Nanopores have emerged as a simple but effective tool to investigate the behavior of polymers in solution. They have shown great potential to simplify expensive and time consuming procedures like DNA sequencing, protein detection, and disease biomarker detection. With the development of in situ fabrication of solid-state nanopores by controlled breakdown (CBD) of a dielectric material, nanomanufacturing of nanopore-based technologies became feasible. However, there are still a lot of challenges to overcome for these applications to become reality. One of the major problems with solid-state nanopores is the rapid passage time of analytes going through the pore, complicating detection and reliable identification of molecules. In this thesis molecular structures are proposed that increase passage times due to increased interactions between analyte and pore wall, and at the same time increase signal amplitude due to increased blockage of the pore. These structures are short, branched DNA molecules that were assembled with built-in modifications and matching sequences to assume their structure. Nanopore experiments reveal that these structurally defined DNA produce higher detection rates than their linear DNA counterparts, making them better candidates for labels in single-molecule sensing experiments.

Riboswitches are regulatory structures present in the 5′-UTR of a wide range of bacterial mRNAs. They consist of a small-molecule binding aptamer domain, which affects the conformation of a nearby expression platform to control gene expression through a transcriptional or translational mechanism. Because of their ability to bind selectively to very small concentrations of ligand, in a protein-independent manner, they have great potential for use as novel small-molecule controllable gene expression systems. This thesis describes how a combination of chemical genetics and genetic selection were used to develop and test a novel riboswitch-based gene-expression system. Several constructs were generated which could respond in vivo to a variety of non-natural small heterocyclic compounds and output via a simple fluorescence based assay in a dose-dependent manner. Methods for controlling the overall protein expression landscape of the riboswitch-based gene-expression system are outlined. In addition, the rational design of mutant riboswitch aptamers with improved ligand-binding capabilities is described alongside attempts to modulate the structural stability of the expression platform. Riboswitches need to be highly discriminatory to function effectively in vivo, binding to one ligand from a cellular pool of thousands. Mutant riboswitches were created that responded specifically to the ligands ammeline or azacytosine, and were found to have no induction in the presence of adenine, the wild-type riboswitch ligand. This in vivo ligand orthogonality was confirmed by subsequent in vitro studies. The ligand-induced structural changes undertaken by the mutant riboswitch aptamer domains were subsequently characterised using a variety of in vitro methods including SHAPE, ITC and x-ray crystallography. Finally, the feasibility of using riboswitch gene-expression systems in fully synthetic applications was demonstrated through the construction and analysis of small synthetic gene clusters and operons. The in vivo expression of two fluorescent proteins under independent riboswitch control was studied under single and dual induction for a range of ligand concentrations. The ability to control the expression of multiple genes is highly desirable in the emerging field of synthetic biology, the results described here indicate that riboswitches are ideally suited to complement current gene expression tools.

