The search for a satisfactory model for complexity, meant as an intermediate condition between total order and total disorder, is still subject of debate in the scientific community. In this dissertation the emergence of non-Poisson renewal processes in several complex systems is investigated. After reviewing the basics of renewal theory, another popular approach to complexity, called modulation, is introduced. I show how these two different approaches, given a suitable choice of the parameter involved, can generate the same macroscopic outcome, namely an inverse power law distribution density of events occurrence. To solve this ambiguity, a numerical instrument, based on the theoretical analysis of the aging properties of renewal systems, is introduced. The application of this method, called renewal aging experiment, allows us to distinguish if a time series has been generated by a renewal or a modulation process. This method of analysis is then applied to several physical systems, from blinking quantum dots, to the human brain activity, to seismic fluctuations. Theoretical conclusions about the underlying nature of the considered complex systems are drawn.

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Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes the application of single molecule spectroscopy and tracking to investigations of the nanoscale properties of thin-film chemical gradients and the transport dynamics of molecules dispersed within and upon these gradients. Chemical gradients are surface bound materials that incorporate gradually changing chemical and/or physical properties. A continuous and gradual change in the properties of gradients are expected and often required for their intended applications, which range from directed growth of cell colonies to combinatorial materials science. In reality, such conditions are almost never met due to spontaneous demixing and dewetting processes that can lead to properties variations on microscopic length scales. A better understanding on the properties of chemical gradients on microscopic length scales will aid in the production of better engineered materials. Single molecule spectroscopy (SMS) allows for gradient properties to be probed on nanometer-to-micrometer length scales. In this dissertation, quantitative measurements of gradient polarity (i.e., dielectric properties) are made along a sol-gel derived thin film that incorporates a macroscopic polarity gradient. These measurements report on the microscopic heterogeneity of the gradient film, and point to the occurrence of phase separation of the polar and nonpolar components along the gradient. Single molecule tracking (SMT) provides an important means to examine the dynamics of molecular mass transport in thin films and on surfaces. In this dissertation, SMT is employed to study mass transport in thin water films condensed over monolayer wettability gradients under ambient environments. The results show that the rate and the mechanism of molecular transport depend on the surface wettability, and on the ambient relative humidity. Finally, wettability gradients have been broadly used to drive the transport of liquid droplets. In this dissertation, these applications are extended to achieve spontaneous stretching of DNA by the propulsion of liquid droplets along the gradient. Single molecule fluorescence imaging of DNA stretched along these gradients demonstrates that hydrophobic surfaces play an important role in DNA stretching. The study also shows the surface tension force acting at the gradient-droplet contact line (interface) to be responsible for DNA elongation and alignment. Overall, single molecule methods have been shown to be highly useful for better understanding the properties of chemical gradients as described in this dissertation.

Doctor of Philosophy / Department of Chemical Engineering / Keith L. Hohn / The autothermal reforming and partial oxidation of hexadecane via Pt/Ni bimetallic nanoparticles on various ceria-based supports were investigated. Nanoparticles with Pt/Ni molar ratios ranging from 0/100 to 10/90 were loaded on ceria-based supports including cerium oxide, gadolinium-doped cerium oxide and cerium-doped zirconium oxide. The effect of the Pt/Ni molar ratio and the promotional effect of the support were studied by comparing the hydrogen yield. TPR and XPS analysis showed that there was a strong interaction between Ni and the CeO₂-ZrO₂ support, which led to enhancement of catalyst performance when the Pt/Ni ratio was low. The strong interaction between Ni and CeO₂-ZrO₂ support was induced by the formation of a solid solution between NiO and ZrO₂. In the case of bimetallic catalysts loaded on Gd₂O₃-CeO₂, no significant improvement in the catalytic activity of autothermal reforming was achieved until the Pt/Ni ratio reached 10/90. With C-snarf-1 as a pH-sensitive fluorescent probe, the local acidity on the surface of gradient aluminosilicate thin films and in the pore structure of mesoporous silicate films was explored. The single molecule emission ratio (I₅₈₀/I₆₄₀) of C-snarf-1 on the gradient aluminosilicate films showed similar results as previously reported for aluminosilicate mesoporous films. As the Al/Si ratio increases, the emission ratio declines, indicative of increased material acidity. In the case the mesoporous silicate films, much broader distributions of emission ratios were observed and are suggestive of significant heterogeneity in the pore structure of these films. The average emission ratio increased with a rise in pH until pH 6 or 7. A further rise in pH leads to a decline in emission ratio. Molecules with high mobility showed a narrow distribution and slightly lower average emission ratio when compared to data from all detected molecules. This observation implies a reduced heterogeneity for mesopores in which the molecules rapidly diffuse. The narrow distribution and lower average value of emission ratio at low pH, combined with the decrease in emission ratio induced by an increase in ionic strength may further indicate that the interaction between dye molecules and the pore surface impacts the emission ratio of the dye molecules.

