In this work organic molecules, namely derivatives of BODIPY and poly-para-phenyls are investigated on different metal surfaces by means of LT-STM. These molecule are important for the development of molecular electronics and spintronics. I show that aza-BODIPY molecules form a weak chemical bond with the Au(111) substrate and the molecular structure significantly changes upon adsorption. Due to the low corrugation of the Au(111) surface, diffusion of the molecule is observed for applied bias in excess of ±1 V. The temperature dependent formation of different molecular nanostructures formed by polyparaphenyls and Au adatoms is discussed. The diffusing Au adatoms act as coordination centers for the cyano groups present on one end of the molecules. The structure of the super molecular assemblies completely changes in a temperature range of only 60 K. Furthermore, I investigate in this work the hybridization of atomic orbitals within the molecular ligand. The Kondo resonance of a Co atom incorporated into an other aza-BODIPY derivative is investigated in detail on Ag(100). The hybridization of the atomic Co orbital with the organic ligands molecular orbitals is shown by spectroscopy measurements with submolecular resolution. The changing line shape of the Kondo resonance for the molecule-substrate system is discussed. This data is compared to measurements of Co incorporated in another molecular binding motive and on different metal samples to show the importance of the local environment for molecular materials.:Introduction 1 1 Basic Principles 5 1.1 The Scanning Tunneling Microscope 6 1.2 Theory of STM/STS 8 1.2.1 Scanning Tunneling Microscopy 8 1.2.2 Scanning Tunneling Spectroscopy 12 1.2.3 dI/dV-maps and SPECGRIDs 15 1.3 Lateral Manipulation of Adsorbates 15 1.4 The Kondo Effect 17 1.4.1 Investigation of Kondo Systems by STM 19 2 Experimental Setup 23 2.1 LT-STM and UHV System 24 2.2 Substrates 26 2.2.1 Au(111) 26 2.2.2 Ag(100) 28 2.2.3 Cu(110) 28 2.2.4 Surface Preparation 29 2.3 The Molecules 29 2.3.1 aza-BODIPY 29 2.3.2 6Ph-CN 30 2.3.3 Co-(BiPADI)2, Co-BiPADI, and BiPADI 31 2.3.4 Sample Preparation 32 2.4 Organic Photovoltaics 32 3 Aza-BODIPY on Au(111) 35 3.1 Experimental Results 36 3.1.1 STM Images 36 3.1.2 Spectroscopy 38 3.1.3 Lateral Manipulation 38 3.2 Discussion 39 4 6Ph-CN on Au(111) 43 4.1 Experimental Results 44 4.1.1 STM Images 44 4.1.2 Temperature Dependent Nanostructure Formation 44 4.1.3 Spectroscopy 46 4.2 Discussion 47 5 Co-BiPADI on Ag(100) and Cu(110) 55 5.1 Experimental Results 56 5.1.1 Co-BiPADI on Ag(100) 56 5.1.1.1 STM Images and Identification 56 5.1.1.2 Adsorption Geometry 56 5.1.1.3 Spectroscopy and Kondo Resonance 59 5.1.1.4 Temperature Dependent Measurements 63 5.1.1.5 SPECGRID Measurements and Comparison with Molecular Structure 64 5.1.1.6 Interaction of Several Co-BiPADI Molecules and Related Changes of Their Kondo Resonances 67 5.1.1.7 Determination of Adsorption Position of Molecules in SPECGRID Measurements 69 5.1.2 Co-BiPADI on Cu(110) 71 5.1.2.1 STM Images and Identification 71 5.1.2.2 STS Measurements and dI/dV Maps 72 5.2 Discussion 74 6 Co-(BiPADI)2 on Au(111) 81 6.1 Experimental Results 82 6.1.1 STM Images 82 6.1.2 Spectroscopy 82 6.2 Discussion 86 7 Conclusions and Outlook 89 Bibliography 93 List of Publications 101 Acknowledgments 103 A Appendices 105 A.1 Fitting Routine for Kondo STS 105 A.2 Background Subtraction in Kondo STS 107 A.3 MATLAB Fitting Tool fit.m 107 A.4 Import Routine and Fitting Script for SPECGRID Files 111 A.5 Calibration of Piezo Constants from Atomically Resolved Images 112 A.5.1 Au(111) 112 A.5.2 Ag(100) 112 Confirmation 113 / In dieser Arbeit werden organische Moleküle, Derivate