L’athérosclérose est une maladie inflammatoire chronique de la paroi artérielle, caractérisée par une infiltration des leucocytes consécutive à l’accumulation de lipoprotéines au sein de l’intima. Les molécules d'adhésion jouent un rôle important dans la progression de l’athérosclérose par leur implication dans l’infiltration des leucocytes au niveau du site de lésion. CD146 est une molécule d’adhésion présente sur les cellules endothéliales et sur certaines populations leucocytaires. Il a été récemment montré une augmentation de sa forme soluble lors de l’athérosclérose et de ses complications. De plus, son expression augmente au sein des plaques d’athérosclérose. En revanche, son implication dans la formation de la plaque d’athérosclérose n’a jamais été étudiée.Nous avons évalué le rôle in vivo de CD146 grâce au modèle murin APOEKO/CD146KO. Nous montrons que CD146 joue un rôle protecteur dans l’athérosclérose en inhibant la sécrétion de la chimiokine RANTES. La régulation de RANTES médiée par CD146 est dépendante de la voie VEGFR2-p38/MAPK. Dans une seconde étude, nous montrons que l’implication de CD146 dans l’athérosclérose est dépendante du régime alimentaire. Enfin dans une étude pilote, nous proposons une nouvelle stratégie thérapeutique de l’athérosclérose via l’injection de microvésicules CD146+. Nous montrons que l’apport du CD146 membranaire conduit à une réduction de RANTES, des neutrophiles circulants et de la plaque d’athérosclérose.En conclusion, l’ensemble de ces travaux apporte de nouvelles données dans la compréhension de la pathologie de l’athérosclérose en identifiant CD146 comme une nouvelle cible thérapeutique dans le traitement de l’athérosclérose. / Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the leukocyte infiltration consecutive to the accumulation of lipoproteins within the intima. Adhesion molecules play an important role in the progression of atherosclerosis since they are involved in the leukocyte infiltration at the lesion site. CD146 is an adhesion molecule detected on all endothelial cells of the vascular tree and also expressed on some subpopulations of leukocytes. Recently, few studies have shown that soluble form of CD146 is increased in atherosclerosis and its clinical complications. In addition, CD146 expression increases in atherosclerotic plaque. However, its involvement in atherosclerotic plaque formation has never been investigated. We evaluate the in vivo role of CD146 thanks to APOE KO/CD146 KO mice model. We demonstrate that CD146 plays a protective role in atherosclerosis by inhibiting the secretion of the RANTES chemokine responsible for neutrophils and monocytes recruitment within atherosclerotic plaque. RANTES regulation mediated by CD146 is dependent on VEGFR2-p38/MAPK pathway. In a second study, we show that the involvement of CD146 in atherosclerosis is dependent on the diet. Finally, in a pilot study, we propose a new therapeutic strategy for atherosclerosis through the injection of CD146 + microvesicles. We demonstrate that bringing CD146 membrane isoform through microvesicles leads to a reduction of RANTES, circulating neutrophils and atherosclerotic plaque.Overall, this work provides advances in atherosclerosis for a better understanding of its mechanisms by identifying CD146 as a new therapeutic target in the treatment of atherosclerosis.

Master of Science / Department of Chemistry / Takashi Ito / Solvent swelling of block copolymer microdomains plays an essential role in the improvement of microdomain alignment by solvent vapor annealing and in chemical separations using block copolymer monoliths. Here, investigation of the effects of solvent swelling on the molecular permeability and dimensions of cylindrical microdomains in polystyrene-block-poly(ethylene oxide) (PS-b-PEO) films is done by using single molecule tracking. These films are prepared by sandwiching benzene (with/without methanol) or THF (with/without methanol) solutions containing 5 nM sulforhodamine B (SRB) between two glass substrates. The PEO microdomains are aligned in the solution flow direction during the film preparation. The diffusional motions of individual SRB molecules are measured at different drying times to assess the microdomain radius and permeability. These parameters, on average, gradually decrease with an increase in drying time; however the trend differs slightly from one solvent system to another. A sharp decrease of microdomain radius is observed for benzene, benzene-methanol, THF and THF-methanol swollen films at initial drying condition (for example 2 days). In contrast, microdomain permeability does not decrease sharply; instead a gradual decreasing trend is seen for all solvent systems. In addition, mixing of a small amount of methanol (14% in PEO microdomains) either with benzene or with THF does not produce noticeable difference in the swelling of PEO microdoamins. Importantly, both benzene and THF offer similar microdomain swelling behavior at the same drying temperature, which is evident from the microdomain radius values, however THF shows comparatively larger microdomain permeability and better correlation between permeability and microdomain radius compared with benzene.

