Mechanics and biological function of nucleic acids are intimately coupled. The DNA double helix must be opened to allow base pairing of RNA during transcription; RNA must bend and fold in its many cellular functions. Presented in this dissertation are two investigations of mechanical deformations of nucleic acids, conducted with optical tweezers.In the introduction, the mechanical properties of DNA and RNA and their relevance to their cellular functions are introduced, to give the reader context for the results presented in the Chapters 2 and 3. This is followed by an introduction to the theory of semiflexible polymer elasticity. The optical tweezers instruments used in conducting these investigations are then presented, along with calibration procedures and a short introduction to optical trapping physics.Chapter 2 presents an investigation of the effect of downstream DNA tension on initiation by T7 RNA polymerase. A hidden Markov model is fit to force-dependent lifetimes obtained from optical tweezers experiments, allowing us to identify which steps in initiation are force-dependent and estimate rates and transition state distances. We find that 1-2 pN of tension is sufficient to turn o gene expression by causing transcription bubble collapse and destabilizing the bound state. Our force-dependence scheme and estimated transition distances provide independent supportfor the \scrunching" model of initiation.The effects of cation binding and screening on single-stranded helix formation in poly(A) RNA are presented in Chapter 3. Magnesium and calcium bind to poly(A), stabilize the helix, and change its mechanical properties. A new model of helix-coil transitions is presented and used to estimate energetics and mechanical properties.Chapter 4 presents the first fully objective algorithm for use in analyzing the noisy staircaselike data that is often produced by single-molecule fluorescence experiments. A test based on the SIC (BIC) statistic is used in conjunction with a progressive step-placement scheme to locate changepoints (steps) in noisy data. Its performance is compared to other step detection algorithms in use by biophysicists by repeating tests performed in a recent review.Experimental protocols and computer codes used in these investigations are presentedin detail in the appendices.

Interfacial charge transfer between metal oxides and organic semiconductors has been found to limit the efficiency of organic optoelectronic devices. Although a number of investigations of inorganic/organic systems exist, very few generally applicable rules for oxide/organic interfaces have been developed and many questions about these systems remain unanswered. Thus the studies presented in this dissertation were designed to improve the understanding of the fundamental interface physics of metal oxide/organic systems. Single molecule fluorescence microscopy was employed to determine the charge transfer mechanism while photoelectron spectroscopy was used to determine the energy level alignment of model systems. Additional computational studies allowed the examination of the properties of the charged organic molecules involved in charge transfer and modeling of the molecule-surface interaction. Calculations of the ground state properties and excited state transitions of the neutral and singly charged states of a modified perylene molecule were performed to provide insight into the orbitals of the initial and final states involved in the interfacial charge transfer process. The design and implementation of a novel UHV single molecule microscope is described. This microscope was used to observe the excited state charge transfer between a modified perylene molecule and Al₂O₃ (0001). The charge transfer mechanism was identified as involving activated trapping and detrapping of the defect derived states within the Al₂O₃ band gap, which resulted in the observation of strongly distributed kinetics for this system. The influence of defects and adsorbates on the electronic structure of ZnO and its interface with organic semiconductors was determined from photoelectron spectroscopy. Modified perylene molecules were found to have strong chemisorptive interactions with the ZnO surface involving charge transfer from defect derived ZnO states to the LUMO, while magnesium phthalocyanine molecules appear to have only weak physisorptive interactions with the ZnO surface. The interfacial investigations of the organic/oxide systems demonstrate the rich defect structure present in metal oxides. In both cases, defects were found to control the interfacial interactions between the metal oxide surface and the modified perylene molecules. Thus the manipulation of these defects states is of fundamental importance for optoelectronic device design.

No description available.

