrices. Septonex was chosen as a surfactant. The release of solubilizate from the agarose hydrogel matrix was studied with a hydrophobic dye Sudan III in order to obtain a model system of hydrophobic solubilized drugs. Sudan III was solubilized via surfactant´s micels. Subsequently, this micellar aqueous solution with the dissolved dye was used to prepare the agarose hydrogel. The system was prepared in the environment of deionized water and physiologic solution. The gradual release of the dye into deionized water and physiologic solution was studied for 22 days using the UV-VIS spectrophotometry method. It was found that after 14 days the concentration of the releasing dyes into the deionized water was decreased. The cause is that after 14 and 22 days there was a slight degradation of gels, which was detected using the rheology method. Using the rheology method it was also found that Septonex does not affect the gel formation process.

The main objective of this thesis is to judge the barrier properties of hydrogels. It was examined in total six kinds of agarose hydrogels, of which five kinds of gels was enriched with humic acids isolated from various sources. Humic acids were selected from IHSS standards, specifically Leonardite, Elliott Soil, Suwannee River, Pahokee Peat and Nordic Lake. Using UV-VIS spectrometry, copper ion concentrations were used to serve as a diffusion solution. Results of diffusion coefficients were evaluated by several diffusion methods. The method of diffusion in the plane of the board and three possible diffusion solutions of the constant source were used.

Presented thesis deals with the preparation and characterization of hydrogels based on positively-charged biopolymer and negatively-charged tenside. As an appropriate representative of positively-charged polymer was chosen chitosan, because of his biocompatibility and biodegrability, and N,N,N-trimethylchitosan, which was synthetised from chitosan in this diploma thesis. The final product of synthesis was characterised by SEC-MALS, elemental analysis, infrared spectroscopy and nuclear magnetic rezonation. Interactions between these polymers and representatives of negatively-charged tensides, which are sodium dodecylsuphate and sodium tetradecylsulphate, were investigated. The main method used in this thesis was rheology, through it were investigated prepared hydrogels based on chitosan and tensides and also prepared microgels based on N,N,N-trimethylchitosan and tensides. The interactions between polymers and tensides have been proved.

abstract: Satellite cells are adult muscle stem cells that activate, proliferate, and differentiate into myofibers upon muscle damage. Satellite cells can be cultured and manipulated in vitro, and thus represent an accessible model for studying skeletal muscle biology, and a potential source of autologous stem cells for regenerative medicine. This work summarizes efforts to further understanding of satellite cell biology, using novel model organisms, bioengineering, and molecular and cellular approaches. Lizards are evolutionarily the closest vertebrates to humans that regenerate entire appendages. An analysis of lizard myoprogenitor cell transcriptome determined they were most transcriptionally similar to mammalian satellite cells. Further examination showed that among genes with the highest level of expression in lizard satellite cells were an increased number of regulators of chondrogenesis. In micromass culture, lizard satellite cells formed nodules that expressed chondrogenic regulatory genes, thus demonstrating increased musculoskeletal plasticity. However, to exploit satellite cells for therapeutics, development of an ex vivo culture is necessary. This work investigates whether substrates composed of extracellular matrix (ECM) proteins, as either coatings or hydrogels, can support expansion of this population whilst maintaining their myogenic potency. Stiffer substrates are necessary for in vitro proliferation and differentiation of satellite cells, while the ECM composition was not significantly important. Additionally, satellite cells on hydrogels entered a quiescent state that could be reversed when the cells were subsequently cultured on Matrigel. Proliferation and gene expression data further indicated that C2C12 cells are not a good proxy for satellite cells. To further understand how different signaling pathways control satellite cell behavior, an investigation of the Notch inhibitor protein Numb was carried out. Numb deficient satellite cells fail to activate, proliferate and participate in muscle repair. Examination of Numb isoform expression in satellite cells and embryonic tissues revealed that while developing limb bud, neural tube, and heart express the long and short isoforms of NUMB, satellite cells predominantly express the short isoforms. A preliminary immunoprecipitation- proteomics experiment suggested that the roles of NUMB in satellite cells are related to cell cycle modulation, cytoskeleton dynamics, and regulation of transcription factors necessary for satellite cell function. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2020