DNA serves as a major target for mainstream drugs used in the treatment of cancer, but current DNA-targeted drugs have significant issues due to their poor selectivity giving rise to adverse effects. Recent research on targeting DNA has focused on DNA interactive compounds with novel mechanisms of action and new cancer-related DNA molecular targets. An understanding of molecular level details of small molecule interactions with their DNA targets is critical for understanding the molecular mechanisms of action and for structure-based rational drug design. This dissertation presents two studies focused on gaining a structural understanding of DNA-targeted small molecules, one with a novel mechanism of action and the second with a cancer-specific DNA molecular target. XR5944 is potent anticancer drug and a novel mechanism of action, DNA bis-intercalation with a major groove binding. It is able to recognize and bind the estrogen response element (ERE) sequence via the major groove to inhibit estrogen receptor-α activity. This mechanism of action may be useful for overcoming drug resistance to currently available antiestrogen treatments for breast cancer, all of which target the hormone-receptor complex. We determined the nuclear magnetic resonance solution structure of the 2:1 complex of XR5944 with the naturally occurring TFF1-ERE, which exhibits important and unexpected features. In the determined structure, each bis-intercalating XR5944 molecule is strongly bound at one of its intercalating site, but weakly at the other. Our results show the sites of intercalation within a native promoter sequence appear to be context and sequence dependent. The binding of one drug molecule influences the binding site of the second. The structure underscores the fact that the DNA binding of a bis-intercalator is directional and differs from the simple addition of two single intercalation sites. Our results provide insights toward future structure-based rational drug design of DNA bis-intercalators to modulate ERα-induced transcriptional activity, as well as for designing bis-intercalators with major groove binding modes in general. Human telomeric DNA G-quadruplex secondary structures have emerged as an attractive molecular target for anticancer drugs. G-quadruplex formation in human telomeres inhibits telomerase, which plays a key role in maintaining the malignant phenotype by stabilizing telomere length and integrity. Under physiologically relevant conditions, human telomeric DNA sequences form two equilibrating G-quadruplex structures, with the hybrid-2 structure being the predominant in an extended sequence Thus, the hybrid-2 human telomeric G-quadruplex is considered to be a potential target for anticancer drugs targeting telomere biology and telomerase. We discovered that epiberberine, a naturally occurring isoquinoline alkaloid, can specifically bind the hybrid-2 telomeric G-quadruplex and induce the conversion of hybrid-1 telomeric G-quadruplex to the hybrid-2 structure. We determined the structure of the hybrid-2 G-quadruplex in complex with epiberberine by NMR in K⁺ solution. This NMR solution structure shows an unexpected, large, drug-induced conformational change in the flanking and loop regions, creating a very well-defined “induced intercalated quasi-triad pocket” with an extensive capping structure. Our result demonstrates the importance of ligand shape as well as the G-quadruplex folding topology and flanking and loop sequences in small molecule targeting the intramolecular hybrid-2 human telomeric G-quadruplex. Our result also indicates that asymmetric compounds containing a crescent-shaped moiety are more likely to bind in a specific manner to an intramolecular G-quadruplex.

An increasing interest in non-canonical nucleic acid structures has drawn the attention of the scientific community during the last few decades. One such structure, the G-quadruplex, has been the focus of an increasing number of scientists as G-quadruplexes are believed to play a role in biological processes such as telomere integrity and gene expression. Their existence in vivo is largely unproven but they have stimulated a lot of research into small molecules that interact with them. The development of a new class of such molecules is described in this thesis. A member of this family showed to be very selective in stabilising one particular G-quadruplex. The further development of another family of G-quadruplex interacting small molecules is also presented in this thesis and some of their effects in cellulo were assessed. Based on the scaffold of this family, an affinity probe was developed, which can mediate the isolation of its nucleic acid targets from human cells. This is the first example of the use of a small molecule with an affinity tag that has been used to isolate a nucleic acid target in a structure specific manner from human cells.

This thesis describes the synthesis, characterization and magnetic investigation of homometallic lanthanide complexes based on two different o-vanillin-derived Schiff base ligands: H2ovph and H2ovgrd. The studies were performed using single crystal X-ray diffractometry, Powder XRD and SQUID magnetometry. Chapter 2 focuses on dinuclear systems 1-8 coordinated to the ligand H2ovph and presents their structural and magnetic properties, mainly with respect to their intramolecular interactions. Chapter 3 describes two hexanuclear systems, 9 (DyIII) and 10 (GdIII), with trigonal prism-assembled core structures. A structural comparison to other similar complexes in the literature is performed. A series of dinuclear complexes, 11-15, based on the ligand H2ovgrd are described in Chapter 4, focusing on the synthetic strategy, crystal structures and magnetism. The presence of the lanthanide contraction is evident in this system of complexes and is consistent with the intrinsic lanthanide contraction property.

