Magneticky řízené samo-uspořádávání v polymerních nanokompozitech je studováno v této dizertační práci. Strukturování polymerních nanokompozitů pomocí relativně slabých magnetických polí (B=0-50 mT) bylo prokázáno jako praktická metoda pro kontrolu jejích nano a mikrostruktury. Vliv intenzity magnetického pole, množství nanočástic, viskozity a času uspořádávání na výslednou strukturu byl studován v různých systémech jako fotopolymer, polyuretan nebo koloidně dispergované nanočástice v acetonu s malým množstvím rozpuštěného polymeru. Samo-uspořádané struktury – bez aplikace vnějšího magnetického pole vykazují vícekrokovou agregaci nanočástic do uskupení s komplexním tvarem. Magnetické interakce byly označené jako odpovědné za agregaci nanočástic v samo-uspořádaných systémech pomocí výpočtů energii mezi-částicových interakcí. S rostoucím magnetickým polem, magnetické nanočástice jsou rychle uspořádané do jednorozměrných částicových řetězů s vysokým aspektním poměrem a homogenní orientaci v polymerní matrici. S prodluženým časem uspořádaní, tyto struktury postupně rostou z malých submikrometrových struktur do velkých mikroskopických super struktur. Táto metoda vykazuje velký potenciál pro kontrolovanou přípravu široké škály struktur v polymerních nanokompozitech vhodných pro technologické aplikace a také pro fundamentální studie. Magneticky uspořádané polymerní nanokompozity vykazují značnou směrovou anisotropii tuhosti kompozitu nad jeho skelným přechodem přičemž, pod skelným přechodem systému není pozorován žádný efekt. Podélně orientované struktury vykazují větší příspěvek k tuhosti kompozitů. Efektivnost vyztužení vykazuje teplotně závislý průběh a maximum je pozorováno přibližně 60 °C nad skelným přechodem. Struktura magneticky uspořádaného polymerního nanokompozitu byla popsána vícero-úrovňovým hierarchickým modelem materiálu. Mikromechanika byla využitá k popisu směrově závislého vyztužení polymerních nanokompozitů a k popisu teplotně závislé tuhosti hybridních struktur složených z nanočástic a polymeru. Schopnost nést napětí, deformovat se a nenulová tuhost hybridních struktur je odpovědná za vyztužení polymerních nanokompozitů. Přítomnost polymerních přemostění mezi nanočásticemi, které přenášejí napěti skrze magnetické struktury je označená jako nezbytná pro mechanickou odezvu polymerních nanokompozitů a pro tuhost hybridních struktur.

The presented doctoral thesis is focused on preparation, characterization and application of organic micro- and nanoparticles as transport systems for active components and some their complex natural sources. Active component were packed into liposomes and polysaccharide particles. As active components were used caffeine, some drugs – clotrimazole and ibuprofen, further antioxidants and vitamins. Antimicrobial herbs and spices extract, antimicrobial peptides lysozyme, nisin and other antimicrobial ingredients were encapsulated too. Encapsulation of selected hydrolytic enzymes was tested, too. Particles were also used for encapsulation of probiotic strains Bifidobacterium breve and Lactobacillus acidophilus and prebiotic components. These prebiotics were co-encapsulated into capsules with probiotic cells. Natural extracts were encapsulated e.g. extracts of guarana, ginseng, goji, green barley, propolis, black, green and white tea, coffee, fruit and vegetable extracts. The efficiency of encapsulation was determined by HPLC/PDA and by spectrophotometry. Long-term stability of particles and amount of released component in model/real foods, in model cosmetic conditions and in a model physiological environment were monitored too. Size of prepared liposomes and polysaccharide particles was determined by dynamic light scattering and by light microscopy and electron microscopy, respectively. Stability of the particles was measured using a zeta potential. Also, analytical centrifugation was used to measurement of sedimentation velocity and stability of the prepared particles. The antimicrobial activity were tested using two Gram-positive (Bacillus subtilis, Micrococcus luteus), two Gram-negative (Escherichia coli, Serratia marcescens) bacteria and one fungal strains (Candida glabrata). For determining the antimicrobial properties of active component and prepared particles two the most widely used methods were used - agar diffusion method and broth dilution method. The viability of probiotic strains were performed using flow cytometry and fluorescence microscopy. Encapsulation of active component was successful in all types of particles. Liposome showed a very good long-term stability mainly in water conditions with neutral pH and polysaccharide particles were stable in acidic conditions. Prepared particles showed a very good stability in model stomach environment, while in model intestines environments particles were disintegrated and active component were released. Prepared particles with encapsulated caffeine as well as other tested antioxidants and vitamins could be used to modern types of energy drinks, food supplements and also for some cosmetics applications. Encapsulated antimicrobial components could be used for food application as well as for cosmetics and pharmaceutical application like antimicrobial wound formulation. Encapsulated enzymes can be used for controlled release of proteases in wound healing, as delivery systems in digestive tract and as a part of pharmaceutical preparative and food supplements for enzyme therapy. The study revealed that encapsulation of probiotics and also co-encapsulation of probiotics with prebiotics exhibited longer stability of particles and survival bacterial cells. So, prepared particles are suitable for use to food product with beneficial effects on the human body.

