The human genome reference sequence has given us a two‐dimensional blueprint of our inherited code of life, but we need to employ modern‐day technology to expand our knowledge into a third dimension. Inter‐individual and intra‐individual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNA‐sequencing platforms has increased a thousand‐fold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions. Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and cost‐efficient method for enrichment and titration of clonally amplified DNA‐libraries on beads. The method uses fluorescent labeling and a flow‐cytometer to separate DNA‐beads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sun‐exposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its well‐documented involvement in the disease progression of cancers, an estimated 35,000 novel sun‐induced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor protein‐altering mutations in multiple individuals.

This work comprises two main parts. The first part outlines our efforts to expand on the recent work of Gütlich et.al. by synthesizing Co(II) based spin crossover systems within a dendritic framework. We wanted to investigate the possibility of synthesizing different first generation, triazole containing dendrimers using “click” type reactions and their coordination ability with Co(II) ions. To this end we have had limited success mainly due to the numerous challenges in synthesizing a pure dendrimer product. The second part details our efforts in the synthesis of a mononuclear Co(II) based single molecule magnet. This comes as an extension to recent reports by Chang and Long where they have successfully obtained mononuclear Fe(II) single molecule magnets by inducing structural distortions within the complexes to amplify the spin-orbit coupling. We postulated that the use of Co(II) in conjunction with a bulky ligand framework would lead to desirable magnetic properties. We chose the known bis(imino)pyridine ligand scaffold due to its rich chemistry and its interesting and unexpected coordination behaviour, as we have seen in previous research efforts by our lab. To this end we were successful in isolating and characterizing 4 compounds, and we have carried out detailed magnetic measurements on the two most magnetically interesting species.

This work comprises two main parts. The first part outlines our efforts to expand on the recent work of Gütlich et.al. by synthesizing Co(II) based spin crossover systems within a dendritic framework. We wanted to investigate the possibility of synthesizing different first generation, triazole containing dendrimers using “click” type reactions and their coordination ability with Co(II) ions. To this end we have had limited success mainly due to the numerous challenges in synthesizing a pure dendrimer product. The second part details our efforts in the synthesis of a mononuclear Co(II) based single molecule magnet. This comes as an extension to recent reports by Chang and Long where they have successfully obtained mononuclear Fe(II) single molecule magnets by inducing structural distortions within the complexes to amplify the spin-orbit coupling. We postulated that the use of Co(II) in conjunction with a bulky ligand framework would lead to desirable magnetic properties. We chose the known bis(imino)pyridine ligand scaffold due to its rich chemistry and its interesting and unexpected coordination behaviour, as we have seen in previous research efforts by our lab. To this end we were successful in isolating and characterizing 4 compounds, and we have carried out detailed magnetic measurements on the two most magnetically interesting species.

<p>Ankyrin repeats (ARs) are polypeptide motifs identified in thousands of proteins. Many AR proteins play a function as scaffolds in protein-protein interactions which may require specific mechanical properties. Also, a number of AR proteins have been proposed to mediate mechanotransduction in a variety of different functional settings. The folding and stability of a number of AR proteins have been studied in detail by chemical and temperature denaturation experiments, yet the mechanic of AR proteins remain largely unknown. In this dissertation, we have researched the mechanical properties of AR proteins by using protein engineering and a combination of atomic force microscopy (AFM)-based single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations. Three kinds of AR proteins were investigated: NI6C (synthetic AR protein), D34 (of ankyrin-R) and gankyrin (oncoprotein). While the main focus of this research was to characterize the response of AR proteins to mechanical forces, our results extended beyond the protein nanomechanics to the understanding of protein folding mechanisms.</p> / Dissertation