This thesis will describe the magnetic behavior found in a scaling array of magnetic nanomaterials that have been uniquely designed, synthesized and characterised in order to better understand their properties with regards to potential future applications. Within Chapter 1 will be a detailed, yet accessible, introduction to nanomagnetism and the fundamental principles and practical techniques essential to the study of this unique mélange of physics and chemistry. This chapter will be designed to give the reader the necessary tools to understand key literature concepts found in Chapter 2, as well as the work presented in the following chapters. Chapter 2 will provide an overview of relevant literature in the field of magnetic nanomaterials, including: nanoparticles, single-molecule magnets, single-chain magnets and metal-organic frameworks. Chapter 3 will describe work performed on nanoparticles doped with lanthanide ions in order to explore their resulting size, shape, crystallinity and magnetic properties. The relevance of the chosen particles (NaYF4) pertains to their proposed use in a variety of applications due to their known luminescent properties, which we sought to hybridize with interesting magnetic properties, thus creating multimodal imaging capabilities. Doping with a variety of desired ratios of lanthanide ions (GdIII, TbIII, DyIII, ErIII and YbIII) was successful, producing crystalline nanoparticles with tunable size and shape. Magnetic measurements displayed a clear absence of superparamagnetic behavior, indicating that these materials have the potential to be well-suited to applications in biomedicine as multimodal imaging probes and MRI contrast agents. Chapter 4 will build on the previously explored doped nanomaterials through creating a hybrid nanomaterial by tethering lanthanide-based magnetic molecules to the surface of nanoparticles. This is performed through the synthetic design of a SMM with two anisotropic DyIII ions, which was synthesized and designed to bear terminal S-groups in order to promote the binding of the magnetic molecule to capping agent free gold nanoparticles. Upon confirmation of the successful surface attachment of the molecules, magnetic measurements displayed that the magnetic molecules maintained their static properties, however, their dynamic properties were altered. This system was the first example of this type of novel approach to the study of magnetic molecules on surfaces for data storage, spintronics, and quantum computing applications. Chapter 5 will expand on the previous study of ordering arrays of magnetic molecules on the surface of nanoparticles by tethering them into 1D chain networks. We successfully synthesized chain networks with YIII, EuIII, GdIII, TbIII and DyIII lanthanide ions. Magnetic characterisation revealed slow relaxation of the magnetization with no significant interactions between magnetic ions, thus these are discrete magnetic molecules in 1D. Rather surprisingly, the isotropic GdIII analogue displayed field induced slow relaxation of the magnetisation, necessitating the use of ab initio calculations in order to shed light on the potential causes of this unexpected behavior. Overall, through the formation and study of these structures, we observed a new potential method of SMM assembly for the study of ordered arrays of molecular magnets. Chapter 6 will focus on ordering of discrete magnetic systems in 3D. With this in mind, we successfully isolated the first Co8 cuboctahedron MOF. Magnetic measurements displayed that each SBU was well-isolated, with significant antiferromagnetic coupling between CoII ions, leading to an S = 0 ground state. These interactions were then modelled using density functional theory. This type of study promotes the future development of novel high-nuclearity MOF structures with interesting and tuneable magnetic properties, as well as the potential for assembly of discrete molecular magnetic units in 3D using MOFs. Chapter 7 utilizes the principles of Chapter 3, wherein magnetic ions are doped into a diamagnetic material; in this case, MOF-5. We sought to isolate one CoII ion in each SBU, and build upon this by adding additional magnetic ions and probing their interactions. Through magnetic measurements we observed a scaling magnetic moment with CoII content, and with higher dopant percentages we began to observe magnetic interactions occurring within the SBUs. Interestingly, we also observed a change in coordination environment with higher dopant percentages, likely as a result of the previously suggested capability of one ZnII ion within the MOF-5 SBU to become hexacoordinate, allowing CoII doping up to a maximum of 25%. Consequently, this study points to the cause of the structural instability that plagues MOF-5 in the presence of air and moisture. We probed this system further in Chapter 8 using FeIII as a dopant ion, and were able to obtain the first crystallographic evidence of the coordination change of ZnII in MOF-5. Furthermore, the structure obtained with FeIII was the first example of metal ion addition within a MOF that bound two interpenetrated frameworks together. This new MOF was found to have the potential to be a more practical material for gas storage and separation, and/or for catalysis. Thus, this study was informative in regards to the inherent instability of the parent framework, as well as a new method of metal addition to a known MOF structure. Chapter 9 will conclude the work with a discussion of what was performed in, and learned from, each thesis section, as well as provide an outlook and perspective on the novel work that may be derived from these projects going forward.