In dieser Arbeit wurde ein Sensorkonzept mit Hilfe der Si-basierten Lichtemitter (MOSLED) zum Östrogennachweis in wässrigen Lösungen entwickelt. Das Sensorkonzept basiert auf einer direkten Fluoreszenzanalyse und besteht aus der Anordnung der Bio-Komponenten und dem Verfahren zu ihrer Herstellung sowie dem eigentlichen Meßverfahren. Die Anordnung besteht aus drei Teilen: die Funktionalisierung der MOSLED-Oberfläche, die Immobilisierung des hER_-Rezeptors und die Herstellung der Referenzlösung. Den Schwerpunkt dieser Arbeit bildet die Ausführung dieser drei Teile. Die Funktionalisierung der SiO2-Oberfläche der MOSLED wurde mit Hilfe eines im Rahmen dieser Arbeit entwickelten SSC (Spraying Spin Coating)- Verfahrens realisiert. Die Ausgangsmaterialien dieses Verfahrens sind organofunktionelle Silangruppen mit drei unterschiedlichen funktionellen Gruppen, nämlich die Amino-, Carboxyl- und die Thiolgruppen. Die Optimierung dieser Methode erfolgte mittels der zwei Silangruppen APMS ((3-Aminopropyl)trimethoxysilane und Triamino-APMS (N-[3-(Trimethoxysilyl)propyl]ethylenediamine mit der gleichen Molekülstruktur, aber mit einer unterschiedlichen Anzahl an funktionellen Gruppen. Diese Resultate wurden mit in der Literatur beschriebenen Verfahren verglichen. Die Optimierung der SSC-Methode wurde zuerst auf einfache SiO2-Oberflächen und dann auf der Oberfläche der MOSLED angewendet. Die Proben wurden mit Hilfe üblicher Methoden der Oberflächenphysik- wie FTIR-, Raman- und XPS-Spektroskopie untersucht. Die Oberflächenrauhigkeit wurde mittels AFM-Spektroskopie ermittelt, deren Aufnahmen eine glatte Oberfläche bei den mit der SSC-Methode silanisierten Proben zeigen. Während die Hydrophobizität der funktionalisierten SiO2-Oberflächen zunimmt, sinkt dabei die Oberflächenenergie, welche die Anbindung eines hER_-Rezeptors mit großer Bindungsenergie begünstigt. Zur Immobilisierung des hER_-Rezeptors wurde dieser erst an das Hüllenmolekül des QDots R-655-Farbstoffs gebunden und anschließend an der SSC-silanisierten SiO2-Oberflächen adsorbiert. Der Anteil der immobilisierten Rezeptoren wurde mittels PL-Messung kontrolliert. Eine andere Immobilisierungstrategie des hER_-Rezeptors an die SiO2-Oberfläche kann mit Hilfe eines Aminosäure-Derivates um den Rezeptor realisiert werden. Eine Adsorption der Lysinaminosäure an die SSC-APMS silanisierten SiO2- Oberflächen als Funktion des pH-Wertes wurde durchgeführt, und der Adsorbatsanteil des Lysins mittels XPS-Messung durch die Bindungsenergien der Energieniveaus C1s und N1s berechnet. Eine Referenzlösung mit QDots R800-Farbstoff markierten Östrogenmolekülen kommt zum Einsatz. Dabei wird die Position 17 des β-Estradiolmoleküls, welches mit einem N-Hydroxysuccinimide Derivat versehen ist, an das Hüllenmolekül des QDots R800-Farbstoff gebunden, sodass der Phenolring des β-Estradiols frei bleibt. Insbesondere ist bei den FTIR-Spektren eine nichtgebunden OH-Gruppe des β-Estradiolmoleküls gut erkennbar. Das gesamte Sensorkonzept wurde an zwei mit Östrogen mit einer Konzentration von 1mM und 1μM versetzten Wasserproben getestet. Die Anordnung der Bio-Komponenten wurde mittels PL nachgewiesen. Der Östrogennachweis wurde mit Hilfe des Ge- und Tb-basierten Lichtemitters demonstriert. / A sensor concept for estrogen detection in waterish solutions by Silicon based light emitters (MOSLED) was developed. This concept is based on direct fluorescence analysis and consists of a certain arrangement of the bio- components and their fabrication methods as well as the measurements protocol, which consists of for main steps: Passing the prepared MOSLED surface by the water sample, a washing step, passing the MOSLED surface by the reference solution, and the final optical measurement. The arrangement consists of three parts: the functionalisation of the MOSLEDs surface, the immobilization of the hERff