Physical exercise is one of the main factors in achieving best results in competition. We have to create products using the most advanced techniques, in this technology age that would help coaches and athletes. One of the technologies allows us to use GPS receivers for collecting and later analysis of training programs. This master thesis is an overview of GPS possibilities, discussing and analysing GPS receivers and systems used for exercise analysis. In this work overview the skeleton and main components of general-purpose system. Collection of the components as a whole makes a system that corresponds to the requirements for such system. We present fully analysed methods for information system of physical exercise analysis. System for automatic storage and analysis of collected exercise data is designed and created.

Current diagnostic and therapeutic nanocarriers, including liposomes, micelles, and polymeric- and protein-based nanoparticles, are designed to have key functional properties such as: (i) longevity in the bloodstream, leading to accumulation of therapeutic cargos in neoplastic areas with leaky vasculatures; (ii) targeting of specific pathological sites through surface modification with targeting ligands; (iii) stimuli-responsive characteristics for controlled drug release under specific conditions. While some of these drug delivery systems have advanced into clinical stages, other nanocarriers remain under development to overcome issues with effective delivery such as lack of target-ability and fast clearance from circulation. Self-assembling peptides have recently shown great potential as nanocarrier materials for drug and gene delivery, owing to their safety, efficiency, and targeting capabilities. An amino acid pairing strategy enables us to design self/co-assembling peptides with multiple functionalities to fulfill drug delivery requirements. This thesis focuses on functionalization and characterization of self/co-assembling peptides as nanocarriers for hydrophobic anticancer drug delivery. Diethylene glycol (DEG) conjugation and protein binding are the two modification strategies used in this thesis to impart longevity and target-ability upon the peptide-based delivery system. The studies include: (i) characterization of self-assembling properties of the diethylene glycol (DEG)-conjugated amino acid pairing peptide AAP8, (ii) investigation of the self/co-assembling features of a model ionic-complementary peptide (EAR8-II) in complex with the hydrophobic drug pirarubicin, and the anticancer activity of the complex, (iii) the interactions between peptide-drug complexes and serum proteins from the thermodynamic viewpoint, (iv) quantification of the effect of protein binding to the peptide-based delivery system on immune responses and biocompatibility, and (v) exploration of the targeting capability of albumin-bound peptide-drug complexes towards lung cancer cells. Uncontrollable aggregation of AAP8 was the first issue to address in order to develop a promising platform for the peptide-based delivery system. Diethylene glycol (DEG), a short segment of polyethylene glycol (PEG), was conjugated to AAP8 either at one or both terminals, and then self-assembling and drug encapsulation properties of both functionalized AAP8s were characterized to evaluate the effect of DEG-modification. The results illustrated a significant reduction in uncontrollable aggregation, and the formation of uniform fibular nanostructures. In addition, DEG conjugation provided the peptide with safer features towards immune cells by reducing cellular toxicity to macrophages. Moreover, DEG-functionalization improved hydrophobic drug stabilization, as demonstrated by sustained cytotoxic efficacy against lung carcinoma cells over a relatively long time compared to the non-functionalized AAP8. Protein binding strategy was the second approach to utilize the peptide-based delivery system with more biocompatibility and target-ability features. EAR8-II was studied as a model ionic-complementary peptide with high capability of pirarubicin encapsulation and anticancer activities against different cancer cells. Albumin as a most abundant protein in serum was selected to assess its binding affinity to the delivery system, and evaluate its binding effect on immune responses and anticancer activities. The results showed a central role of albumin in the in vitro delivery of peptide-drug complexes to target lung cancer cells based on the following characteristics: (a) Non-covalent binding of albumin to the complex through hydrogen bonding and Van der Waals interactions. The interaction was confirmed by physicochemical methods such as fluorescence quenching and isothermal titration calorimeter (ITC). (b) Shielding properties of albumin for the complex against macrophages and blood components (erythrocytes and complement protein C5b-9). In the presence of albumin, phagocytosis and cytokine expression level of macrophages and hemolytic activity of the peptide-drug complex reduced significantly due to the smaller particle size of the albumin-bound complexes compared to unprotected ones. (c) Targeting the lung cancer cells, possibly because of the inhibition of the albumin-binding protein SPARC (secreted protein, acidic and rich in cysteine). SPARC is a glycoprotein over expressed in lung cancer cells with high affinity to albumin. The results from in vitro SPARC expression in A549 cells, a type of human non-small cell lung carcinoma (NSCLC), showed a significant drop by the albumin-bound complex at the mRNA level evaluated by qRT-PCR. This effect can be explained by transporting the albumin-bound complex into the cell surface, binding to the SPARC proteins, and so inhibiting the SPARC expressions. This work lays out a foundation for modification and characterization of the self/co-assembly peptide-based nanocarriers for hydrophobic anticancer drug delivery, especially to improve longevity and target-ability properties.