Visualization and study of reconformation of polyelectrolytes (PEs) of different architecture is of great fundamental and practical interest. Verification of theoretical predictions with experiment is of essential importance. On the other hand, a wide range of bottom-up techniques based on patterning of matter on the length scale of a few nanometers have been recently developed. Particularly interesting is the possibility of using self-assembled single molecule structures as templates for the deposition of inorganic matter, in particular metals. Synthetic &amp;quot;normal-sized&amp;quot; polymers of various architecture, like poly-2-vinylpyridine (P2VP) or polystyrene-poly(2-vynil pyridine) P2VP7-PS7 star-like block copolymer, adsorbed on solid substrates have been visualized for the first time with molecular resolution by AFM in different conformation. This finding allowed us to study largely discussed problem, a coil-to-globule transition of PEs. It was found that PE molecules undergo conformational transitions from stretched worm-like coil to compact globule via set of necklace-like globules, as the fraction of charged monomers decreases with an increase of pH and ionic strength. These results are in good agreement with recently developed DRO theory for weakly charged flexible PEs in poor solvent. The size of the deposited single molecules correlates very well with molecular dimensions in solution obtained in light scattering experiments. PE single molecules of various architectures was mineralized in different conformations that constitutes the route to nanoparticles with desired shape (including wire-shape and star-shaped), size, and composition (including metallic, magnetic and semiconductive nanoparticles). It was shown that molecular details of the adsorbed linear flexible PE molecules determine the dimensions of the nanostructures after metallization and that observed sizes are consistent with the decoration of single molecules with nanoclusters. Thus those metallized nanoparticles (cluster assembles) reflect the conformation of original adsorbed PE molecules. The dimensions of the obtained nanowires are significantly smaller than those previously reported. All of these features are of the potential benefit in applications for nanodevices. Metallization of the PS7-P2VP7 improves AFM resolution due to the selective deposition of Pd clusters along the P2VP chains. For the first time, the number of the P2VP second generation arms of the heteroarm block-copolymer was directly counted in the single molecule AFM experiment. Simple contrasting procedure was developed to improve AFM visualization of positively charged polymer chains deposited on the substrates of relatively high roughness. This method allows increasing the thickness of the resulting structures up to 10 nm, and, consequently, provide visualization of polymer chains on rough surfaces. This innovation is important for the development of single molecule experiments with polymer chains. The reaction of HCF-anion could be used for recognition of polycation molecules, when polycations, polyanions and neutral molecules coexist on the surface. Recently, the study was strongly restricted to atomically smooth surfaces. The contrasting procedure extends the range of substrates (Si-wafers, chemically modified or patterned Si-wafers, polished glasses, polymer films, etc) appropriate for the experiments. Thus, polymer single molecules can be considered not only as representative of the ensemble molecules, but also as individual nanoscale objects which can be used for future nanotechnology for the fabrication of single molecule electronic devices. Also these findings are important from fundamental point of view, since developed approach can be successfully applied for investigation of various &amp;quot;classical&amp;quot; problems in polymer science, such as polymer reconformation, interpolyelectrolyte complex formation, polymer diffusion, adsorption, etc.

No description available.