receptor und finally the fabrication of the reference solution. The focus of this work is set on the achievement of these three parts. The functionalisation of the SiO2-surface of the MOSLED was realized by means of the new developed SSC (Spraying Spin Coating) method. The chemical precursor of this method are the organofunctional silane groups with three different functional groups, namely the amino-, carboxyl-, and thiolgroups. The optimization of the procedure was investigated with two types of silane groups APMS ((3-Aminopropyl)trimethoxysilane und Triamino-APMS (N-[3-(Trimethoxysilyl)propyl]ethylenediamine), which have the same molecular structure but a different number of functional groups per molecule. These results have been compared with those of the literature. The optimization of the SSC-method was analyzed by means of standard surface science techniques like FTIR-, Raman-, and XPS-spectroscopy. The surface roughness was applied by using AFM-spectroscopy, which showed a smooth surface by the samples treated with the SSC-method. Whereas the hydrophobicity of the functionalized SiO2 surface increases, the surface energy decreases, which favours the binding of a hERff receptor with large binding energy. In order to immobilize the hERff receptor at the surface, the receptor was bound to the molecular shell of the QDots655-dye and finally adsorbed to the silanized SiO2 surfaces. The fraction of the immobilized hERff receptors was controlled via PL-measurements. Another labelling strategy to immobilize the receptor at the SiO2 surface can be realized by using the amino acid as derivate to modify the receptor. For this aim the adsorption of the lysine at silanized SiO2 surfaces was investigated as function of the pH-value. The adsorbent part of the lysine was calculated via XPS by measuring the binding energy of both energy levels C1s and N1s . The reference solution with QDots800-dye marked estrogen molecules was used. The optimal binding was achieved by attaching the molecular shell of the QDots 800-dye to position 17 of the β-Estradiol molecule, which contains of a N-Hydroxysuccinimid derivate so that the phenol ring of the β-Estradiol remains free. In particular the FTIR-spectra showed the non-binding OH-groups of the β-Estradiol molecule. The whole concept of the sensor was tested at two water samples containing estrogen in a concentration of 1mM and 1μM. The adjustment of the Biokomponents was proven by PL, and the estrogen detection was demonstrated by using the Ge- and Tb-based light emitters.

Title: Falls in the eldery Objectives: The aim of this diploma thesis is to analyze and classify knowlage about falls in the eldery during usual daily activities with focus on prevencion and detection of falls by useing wearable and non wearable systems for detecting falls in the eldery. Methods: This diploma thesis is wrote with the metod of literary research through professional literature, books and articles in both Czech and English was used in the work. To meet the objectives that have been provided were selected examples that helped analyze and clarify the issue of falls in eldery. Results: The sources of professional literature have shown that the falls in the eldery are very serious issue of society. At the same time, it is important to be aware of their possible health and economic impacts, as well as how to prevent them. The correct use of fixation shoes has been shown to reduce the risk of fall. Many fall detection devices are able to call for timely help. Last but not least, knowlage of this issue could help to improve and innovate the fall detecion and prevention systems. Keywords: falls in eldery, senior immobilization, biomechanics of falls, detection and prevention of senior falls, osteoporosis, senior bone aging, senior cartilage aging, biomechanics of senior walking, walking...