O celulossomo é um complexo multienzimático extracelular utilizado por bactérias anaeróbias para a degradação de biomassa vegetal. Ele é composto por escafoldinas, estruturas alongadas que abrigam diversos módulos cohesina, às quais se ligam dockerinas, seus parceiros de interação específica de alta afinidade, fusionados às enzimas celulolíticas. Os módulos cohesina e dockerina compõem o elemento central da interação entre todos os componentes que integram o celulossomo. Esses módulos são divididos em tipos, de acordo com sua sequência primária. Essa divisão reflete efeitos funcionais distintos, sendo o tipo I responsável pela ligação de enzimas às escafoldinas, enquanto o tipo II medeia a ligação de escafoldinas à célula. O celulossomo de Ruminococcus flavefaciens é o mais complexo conhecido, e na classificação por tipos, suas sequências divergem, formando o tipo III, que foi posteriormente subdividido em 6 grupos para significância funcional. Nesse sistema, o principal responsável pela integração de enzimas ao sistema é a escafoldina primária ScaA, a qual interage com escafoldina adaptadora ScaB. A especificidade dessa ligação - dockerina de ScaA (Rf-DocA) com cohesinas de ScaB (Rf-CohB1-7) - é classificada como único membro do grupo 5, na divisão de grupos que compõem o tipo III. Assim, essa interação é de suma importância para a organização do celulossomo desse organismo, tendo sido estudada por meio de experimentos biofísicos e bioquímicos. Porém a falta de uma estrutura cristalina resolvida desses componentes limita a compreensão que podemos ter sobre a interação. 1-2 Nesse trabalho, apresentamos as estruturas cristalográficas de Rf-DocA, em complexo com a Rf-CohB4, além da estrutura dessa cohesina isolada, e ainda, a Rf-CohB1, e alguns de seus mutantes pontuais. Com isso, esclarecemos aspectos estruturais desses módulos, como a presença de dois sítios funcionais de ligação a cálcio em Rf-DocA. Também é observável pelos modelos gerados, detalhes da ligação entre eles, como os resíduos participantes da interação. Estudos de afinidade entre esses módulos foram conduzidos para a elucidar algumas propriedades da ligação entre esses módulos, de forma que descobrimos que ela ocorre de uma única maneira, e que há um loop na cohesina cuja flexibilidade afeta a afinidade da ligação. Isso sugere um mecanismo de alteração conformacional que regula a ligação à dockerina. Adicionalmente, buscamos o emprego desses módulos em uma aplicação tecnológica, desenhando redes automontáveis de proteínas, visando a construção de um nanomaterial. Essas redes são formadas por características intrínsecas das proteínas que os compõem, sendo o principal fator considerado sua simetria rotacional.3 Nesse sentido, as dockerinas e cohesinas foram utilizadas para ligação entre proteínas de diferentes simetrias. Utilizamos proteínas de simetrias C3, C4 e C6 com fusão a dockerinas, que se conectam às cohesinas fusionadas a proteínas de simetria C2, as quais formam o elemento linear da ligação entre os diferentes módulos. Esse desenho experimental permite a expressão e purificação independentes dos componentes, o que facilita a obtenção das redes, a partir da mistura dos dois componentes. Através de análises preliminares por