The miniaturization afforded by the integration of microfluidic technologies within lab-on-a-chip devices has greatly enhanced analytical capabilities in several key applications. Microfluidics has been utilized in a wide range of areas including sample preparation and analysis, DNA microarrays, cell detection, as well as environmental monitoring. The use of microfluidics in these applications offer many unique advantages: reduction in the required sample size, reduction in analysis time, lowered cost through batch fabrication, potentially higher throughput and the vision of having such devices used in portable systems. Nanopore sensors are a relatively new technology capable of detection and analysis with single-molecule sensitivity, and show promise in many applications related to the diagnosis and treatment of many diseases. Recently, some research groups demonstrated the integration of nanopores within microfluidic devices to increase analytical throughput. This thesis describes a methodology for integrating nanopore sensors within microfluidic devices with the aim of enhancing the analytical capabilities required to analyze biomolecular samples. In this work, the first generation of an integrated nanopore-microfluidic device contained multiple independently addressable microfluidic channels to fabricate an array of nanopore sensors using controlled breakdown (CBD). Next, for the second generation, we added pneumatic microvalves to manipulate electrical and fluidic access through connected microfluidic channels. As a proof-of-concept, single molecules (single- and double-stranded DNA, proteins) were successfully detected in the devices. It is also demonstrated that inclusion of the microfluidic via (microvia) limited the exposed area of the embedded silicon nitride membrane to the solution. This helped in localizing nanopore formation by confining the electric field to specific regions of the insulating membrane while significantly reducing high frequency noise in the ionic current signal through the reduction of chip capacitance. The devices highlighted in this thesis were designed and fabricated using soft lithography techniques which are available in most biotechnology laboratories. The core of this thesis is based on two scientific articles (Chapters 3 and 4), which are published in peer-reviewed scientific journals. These chapters are preceded by an introductory chapter and another chapter detailing the experimental setup and the methods used during the course of this study.

Hepatitis C virus (HCV) is remarkably capable of efficiently hijacking host cell pathways including lipid metabolism in the liver in order to create pro-viral environments for pathogenesis. It is becoming increasingly clear that identifying small molecule inhibitors that target host factors exploited by the virus will expand available HCV treatment options. As such, a thorough understanding of host-virus interactions is critical to the development of alternative therapeutic strategies. Hepatic lipid droplets (LDs) are recruited by HCV to play essential roles in the viral lifecycle. The intracellular location of LDs is modified upon interacting with viral structural core protein. This enables formation of platforms that support viral particle assembly. Because these interactions are non-static, capturing its dynamic processes in order to better understand viral assembly can be achieved with label-free molecular imaging enhanced with live-cell capabilities. Chemical biology approaches that includes CARS microscopy employed in a multi-modal imaging system was used to probe interactions between HCV and host LDs. By successfully tracking LD trajectories, we identified core protein’s ability to alter LD speed and control for LD directionality. Using protein expression model systems that allowed for simultaneous tracking of core protein and LDs, our data revealed that mutations in the core protein region that vary in hydrophobicity and LD binding strengths, are factors that control for differential modulation of LD kinetics. Furthermore, we measured bidirectional LD travels runs and velocities, and observed critical properties by which core protein induces LD migration towards regions of viral particle assembly. Given that many steps in the HCV lifecycle are directly linked to host lipid metabolism, it is not surprising that disrupting lipid biosynthetic pathways would negatively affect viral replication. From this outlook, we explored small molecule inhibitors that targeted several lipid metabolic pathways to study its antiviral properties. Using fluorescent probes covalently labeled to viral RNA, we captured the visualization of disrupted replication complexes upon antagonizing nuclear hormone receptors that are linked to regulating lipid homeostasis. Correspondingly, biochemistry and molecular imaging techniques were also employed to identify novel antiviral mechanisms of small molecule inhibitors that target additional HCV-dependent lipid metabolic pathways.