The neural cell adhesion molecule L1 is a member of the immunoglobulin superfamily and it mediates many adhesive interactions during brain development. Mutations in the L1 gene are associated with a spectrum of X-linked neurological disorders known as CRASH or L1 syndrome. The objective of this thesis was to use the zebrafish model to investigate the molecular mechanisms of L1 functions and the pathological effects of its mutations. Zebrafish has two L1 homologs, L1.1 and L1.2. Inhibition of L1.1 expression by antisense morpholino oligonucleotides resulted in phenotypes that showed resemblances to L1 patients. However, knockdown of L1.2 expression did not result in notable neural defects. Furthermore, analysis of the expression pattern of L1.1 has led to the discovery of a novel soluble L1.1 isoform, L1.1s. L1.1s is an alternatively spliced form of L1.1, consisting of the first four Ig-like domains and thus a soluble secreted protein. L1.1 morphants exhibited disorganized brain structures with many having an enlarged fourth/hindbrain ventricle. Further characterization revealed aberrations in ventricular polarity, cell patterning and proliferation and helped differentiate the functions of L1.1 and L1.1s. While L1.1 plays a pivotal role in axonal outgrowth and guidance, L1.1s is crucial to brain ventricle formation. Significantly, L1.1s mRNA rescued many anomalies in the morphant brain, but not the trunk phenotypes. Receptor analysis confirmed that L1.1 undergoes heterophilic interactions with neuropilin-1a (Nrp1a). Peptide inhibition studies demonstrated further the involvement of L1.1s in neuroepithelial cell migration during ventricle formation. In the spinal cord, spinal primary motoneurons expressed exclusively the full-length L1.1, and abnormalities in axonal projections of morphants could be rescued only by L1.1 mRNA. Further studies showed that a novel interaction between the Ig3 domain of L1.1 and Unplugged, the zebrafish muscle specific kinase (MuSK), is crucial to motor axonal growth. Together, these results demonstrate that the different parts of L1.1 contribute to the diverse functions of L1.1 in neural development.

A crucial step in the biosynthesis of membrane proteins is their incorporation into the hydrophobic environment of the lipid bilayer. In eukaryotic cells this event occurs largely in concert with translation on ribosomes bound to the membrane of the endoplasmic reticulum (ER) at a site termed the ER translocon. This dynamic proteinaceous complex forms an aqueous conduit across the ER membrane and is laterally gated to allow transmembrane (TM) segments to partition into the lipid phase. In the case of polytopic membrane proteins, the coordinated release of multiple TM segments by the ER translocon is a poorly defined process and appears to be highly substratespecific. In this study, the ion channel subunit P2X2 was used as a novel model to examine themolecular details of membrane protein integration at the ER translocon. A primarily in vitro approach was taken using stable biosynthetic intermediates to simulate each stage of the membrane translocation and integration of P2X2. Chemical and photoreactive site-specific cross-linking analyses were then conducted to determine the molecular environment of the P2X2 TM segments throughout biosynthesis. Remarkably, both TM1 and TM2 of P2X2 were found to remain directly adjacent to the ER translocon throughout P2X2 biosynthesis and were only dislocated into the lipid phase by artificial termination of translation and disruption of the ribosome-translocon interaction. Retention of P2X2 TM1 at the ER translocon is maintained despite the synthesis of over 300 amino acid residues separating it from the ribosome peptidyl transferase centre. Premature dislocation of TM1 from the ER translocon site resulted in a pronounced aggregation of TM1 fragments both in vitro and in vivo. This is in stark contrast to previous passive-partitioning models of membrane integration and suggests that the ER translocon regulates the integration of polytopic membrane proteins in order to accommodate the specific requirementsof the substrate protein itself. The detailed characterisation of P2X2 biosynthesis was then exploited in order to examine the effect of a novel small inhibitor of ER translocon function. Eeyarestatin 1 (ESI) was found to cause a substantial inhibition of protein secretion in vivo and dramatically reduced the ER translocation of three distinct classes of substrate, including P2X2, in vitro. Using both a cross-linking analysis and a protease-protection assay for a specialised translocation reaction, ESI was shown to prevent the transfer of the nascent polypeptide chain from the membrane delivery machinery to the ER translocon complex. Further evidence that ESI targets the Sec61 complex is presented and a model for ESI-mediated inhibition of ER translocation is suggested. Taken together these data establish ESI as a novel small molecule inhibitor that selectively inhibits protein translocation both in vitroand in vivo.

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.