microscopia eletrônica de transmissão, observamos a formação de filmes bidimensionais extensos e nanotubos com a construção testada. / The cellulosome is an intricate multienzyme extracelular complexes evolved by anaerobic bacteria for degradation of cellulosic biomass. It is composed of scaffoldins, elongated structures, which bare numerous cohesin modules, which bind to dockerin modules, their high affinity and specificity partners, borne by cellulolytic enzymes. The cohesin and dockerina modules constitute the central element of the interaction between every component of the cellulosome. These modules are categorized in types, according to their primary sequence. That distribution reflects distinct functions, in which the type I is responsible for integration of enzymes to scaffoldins, while type II mediates anchoring of scaffoldins to the cell wall. The cellulosome of Ruminococcus flavefaciens is the most intricate known to date, which is categorized into a third type of cohesins and dockerins, due to sequence diversion. The type III was further divided into 6 groups to impart functional significance. In that system, the main enzyme integrating component is the primary scaffoldin ScaA, which interacts to the adaptor scaffoldin ScaB. The specificity of this interaction - dockerina of ScaA (Rf-DocA) to ScaB cohesins (Rf-CohB1-7) - is sorted as a single member of group 5, in the subtypes of type III. Thus, this interaction is essential for cellulosome organization, having been studied by biophysical and biochemical experiments. However, the lack of a solved crystalline structure of these components narrows our understanding on this interaction. In the present study, we present the structures of Rf-DocA, complexed to Rf-CohB4, besides the structure of this isolated cohesin, and also Rf-CohB1 and its point mutants. Due to these data, we clarify structural aspects of these modules, such as the occurrence of two functioning calcium binding sites in Rf-DocA. We also identified details of their binding, such as the interacting residues. Through binding affinity studies, we concluded that the interaction between these modules occurs in a single mode, and that there is a loop in the cohesin module whose flexibility has direct effects on the binding affinity to dockerin. Additionally, we sought to utilize these modules in a downstream application, by designing self-assembling arrays of proteins, aiming for the construction of a nanomaterial. These arrays are constructed from the intrinsic properties of its constituent proteins, in which the main factor is rotational symmetry. In this context, dockerina and cohesin modules were used of binding different symmetry proteins. We utilized C3, C4 and C6 point symmetry proteins fused to dockerin modules, which bind to the cohesin modules fused to C2 point symmetry proteins, which establish the linear connection between the distinct components. This experimental design allows for the independent expression and purification of the components, which facilitates the achievement of the arrays, by simple mixture of the two components. Through preliminary analysis by transmission election microscopy, we observed the construction of two-dimensional films and nanotubes.