Nanoporous materials have been widely used in the fields of biological and chemical sensing, chemical separation, heterogeneous catalysis and biomedicine due to their merits of high surface area-to-volume ratio, chemical and thermal stabilities, and flexible surface modification. However, as the nature of nanoporous materials, they are inherently heterogeneous in the micro- and nanoenvironments. The environmental heterogeneity plays a decisive role in determining the performance of various applications of nanoporous materials. In order to provide an in-depth understanding of the nanoporous materials, it is of great interest to investigate the environmental heterogeneity in them. Single molecule spectroscopy, combined the quantitative confocal fluorescence imaging which possesses the capability of optical sectioning, has demonstrated to be a powerful tool to approach the environmental heterogeneity inside nanoporous materials. Single molecule spectroscopy is an ultrasensitive technique for probing molecular transport and properties of individual molecules. This technique has been extensively used in the research of environmental heterogeneity in nanoporous materials since it removes the issues of ensemble averaging and directly approaches detailed information that is obscured in ensemble measurements. In order to proficiently interpret single molecule data, we developed a comprehensive methodology – single molecule counting – for characterizing molecular transport in nanoporous silica. With this methodology as a tool, the nanoenvironmental heterogeneity inside the nanopores of C18-derivatized silica particles was explored by probing single molecular diffusion inside the pores. By employing single molecule ratiometric spectroscopy and a solvatochromic fluorophore as viii reporter of local environment, the gradient in nanopolarity as well as the nanoviscosity along the C18 layer after the inclusion of solvent was uncovered. The chemical properties of solute molecules at the nanopore surface are ultimately controlled by the energetics of the solute-interface interactions. The imaging of distribution of energies would be a decisive approach to assess the fundamental heterogeneity of the interface. To this end, we investigated the ΔG distribution of C18-derivatized nanoporous silica particles with quantitative confocal imaging. The pixel-to-pixel and particle-to-particle analysis showed the existence of ΔG heterogeneity between particles as well as within individual particles. The heterogeneity in ΔG could be partially responsible for band broadening in chemical separations and significantly affect overall reaction yield when using nanoporous materials as solid support for heterogeneous catalysis.

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In dieser Dissertation werden die Bindungseigenschaften von Rezeptor-Ligand-Komplexen mit Hilfe von Optischen Pinzetten untersucht. Aufgrund ihrer außerordentlichen Orts- (2nm) und Kraftauflösung (0,2pN) ist es möglich, diese spezifischen Interaktionen anhand einzelner Bindungsereignisse zu charakterisieren. Als Modellsysteme dienen die Wechselwirkungen zwischen den phosphorylierungsspezifischen, monoklonalen Antikörpern HPT-101 und HPT-104 und dem Morbus Alzheimer relevanten Tau-Peptid. Dieses pathogen veränderte Peptid wird krankheitsspezifisch an den Aminosäuren Threonin231 und Serin235 phosphoryliert, sodass die Detektion dieses Phosphorylierungsmusters mit Hilfe von monoklonalen Antikörpern eine mögliche Früherkennung der Alzheimer-Krankheit darstellt. Eine notwendige Voraussetzung dafür ist jedoch die exakte Kenntnis der Bindungsstellen des Liganden am Rezeptor. Ziel des ersten Teils dieser Arbeit ist es, das Epitop des monoklonalen Antikörpers HPT-101 zu bestimmen. Dazu werden mögliche bindungsrelevante Aminosäuren durch ein Alanin ausgetauscht (Alanin-Scan) und so insgesamt sieben neue Tau-Isoformen aus dem ursprünglichen doppelt-phosphorylierten Peptid Tau[pThr231/pSer235] hergestellt. Die jeweiligen Interaktionen zwischen den modifizierten Peptiden und dem Antikörper werden mit der dynamischen Kraftspektroskopie untersucht und mit Hilfe eines literaturbekannten Modells analysiert. Die sich daraus ergebenden Bindungsparameter (Lebensdauer der Bindung, charakteristische Bindungslänge, freie Aktivierungsenergie und Affinitätskonstante) werden zusammen mit den relativen Bindungshäufigkeiten erstmals genutzt, um Kriterien für essentielle, sekundäre und nicht-essentielle Aminosäuren im Tau-Peptid zu definieren. Bemerkenswerterweise existieren für insgesamt drei dieser Parameter (Bindungslebensdauer, Bindungslänge und Affinitätskonstante) scharfe Klassengrenzen, mit denen eine objektive Einteilung des Epitops von Antikörper HPT-101 möglich ist. Die erhaltenen Ergebnisse sind in überzeugender Weise im Einklang mit ELISA-Messungen zu diesem Antikörper-Peptid-Komplexen, sie liefern jedoch einen tieferen Einblick in die Natur einer spezifischen Bindung, da den kraftspektroskopischen Messungen auch die Bindungskinetik zugänglich ist. Das zweite Projekt der vorliegenden Dissertation etabliert eine Methodik, um die Datenvarianz in der Bestimmung der relativen Bindungshäufigkeit zu reduzieren. Anhand einer Kombination aus Fluoreszenz- und kraftspektroskopischen Messungen werden die Wechselwirkungen zwischen dem monoklonalen Antikörper HPT-104 und dem fluoreszenzmarkierten Peptid Tau[Fl-pThr231] untersucht. Es wird gezeigt, dass durch Vorsortieren der Peptid-beschichteten Kolloide, entsprechend ihrer Oberflächenbeladung, die Datenvarianz in den Bindungshäufigkeitsmessungen signifikant reduziert wird.