Orientador: Fauze Ahmad Aouada / Resumo: Com o objetivo de aplicar agroquímicos de uma forma controlada, e reduzir os danos à saúde e ao meio ambiente causados por estes, foram desenvolvidos neste trabalho, dois novos hidrogéis a partir de dois polissacarídeos diferentes carboximetilcelulose (CMC) e quitosana (Qs), suportados em redes de poli (ácido metacrílico)-co-poliacrilamida (PMAA-co-PAAm). Também foi investigada a influência da zeólita nas principais propriedades agroquímicas desses materiais como sorção e dessorção de herbicidas. A estabilidade da zeólita na solução polimérica foi estudada por meio de medidas de viscosidade. Estes comprovaram que a adição de polissacarídeo na solução formadora dos hidrogéis possibilita a obtenção de um hidrogel nanocompósito homogêneo. A incorporação da zeólita nos hidrogéis foi confirmada pela presença das bandas características da zeólita em 798 cm-1 e 600 cm-1 nos espectros de FTIR dos hidrogéis nanoestruturados. Outros indícios de incorporação foram a redução no volume dos poros e a presença de microporos nas paredes dos poros dos hidrogéis, aumento na massa residual dos hidrogéis nanoestruturados a 800ºC, observados nos resultados obtidos por microscopia eletrônica de varredura (MEV) e análise termogravimétrica (TG), respectivamente. Constatou-se também que a presença da zeólita reduziu o grau de intumescimento desses materiais, devido ao decréscimo na elasticidade das cadeias poliméricas. Entretanto, nos resultados de dessorção observou-se que a presença da zeólita pode... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In order to apply agrochemicals in a controlled manner, and to reduce the damages to the health and environmental caused by these, two new hydrogels based on carboxymethylcellulose (CMC) and chitosan (Qs) polysaccharides supported on poly(methacrylic acid)-co-polyacrylamide (PMAA-co-PAAm) networks were synthesized. The influence of zeolite on the main properties of these materials such as sorption and desorption was also investigated. The stability of zeolite in the polymeric solution was studied by viscosimetry. These results showed that the addition of polysaccharide in the hydrogel-forming solution enables the obtaining of homogeneous nanocomposite hydrogels. The incorporation of zeolite into hydrogels was confirmed by the presence of spectroscopic bands at 798 cm-1 and 600 cm-1 in the FTIR nanocomposite spectra. Other indicatives were the reduction in pore volume and the presence of micropores in the pore walls of zeolite hydrogels, and increase in the residual mass of nanostructured hydrogels at 800ºC, observed by scanning electron microscopy (SEM) and thermogravimetric analysis (TG), respectively. It was also observed that the presence of zeolite reduced the degree of swelling of these materials, due to the decrease in the elasticity of the polymeric chains. The results of desorption in saline medium show that the hydrogels studied in this work can reduce the presence of Al+3 ions, which in acidic soils are harmful to some crops. These results indicate that the hydrogel... (Complete abstract click electronic access below) / Doutor

<p>The pancreas is a vital organ that secretes key metabolic hormones and digestive enzymes. In pancreatic ductal adenocarcinoma (PDAC), one of the leading causes of cancer-related death in the world, limited advances in diagnosis or therapies have been made over decades. Key features of PDAC progression include an elevated matrix stiffness and an increased deposition of extracellular matrices (ECM), such as hyaluronic acid (HA). Understanding how cells interact with components in the tumor microenvironment (TME) as PDAC progresses can assist in developing diagnostic tools and therapeutic treatment options. In recent years, hydrogels have proven to be an excellent platform for studying cell-cell and cell-matrix interactions. Utilizing chemically modified and naturally derived materials, hydrogel networks can be formed to encompass not only the components, but also the physicochemical properties of the dynamic TME. In this work, a dynamic hydrogel system that integrates multiple click chemistries was developed for tuning matrix physicochemical properties in a manner similar to the temporally increased matrix stiffness and depositions of HA. Subsequently, these dynamic hydrogels were used to investigate how matrix stiffening and increased HA presentation might affect survival of PDAC cells and their response to chemotherapeutics. </p>

Nicht behandelte Knorpelschäden führen langfristig zum Verschleiß des Gelenks – der Arthrose. Eine vielversprechende Möglichkeit zur Behandlung von Knorpelschäden ist die Matrix-assoziierte Chondrozytenimplantation. In der vorliegenden Dissertationsarbeit sollte die neuartige Matrix NOVOCART® Inject der Firma TETEC® auf ihre Eignung für die Therapie von Knorpeldefekten untersucht und ihre potentiellen Effekte im Vergleich zu dem bereits in der klinischen Anwendung befindlichen Biomaterial Fibrin charakterisiert werden. Für diese Studie wurden bei insgesamt 12 Göttinger Minipigs jeweils zwei vollschichtige Defekte (Ø 6mm) auf den medialen Femurkondylen beider Knie gesetzt und mit humanen Chondrozyten, die entweder in NOVOCART® Inject oder Fibringel eingebettet waren, gefüllt. Zellfreie Träger dienten jeweils als Kontrolle. Nach zwei und vier Wochen wurde die Defektfüllung, Biokompatibilität, Wirtszellanlockung und der Verbleib der implantierten Zellen sowie die Regeneratqualität histologisch und auf Genexpressionsebene evaluiert. Es konnte nachgewiesen werden, dass NOVOCART® Inject auch ohne Membranabdeckung eine gute Defektfüllung erzielt. Es ist biokompatibel und weist keine signifikant erhöhten Entzündungszeichen gegenüber Fibringel auf. Des Weiteren konnte eine vergleichbare Regeneratqualität im Vergleich zum Fibringel im porzinen Tiermodell erreicht werden. Unabhängig vom verwendeten Trägermaterial überdauerten die xenogen transplantierten Zellen im vorliegenden Defektmodell nur kurzfristig. Wir fanden Hinweise auf eine Immunreaktion unter Beteiligung von Makrophagen, die ursächlich für das Verschwinden der implantierten Zellen sein könnten. Die Ergebnisse unserer Untersuchungen stellen folglich die häufig diskutierte Theorie des immunpriviligierten Status der Chondrozyten in Frage.