Hydrogels based on self-assembling peptides are believed to have potential for use in a wide range of biomedical and biodiagnostic applications. For many of these, control over various properties of the gels is essential for tuning the gels to fit certain constraints or requirements in terms physical properties such as diffusive properties and swelling. One important property to control for applications such as cell culture and drug delivery is its mechanical strength, and this study investigates three different strategies by which the individual peptide monomers can be modified in order to effect a change in the macromolecular self-assembled structure and therefore a bulk change in the mechanical stiffness. In chapter 4, two ionic-complementary octapeptides, FEFKFKFK and FEFQFKFK are described, with monomer charges of +2 and +1, respectively at physiological pH. FEFKFKFK was observed to form largely discrete fibrils, characteristic of similar systems, while FEFQFKFK formed fibril bundles – believed to be a limited form of an aggregation effect frequently seen in similar peptides with neutral charge. As a result of this structural change, FEFQFKFK was found to have values for the elastic and viscous moduli (which are often used to measure the ‘strength’ of a gel) between 5 and 10 times larger than those of FEFKFKFK at the same concentration. The same behaviour was seen in FEFKFKFK when the monomer charges were reversed by adjusting pH, suggesting that the monomer charge is indeed responsible for the bundling effect. In chapter 5, two branched peptides were designed and synthesized: KG17, with two arms consisting of self-assembling FKFEFKFK-motifs, and KG28 which had three such arms. Each branched peptide was doped into pure FKFEFKFK and the resulting gels investigated. While no obvious structural changes were observed for either dopant (save for a potential fibril parallelisation effect with KG17 observed in Small-Angle Neutron Scattering (SANS)), both were observed to increase the elastic and viscous moduli of the gels at overall peptide concentrations of 30 and 50 mg mL-1 (gels), but not at 10 mg mL-1 (viscous liquid). The most dramatic change was observed in the 50 mg mL-1 gels, suggesting that higher concentrations could enhance the effect of the dopants. In chapter 6, three thermo-responsive polymers (pTEGMA), of Degrees of polymerisation (DPs) 17, 47 and 142 were conjugated to CGFKFEFKFK and incorporated into a peptide hydrogel. Gels containing the non-conjugated versions of each polymer were also tested. While no changes in morphology were observed at the fibillar level, the polymer Lower Critical Solution Temperature (LCST) behaviour could be observed in SANS in all samples apart from the DP17 conjugate. However, in rheological tests gels doped with this conjugate appeared to show the strongest the elastic and viscous moduli. In general the conjugates appeared to increase the elastic and viscous moduli, particularly at temperatures above ca. 50°C. Rather than this being LCST behaviour, it was suggested that the polymers can act to enhance a natural thermo-response that was observed in the peptide, with the shortest polymer (DP17) experiencing the least steric hindrance and therefore having the strongest effect. It was postulated that this interaction could involve the screening of charge on the peptide fibril. Non-conjugated polymer appeared to have little effect on the mechanical properties, with elastic modulus values correlating strongly to the overall peptide concentration.

L'insertion de couches minces d'oxyde métallique (MO) à l'interface entre les électrodes conductrices (FTO / ITO, Métaux) et la couche active (polymère, pérovskite) constitue une solution prometteuse pour améliorer les performances des dispositifs photovoltaïques organiques et hybrides. La procédure consiste à introduire des couches MO fonctionnalisées par des monocouches auto-assemblées dipolaires (SAMs) à l'interface entre l'électrode conductrice et la couche active. Les couches SAMs supportant des dipôles perpendiculaires à la surface peuvent avoir un impact important sur les dispositifs électroniques à la fois en affectant la croissance et l'organisation de la couche organique active et en accordant le travail de sortie des couches MO. Dans ce travail, nous montrons que le greffage des molécules dipolaires sur des couches minces de MO peut affecter considérablement les performances des cellules photovoltaïques. Cet impact dépend fortement de l'orientation du dipôle permanent situé sur la molécule SAM. / The insertion of very thin metal-oxide (MO) layers at the interface between the conductive electrodes (FTO/ITO, Metals) and the active layer (polymer, perovskite) presents a promising solution to improve the performances of organic and hybrid photovoltaic devices. The procedure is about introducing MO layers functionalized by dipolar self-assembling monolayer’s (SAMs) at the interface between the conductive electrode and the active layer. The SAM layers bearing dipoles perpendicular to the surface that can have a large impact on the electronic devices both by affecting the growth and organization of active organic layer and by tuning the work function of the MO layers. In this work we show that the grafting of dipole molecular on top of MO thin films can considerably affect the performance of the photovoltaic cell. The impact on these performances depends strongly on the orientation of the permanent dipole lying on the SAM molecule.