Im Mittelpunkt dieser Arbeit steht die Verwendung verschiedener Fluoreszenzfarbstoffe in Form photostabiler Perylenbisimide sowie etallischer Nanopartikel zur Untersuchung von Polymeren und nanoskopischen Flüssigkeitsfilmen. Einzelmoleküluntersuchungen zeigen, dass eine chemische Modifizierung der Farbstoffe durch löslichkeitserhöhende Seitengruppen, Molekülkonformationen mit stark variierenden Emissionswellenlängen je nach Seitengruppen-orientierung zur Folge hat. Zeitabhängige Fluoreszenzmessungen an einzelnen Molekülen ermöglichen eine direkte Beobachtung von Übergängen zwischen diesen molekularen Konformationen deren Dynamik vorwiegend durch die Eigenschaften der umgebenden Polymermatrix bestimmt wird. Die gewonnenen Ergebnisse lassen somit Rückschlüsse auf die nanoskopische Umgebung des Moleküls zu. Es werden diskrete Zustände innerhalb der Molekülumgebung sowie eine erhöhte Konformationsdynamik im Falle von alkylsubstituierten Perylenbisimiden beobachtet. Darüber hinaus werden die nanoskopischen Auswirkungen von makroskopischen, mechanischen Deformationen auf amorphe Polymerfilme mikrorheologisch mit Hilfe von stäbchenförmigen Perylenbisimiden studiert. Die gewonnenen Einzelmoleküldaten ermöglichen die Berechnung der lokalen, mikroskopischen Deformation sowie der Orientierung der Sondenmoleküle, welche gut mit einem Model für stäbchenförmige Objekte in einem uniaxial deformierten Kontinuum übereinstimmt. In weiteren Experimenten gelingt der Nachweis ultradünner Wasserfilme auf SiO2-Oberflächen durch Messung der Diffusion von Silbernanopartikeln. Die verwendeten Nanopartikel weisen hierbei eine monodisperse Größenverteilung im Bereich von einem Nanometer als Resultat ihrer Synthese in Y-Zeolith-Kristallen auf. Die Untersuchungen ergeben eine Filmdickenabhängigkeit des Diffusionsverhaltens sowie einen starken Einfluss durch Oberflächensilanisierung bzw. Hydroxylierung.

In the first part of this thesis, we study the kinetics of the complexation of a double-stranded DNA byNCp7 protein. To do this, we study the evolution of mechanical properties of DNA and its complexation by stretching the DNA/NCp7 complex with a optical trap. We observed that the persistence length of the complex decreases progressively during the complexation. Using astatistical model we describe the evolution of the flexibility of DNA complexed with NCp7. Our main result is that the fraction phi of base pairs that have reacted is not a linear function of time at low phi.We interpret our results assuming that the adsorption of NCp7 on DNA is highly cooperative. In the second chapter, we describe the dynamics of probe particles in a colloidal glassy suspension of Laponite. Laponite is a colloidal discoidal particle of 25 nm in diameter and 0.92 nm thick. We take advantage of evanescent wave microscopy, and follow the movement of fluorescent latex particles.Then we image these particles. We show that for a movement that has a single characteristic time scale, it is simply a linear function of time. We find that, what ever their size, the motion of probe particles can be described by a succession of two dynamic modes, where the fastest mode corresponds to the diffusion of particles in a viscoelastic fluid.

Heterogeneous line broadening and spectral diffusion of the fluorescence emission spectra of perylene diimide molecules have been investigated by means of time dependent single molecule spectroscopy. The influence of temperature and environment has been studied and reveals strong correlation to spectral diffusion processes. We followed the freezing of the molecular mobility of quasi free molecules on the surface upon temperature lowering and by embedding into a poly(methyl methacrylate) (PMMA) polymer. Thereby changes of optical transition energies as a result of both intramolecular changes of conformation and external induced dynamics by the surrounding polymer matrix could be observed. Simulations of spectral fluctuations within a two-level system (TLS) model showed good agreement with the experimental findings.

This work comprises two main parts. The first part outlines our efforts to expand on the recent work of Gütlich et.al. by synthesizing Co(II) based spin crossover systems within a dendritic framework. We wanted to investigate the possibility of synthesizing different first generation, triazole containing dendrimers using “click” type reactions and their coordination ability with Co(II) ions. To this end we have had limited success mainly due to the numerous challenges in synthesizing a pure dendrimer product. The second part details our efforts in the synthesis of a mononuclear Co(II) based single molecule magnet. This comes as an extension to recent reports by Chang and Long where they have successfully obtained mononuclear Fe(II) single molecule magnets by inducing structural distortions within the complexes to amplify the spin-orbit coupling. We postulated that the use of Co(II) in conjunction with a bulky ligand framework would lead to desirable magnetic properties. We chose the known bis(imino)pyridine ligand scaffold due to its rich chemistry and its interesting and unexpected coordination behaviour, as we have seen in previous research efforts by our lab. To this end we were successful in isolating and characterizing 4 compounds, and we have carried out detailed magnetic measurements on the two most magnetically interesting species.