This thesis describes the development of sensitive and high-resolution fluorescence spectroscopic and microscopic techniques and their application to probe biomolecules and their interactions in solution, lipid membrane model systems and in cells. Paper I-IV are largely focused on methodological developments. In paper I, a new fluorescence method based on fluorescence correlation spectroscopy (FCS) for detecting single particles was realized, requiring no fluorescent labeling of the particles. The method can yield information both about the diffusion properties of the particles as well as about their volumes. In paper II, a modified fluorescence cross correlation spectroscopy procedure with well characterized instrumental calibration was developed and applied to study cis interactions between an inhibitory receptor and its Major Histocompatibility Complex class I ligand molecule, both within the same cellular membranes. The quantitative analysis brought new insights into the Nature killer cell’s self-regulating of tolerance and aggressiveness for immune responses. Paper III describes a multi-color STED (STimulated Emission Depletion) microscopy procedure, capable of imaging four different targets in the same cells at 40nm optical resolution, which was developed and successfully demonstrated on platelets. In paper IV, a modified co-localization algorithm for fluorescence images analysis was proposed, which is essentially insensitive to resolutions and molecule densities. Further, the performance of this algorithm and of using STED microscopy for co-localization analysis was evaluated using both simulated and experimentally acquired images. Papers V-VII have their main emphasis on the application side. In paper V, transient state imaging was demonstrated on live cells to image intracellular oxygen concentration and successfully differentiated different breast cancer cell lines and the different metabolic pathways they adopted to under different culturing conditions. Paper VI describes a FCS-based study of proton exchange at biological membranes, the size-dependence of the membrane proton collecting antenna effect as well as effects of external buffer solutions on the proton exchange, in a nanodisc lipid membrane model system. These findings provide insights for understanding proton transport at and across membranes of live cells, which has a central biological relevance. In paper VII, STED imaging and co-localization analysis was applied to analyze cell adhesion related protein interactions, which are believed to have an important modulating role for the proliferation, differentiation, survival and motility of the cells. The outcome of efforts taken to develop means for early cancer diagnosis are also presented. It is based on single cells extracted by fine needle aspiration and the use of multi-parameter fluorescence detection and STED imaging to detect protein interactions in the clinical samples. Taken together, detailed studies at a molecular level are critical to understand complex systems such as living organisms. It is the hope that the methodologies developed and applied in this thesis can contribute not only to the development of fundamental science, but also that they can be of benefit to mankind in the field of biomedicine, especially with an ultimate goal of developing novel techniques for cancer diagnosis. / <p>QC 20140609</p>

ROLE ASOVÉ 'KÁLY INTERAKCE SYSTÉM-LÁZE VE FOTOSYNTETICKÉM P ENOSU EXCITANÍ ENERGIE Tato práce se věnuje vlivu rychlého a pomalého molekulárno pohybu na přenos excitační energie ve fotosyntetick- ých světlosběrných komplexech. Vyvinuli jsme nový teoretický popis vnitromolekulárních vibračních mod· a zjistili jsme, že jejich resonance s energetickými rozdíly mezi fotosyntetickými pigmenty m·že vést ke zrychlení přenosu energie. Použitím jednomolekulární spektroskopie jsme pozorovali jak pomalé změny bílkovinné konformace mohou zcela změnit stav světlosběrného komplexu LHCII vyšších rostlin. Také jsme vyvinuli novou experimentální techniku, dvoupulzní ultrarychlou jednomolekulární spektroskopii. S její pomocí m·žeme pozorovat jak pomalý pohyb bílkoviny bakteriální antény LH2 ovlivňuje ultrarychlou relaxaci energie uvnitř komplexu. Konstrukcí jednotného modelu pro ultrarychlé objemové a jednomolekulární experimenty se nám podařilo zakomponovat rychlou a pomalou časovou škálu molekulárního pohybu do jednoho pohledu na fotosyntetický sběr světla.