Thesis: M.S., Massachusetts Institute of Technology, Department of Mechanical Engineering, 1976 / MICROFICHE COPY AVAILABLE IN ARCHIVES AND ENGINEERING. At head of title: T-624. / Includes bibliographical references. / by Sergio N. Simunovic S. / M.S. / M.S. Massachusetts Institute of Technology, Department of Mechanical Engineering

The objective of the thesis is rationalization of current process of assembling of manifold assemblies from the reduction of operating costs point of view. The reason for rationalization is uncompetitiveness of actual solution due to high manufacturing cost of manifold assemblies coming from this process. After analysis of current operations, taking into account presumed production plan, the capacitive calculation of assembly line was done. Then alternative solutions were proposed based on above mentioned criterion, and from these the optimal variant was picked and developed. The assessment of resultant solution was done in the end.

Gerbstoffe stabilisieren die Kollagenmatrix der Haut, in dem sie auf unterschiedlichste Art und Weise chemische Quervernetzungen herstellen. Es bleibt jedoch bis heute weitgehend unbeantwortet, auf welcher hierarchischen Ebene der Kollagenstruktur diese Wechselwirkung stattfindet und wie stringent eine solche Bindung mindestens sein muss, um einer Substanz den Charakter eines Gerbstoffes zu verleihen. Im Rahmen der vorliegenden Arbeit wurde ein Modellsystem entwickelt, das es gestattet, Aussagen darüber zu treffen, auf welcher Strukturebene des Kollagens diese Wechselwirkung stattfindet. Dazu wurde auf ein „bottom-up“ Verfahren zurückgegriffen, bei dem der Gerbstoff nicht auf Haut aufgebracht, sondern die Fibrillen in Anwesenheit von verschiedenen Wirkstoffen neu gebildet werden. Für die Untersuchungen wurden Vertreter aus der Substanzklasse der Polymere ausgewählt. Es kam Polyacrylsäure zum Einsatz, die als Fettungsmittel genutzt wird, und Polymethacrylsäure, welche in der Produktion als Nachgerbstoff verwendet wird. Vertreter ungeladener Polymere waren Ethylen-, Diethylen- und Polyethylenglycol, wobei hier die unterschiedlichen Molekülgrößen im Vergleich von Bedeutung waren. Des Weiteren wurde Glutaraldehyd als Vertreter eines gerbenden kovalenten Vernetzers untersucht. Kollagen Typ I Fibrillen wurden in vitro ausgehend von der Monomerform assembliert, und mit UV/Vis-Spektroskopie wurde verfolgt, ob und gegebenenfalls wie die Polymere die Kinetik der in vitro Fibrillogenese verändern. Dabei wurde beobachtet, dass bis auf Polyethylenglycol alle eingesetzten Substanzen auf unterschiedlichste Art schon auf der kleinsten Kollageneinheit - der Tripelhelix wirken. Diese Daten wurden mittels eines mathematischen Modells ausgewertet, das es ermöglicht, die Fibrillogenese in Teilreaktionen zu gliedern und die geschwindigkeitsbestimmenden Schritte zu evaluieren. So konnte ermittelt werden, dass trotz ähnlich erscheinender Fibrillenbildungskinetiken große Unterschiede zwischen Polyacrylsäure und Polymethacrylsäure bezüglich ihres Hauptwirkortes in den hierarchischen Strukturebenen des Kollagens auftreten. Während der Nachgerbstoff Polymethacrylsäure schon in geringen Konzentrationen in großem Maße in alle Teilprozesse der Fibrillogenese eingreift, zeigt Polyacrylsäure die größten Effekte auf mikrofibrillärer Ebene - einer Fibrillensubstruktur. Dieser Einfluss spiegelt sich in Morphologieänderungen in Form von so genannten gesplitteten Fibrillen wider, welche mittels atomkraftmikroskopischen Untersuchungen beobachtet werden konnten. Zusätzlich scheint Polymethacrylsäure ab einer kritischen Konzentration die Fibrillenbildung über einen alternativen Weg ablaufen zu lassen, was sich in der morphologischen Betrachtung in Form von langen, dünnen Fibrillen äußert, welche nicht mehr in der Lage sind zu höheren Strukturen zu verdrillen. Um zusätzlich die Art der Bindung näher zu charakterisieren, wurde ein weiteres Verfahren entwickelt, welches die Umkehrreaktion der in vitro Fibrillogenese