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<p>Small nematode model organisms such as <em>Caenorhabditis elegans</em> are widely used in the fields of neurobiology, toxicology, drug discovery, etc. They are advantageous due to their fully characterized genomic and cellular system. Traditional screening methods involve the exposure of animals to chemicals/drugs inside multiwell-plates while its effects on growth, movement and other cellular/sub-cellular processes are monitored by visual inspection. Yet, these methods are time-consuming, low-throughput, expensive, tedious, difficult to control, hard to modulate instantaneously, prone to subjectivity and not suitable for movement-based behavioural assays. Hence, a method to induce and to quantify movement on-demand in a rapid, sensitive, precise and reversible manner would greatly facilitate biological studies. In this thesis, microfluidic engineering approaches have been utilized in nematode-based assays due to their potential to obtain high precision measurements in a low-cost, rapid and automated manner. Movement response of worms to a diverse range of electric signals has been quantitatively characterized. DC and pulse-DC electric fields have been shown to stimulate worms’ swimming towards the negative electrode inside a microchannel (electrotaxis). AC electric fields were used to inhibit movement on-demand. Animals’ movement has been characterized in terms of speed and range of motion, body-bend frequency and turning time. Electrotaxis was shown to be mediated by neuronal activities and correlations between animal’s behaviour and neuronal signalling has also been demonstrated. Using this basic understanding, multiple microfluidic components such as position sensors and electric immobilizers have been developed. Electrotaxis has then been applied as a technique to sort worms in accordance to their size/age and phenotype as well as to perform drug screening at a single-animal level. Integration of the techniques and components developed during this research is expected to have a significant impact on the development of an integrated microfluidic platform for high throughput automated behavioural screening of nematodes with applications in drug discovery, toxicology, neurobiology and genetics.</p> / Doctor of Philosophy (PhD)

Der Sprengstoff PETN ist äußerst schwer zu detektieren. Ein verbesserter anti-PETN-Antikörper wurde durch Anwendung des Bioisosterie-Konzepts entwickelt. Diese polyklonalen IgGs sind sehr selektiv und sensitiv. Die Nachweisgrenze des ELISAs beträgt 0,15 µg/L. Der Messbereich des Immunoassays liegt zwischen 1 und 1000 µg/L. Die Antikörper sind recht pH-stabil als auch robust gegen Lösungsmittelzusätze. Für die Umweltanalytik von TNT wurde eine HPLC-kompatible Affinitätssäule mit porösem Glas als Trägermaterial hergestellt. Um die anti-TNT-Antikörper selektiv aus den TNT-Seren zu isolieren, wurde eine Trennung an einer Dinitrophenyl-Affinitätssäule durchgeführt. Zur Optimierung der Kopplungsmethode wurden orangefarbene Dabsyl-Proteine synthetisiert und auf der Oberfläche gebunden. Die Färbung wurde als Indikator für die Ligandendichte verwendet. Wegen der hohen Affinitätskonstanten der anti-TNT-IgGs lässt sich TNT nicht reversibel von der TNT-Affinitätssäule eluieren. Daher wurde eine neuartige Elutionsmethode entwickelt, die thermische Online-Elution. Die maximale Kapazität einer TNT- Affinitätssäule betrug 650 ng TNT bzw. 10 µg/mL Säulenvolumen. Um die Ligandendichte der TNT-Affinitätssäulen zu bestimmen, wurde ein neues Verfahren entwickelt, da die spektroskopischen Proteinbestimmungsmethoden nicht geeignet waren. Zur Proteinbestimmung wurde eine HPLC-Trennung der Aminosäuren Tyr und Phe ohne vorherige Derivatisierung entwickelt. Die Proteinhydrolysezeit wurde durch Einsatz einer Mikrowelle von 22 h auf 30 min verkürzt. Zur internen Kalibrierung wurden HTyr und FPhe verwendet. Die Nachweisgrenze bei 215 nm ist sowohl für Tyr als auch für Phe 0,05 µM (~ 10 µg/L). Dieses neue Verfahren, das als Aromatische Aminosäureanalyse (AAAA) bezeichnet werden kann, wurde zur Proteinbestimmung von homogenen Proben mit NIST-BSA validiert, wobei die Nachweisgrenze für Proteine 16 mg/L (~ 300 ng BSA) ist. Die relative