<p>Just as heat, light and electricity do, mechanical forces can also stimulate reactions. Conventionally, these processes - known as mechanochemistry - were viewed as comprising only destructive events, such as bond scission and material failure. Recently, Moore and coworkers demonstrated that the incorporation of mechanophores, i.e., mechanochemically active moieties, can bring new types of chemistry. This demonstration has inspired a series of fruitful works, at both the molecular and material levels, in both theoretical and experimental aspects, for both fundamental research and applications. This dissertation evaluates mechanochemical behavior in all of these contexts. </p><p>At the level of fundamental reactivity, forbidden reactions, such as those that violate orbital symmetry effects as captured in the Woodward-Hoffman rules, remain an ongoing challenge for experimental characterization, because when the competing allowed pathway is available, the reactions are intrinsically difficult to trigger. Recent developments in covalent mechanochemistry have opened the door to activating otherwise inaccessible reactions. This dissertation describes the first real-time observation and quantified measurement of four mechanically activated forbidden reactions. The results provide the experimental benchmarks for mechanically induced forbidden reactions, including those that violate the Woodward-Hoffmann and Woodward-Hoffmann-DePuy rules, and in some cases suggest revisions to prior computational predictions. The single-molecule measurement also captured competing reactions between isomerization and bimolecular reaction, which to the best of our knowledge, is the first time that competing reactions are probed by force spectroscopy. </p><p> Most characterization for mechanochemistry has been focused on the reactivity of mechanophores, and investigations of the force coupling efficiency are much less reported. We discovered that the stereochemistry of a non-reactive alkene pendant to a reacting mechanophore has a dramatic effect on the magnitude of the force required to trigger reactivity on a given timescale (here, a 400 pN difference for reactivity on the timescale of 100 ms). The stereochemical perturbation has essentially no measurable effect on the force-free reactivity, providing an almost perfectly orthogonal handle for tuning mechanochemical reactivity independently of intrinsic reactivity. </p><p>Mechanochemical coupling is also applied here to the study of reaction dynamics. The dynamics of reactions at or in the immediate vicinity of transition states are critical to reaction rates and product distributions, but direct experimental probes of those dynamics are rare. The s-trans, s-trans 1,3-diradicaloid transition states are trapped by tension along the backbone of purely cis-substituted gem-difluorocyclopropanated polybutadiene using the extensional forces generated by pulsed sonication of dilute polymer solutions. Once released, the branching ratio between symmetry-allowed disrotatory ring closing (of which the trapped diradicaloid structure is the transition state) and symmetry-forbidden conrotatory ring closing (whose transition state is nearby) can be inferred. Net conrotatory ring closing occurred in 5.0 ± 0.5% of the released transition states, as compared to 19 out of 400 such events in molecular dynamics simulations.</p><p>On the materials level, the inevitable stress in materials during usage causes bond breakage, materials aging and failure. A strategy for solving this problem is to learn from biological materials, which are capable to remodel and become stronger in response to the otherwise destructive forces. Benzocyclobutene has been demonstrated to mechanically active to ortho-quinodimethide, an intermediate capable for [4+4] dimerization and [4+2] cycloaddition. These features make it an excellent candidate for and synthesis of mechanochemical remodeling. A polymer containing hundreds of benzocyclobutene on the backbone was synthesized. When the polymer was exposed to otherwise destructive shear forces generated by pulsed ultrasound, its molecular weight increased as oppose to other mechanophore-containing polymers. When a solution of the polymer with bismaleimide was subjected to pulsed ultrasonication, crosslink occurred and the modulus increased by two orders of magnitude.</p> / Dissertation

No description available.

Solid-state dye-sensitized solar cells (ssDSSCs) and recently developed perovskite solar cells (PSCs) have attracted a great attention in the scientific field of photovoltaics due to their low cost, absence of solvent, simple fabrication and promising power conversion efficiency (PCE). In these types of solar cell, the dye molecule or the perovskite can harvest the light on the basis of electron excitation. Afterwards, the electron and hole are collected at the charge transport materials. Photoelectrochemical polymerization (PEP) is employed in this thesis to synthesize conducting polymer hole transport materials (HTMs) for ssDSSCs. We have for the first time developed aqueous PEP in comparison with the conventional organic PEP with acetonitrile as solvent. This water-based PEP could potentially provide a low-cost, environmental-friendly method for efficient deposition of polymer HTM for ssDSSCs. In addition, new and simple precursors have been tested with PEP method. The effects of dye molecules on the PEP were also systematically studied, and we found that (a) the bulky structure of dye is of key importance for blocking the interfacial charge recombination; and (b) the matching of the energy levels between the dye and the precursor plays a key role in determining the kinetics of the PEP process. In PSCs, the HTM layer is crucial for efficient charge collection and its long term stability. We have studied different series of new molecular HTMs in order to understand fundamentally the influence of alkyl chains, molecular energy levels, and molecular geometry of the HTM on the photovoltaic performance. We have identified several important factors of the HTMs for efficient PSCs, including high uniformity of the HTM capping layer, perovskite-HTM energy level matching, good HTM solubility, and high conductivity. These factors affect interfacial hole injection, hole transport, and charge recombination in PSCs. By systematical optimization, a promising PCE of 19.8% has been achieved by employing a new HTM H11. We believe that this work could provide important guidance for the future development of new and efficient HTMs for PSCs.

We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett. 2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-lM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.