beschreibt. Diese Methode der so genannten Deassemblierung ermöglicht eine Unterscheidung zwischen elektrostatischen Wechselwirkungen und kovalenten Bindungen zwischen Kollagen und Polymer, indem die Fibrille wieder in ihre nativen Monomeruntereinheiten zerlegt und gleichzeitig studiert wird, ob sich die Polymere vom Kollagen durch Ladungseintrag lösen lassen. Polyethylenglycol und seine niedermolekularen Äquivalente lassen sich in saurem Milieu problemlos von Kollagen abwaschen, was für sehr schwache Wechselwirkungen spricht. Kovalente Bindungen, wie etwa zwischen Kollagen und Glutaraldehyd lassen sich mit dieser Methode nicht lösen. Polyacrylsäure und Polymethacrylsäure lassen sich nur zu einem geringen Anteil von Kollagen lösen, was auf unterschiedlich affine Bindungsplätze der Polymere an Kollagen deutet. Beeindruckender Weise ist es für ausschließlich mit Polymethacrylsäure vernetztes Kollagen nicht möglich, dieses wieder in seine Untereinheiten zu zerlegen. Polymethacrylsäure ist demnach in der Lage die Kollagenmatrix auch ohne die Ausbildung kovalenter Bindungen ausreichend zu stabilisieren. Damit können die eingesetzten und entwickelten Verfahren als Screeningmethoden gesehen werden, welche schon vor dem eigentlichen Gerbversuch weit reichende Auskünfte über ein mögliches Gerbverhalten der zu Untersuchung eingesetzten Substanz liefern. Die vorliegenden Ergebnisse erlauben Aussagen über den molekularen Charakter der Kollagen - Polymer Wechselwirkung und stellen somit einen Beitrag zum Verständnis der Gerbung dar.

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The interactions between cells and their microenvironment influence their morphological features and regulate important cellular processes. To understand deleterious neurological disorders such as Parkinson’s disease, there is an immense need to develop efficient in vitro 3D models that can recapitulate complex organs such as the brain. Ultrashort self- assembling peptides offer a revolutionary tool for generating tunable and well-defined 3D in vitro neural tissues capable of recreating complex cellular characteristics, and tissue-level responses. Herein, we describe the use of ultrashort self-assembling peptide-based scaffolds for the development of functional 3D neuronal models including an in vitro model for Parkinson’s disease. Both primary mouse embryonic dopaminergic neurons and human dopaminergic neurons derived from human embryonic stem cells were found biocompatible in our peptide-based models. Using microelectrode arrays, we recorded spontaneous activity in dopaminergic neurons encapsulated within these 3D peptide scaffolds for more than 1 month without a decrease in signal intensity. In addition, we demonstrate a 3D bioprinted model of dopaminergic neurons inspired by the mouse brain using an extrusion-based 3D robotic bioprinting technology. We used our 3D in vitro neuronal models to study the effect of both gabapentin and pregabalin on the development of dopaminergic neurons. Pregabalin and gabapentin are frequently regarded as first-line therapies for a variety of neuropathic pain syndromes, regardless of the underlying cause. Our results showed that both drugs can interfere with the neurogenesis and morphogenesis of ventral midbrain dopaminergic neurons during early brain development. Finally, to gain a better understanding of the influence of cell-cell and cell- matrix interactions on cellular behavior and function in 3D cultured cells within our peptide-based scaffolds compared to the ones cultured in 2D, we studied the metabolic and transcriptomic profiles of 2D and 3D cultured cells. 2D cultured cells exhibited distinct metabolic and transcriptomic profiles compared to the 3D cultured cells. Advancements in the fields of 3D in vitro modeling, 3D bioprinting, and biomaterials are of extreme value for the development of efficient models suitable for investigating disease-specific pathways, aiding the discovery of novel treatments, and promoting tissue regeneration.