Standardabweichung incl. der Hydrolysestufe beträgt 5%. / The explosive Pentaerythritol tetranitrate (PETN) is extremely difficult to detect. An improved antibody against PETN was developed by using the bioisosteric concept. These polyclonal antibodies are highly selective and sensitive. The limit of detection (LOD) of the ELISA was determined to be 0.15 µg/L. The dynamic range of the assay was found to be between 1 and 1000 µg/L. The antibodies are sufficiently pH-stable and resistant to solvent additives. An HPLC-compatible TNT-affinity column with porous glass as support material was prepared for the environmental analysis. In order to isolate the anti-TNT antibodies of the TNT sera a separation was carried out on a dinitrophenyl-affinity column. To optimize the immobilization method, orange-coloured dabsyl proteins were synthesized and bound to the surface. The colour intensity was found to be an indicator for the immobilization rate. In consequence of the high affinity constants of the anti-TNT antibodies, TNT can''t elute by a typical acidic elution step. Therefore, a novel separation approach, the thermal online-elution was developed. The maximum capacity of an affinity column was 650 ng TNT or 10 µg/mL of column volume. To quantify the immobilization rate of proteins, a new method has been developed, because the usual protein determination methods were unsuitable. Therefore an HPLC separation method of Tyr and Phe was developed without prior derivatization. Two internal standard compounds, HTyr and FPhe, were used for calibration. The LOD was estimated to be 0.05 µM (~ 10 µg/L) for Tyr and Phe at 215 nm. The protein hydrolysis time was reduced from 22 h to 30 min using microwave technique. This procedure, that was termed aromatic amino acid analysis (AAAA), has been validated for protein determination of homogeneous samples with NIST-BSA. The LOD for proteins was calculated to be below 16 mg/L (~ 300 ng BSA absolute). The relative standard deviation, including the hydrolysis step, is 5%.

The open nature of Cell-Free Protein Synthesis (CFPS) systems has enabled flexible design, easy manipulation, and novel applications of protein engineering in therapeutic production, biocatalysis, and biosensors. This dissertation reports on three advances in the application of CFPS systems for 1) improving biocatalysis performance in industrial applications by site-specific covalent enzyme immobilization, 2) expressing and optimizing a difficult to express a mammalian protein in bacterial-based CFPS systems and its application for cost-effective, on-demand biosensors compatible with human body fluids, and 3) streamlining the procedure of an E. coli extract with built-in compatibility with human body fluid biosensors. Site-specific covalent immobilization stabilizes enzymes and facilitates recovery and reuse of enzymes which improves the net profit margin of industrial enzymes. Yet, the suitability of a given site on the enzyme for immobilization remains a trial-and-error procedure. This dissertation reports the reliability of several design heuristics and a coarse-grain molecular simulation in predicting the optimum sites for covalent immobilization of a target enzyme, TEM-1 ?-lactamase. This work demonstrates that the design heuristics can successfully identify a subset of favorable locations for experimental validation. This approach highlights the advantages of combining coarse-grain simulation and high-throughput experimentation using CFPS to efficiently identify optimal enzyme immobilization sites. Additionally, this dissertation reports high-yield soluble expression of a difficult-to-express protein (murine RNase Inhibitor or m-RI) in E. coli-lysate-based CFPS. Several factors including reaction temperature, reaction time, redox potential, and presence of folding chaperones in CFPS reactions were altered to find suitable conditions for m-RI expression. m-RI with the highest activity and stability was used to develop a lyophilized CFPS biosensor in human body fluids which reduced the cost of biosensor test by ~90%. Moreover, an E. coli extract with RNase inhibition activity was developed and tested which further streamlines the production of CFPS biosensors compatible with human body fluids.