von BODIPY und poly-para-Phenyl, auf verschiedenen Metalloberflächen mittels Tief-Temperatur Rastertunnelmikroskopie (LT-STM) untersucht. Diese Moleküle sind wichtig für die Entwicklung von molekularer Elektronik und Spintronik. Ich zeige, dass aza-BODIPY-Moleküle eine schwache chemische Bindung mit dem Au(111)- Substrat eingehen und die molekulare Struktur bei der Adsorption deutlich verändert wird. Wegen der geringen Rauigkeit der Au(111)-Oberfläche wird bereits bei einer angelegten Spannungen über ±1 V die Diffusion der Moleküle beobachtet. Die temperaturabhängige Bildung verschiedener molekularer Nanostrukturen aus poly-para-Phenyl und frei beweglichen Goldatomen wird diskutiert. Die diffundierenden Goldatome agieren hierbei als Koordinationszentren für die Cyanogruppen am einen Ende der Moleküle. Die Struktur der supramolekularen Anordnungen verändert sich dabei in einem Temperaturbereich von nur 60 K vollkommen. Außerdem beschäftige ich mich in dieser Arbeit mit der Hybridisierung atomare Orbitale im molekularen Verbund. Die Kondo-Resonanz eine Co-Atoms, welches in einem anderen aza-BODIPY-Derivat gebunden ist, wird detailliert auf der Ag(100)-Oberfläche untersucht. Die Hybridisierung des atomaren Co-Orbitals mit den molekularen Orbitalen des organischen Liganden wird an Hand von Spektroskopiemessungen mit submolekularer Auflösung gezeigt. Die veränderte Form der Kondo-Resonanz für dieses Molekül-Substrat-System wird diskutiert. Diese Daten werden mit Messungen an Co-Atomen in anderen molekularen Bindungsschemen und auf anderen Substraten verglichen um dieWichtigkeit der lokalen Umgebung für molekulare Materialien zu verdeutlichen.:Introduction 1 1 Basic Principles 5 1.1 The Scanning Tunneling Microscope 6 1.2 Theory of STM/STS 8 1.2.1 Scanning Tunneling Microscopy 8 1.2.2 Scanning Tunneling Spectroscopy 12 1.2.3 dI/dV-maps and SPECGRIDs 15 1.3 Lateral Manipulation of Adsorbates 15 1.4 The Kondo Effect 17 1.4.1 Investigation of Kondo Systems by STM 19 2 Experimental Setup 23 2.1 LT-STM and UHV System 24 2.2 Substrates 26 2.2.1 Au(111) 26 2.2.2 Ag(100) 28 2.2.3 Cu(110) 28 2.2.4 Surface Preparation 29 2.3 The Molecules 29 2.3.1 aza-BODIPY 29 2.3.2 6Ph-CN 30 2.3.3 Co-(BiPADI)2, Co-BiPADI, and BiPADI 31 2.3.4 Sample Preparation 32 2.4 Organic Photovoltaics 32 3 Aza-BODIPY on Au(111) 35 3.1 Experimental Results 36 3.1.1 STM Images 36 3.1.2 Spectroscopy 38 3.1.3 Lateral Manipulation 38 3.2 Discussion 39 4 6Ph-CN on Au(111) 43 4.1 Experimental Results 44 4.1.1 STM Images 44 4.1.2 Temperature Dependent Nanostructure Formation 44 4.1.3 Spectroscopy 46 4.2 Discussion 47 5 Co-BiPADI on Ag(100) and Cu(110) 55 5.1 Experimental Results 56 5.1.1 Co-BiPADI on Ag(100) 56 5.1.1.1 STM Images and Identification 56 5.1.1.2 Adsorption Geometry 56 5.1.1.3 Spectroscopy and Kondo Resonance 59 5.1.1.4 Temperature Dependent Measurements 63 5.1.1.5 SPECGRID Measurements and Comparison with Molecular Structure 64 5.1.1.6 Interaction of Several Co-BiPADI Molecules and Related Changes of Their Kondo Resonances 67 5.1.1.7 Determination of Adsorption Position of Molecules in SPECGRID Measurements 69 5.1.2 Co-BiPADI on Cu(110) 71 5.1.2.1 STM Images and Identification 71 5.1.2.2 STS Measurements and dI/dV Maps 72 5.2 Discussion 74 6 Co-(BiPADI)2 on Au(111) 81 6.1 Experimental Results 82 6.1.1 STM Images 82 6.1.2 Spectroscopy 82 6.2 Discussion 86 7 Conclusions and Outlook 89 Bibliography 93 List of Publications 101 Acknowledgments 103 A Appendices 105 A.1 Fitting Routine for Kondo STS 105 A.2 Background Subtraction in Kondo STS 107 A.3 MATLAB Fitting Tool fit.m 107 A.4 Import Routine and Fitting Script for SPECGRID Files 111 A.5 Calibration of Piezo Constants from Atomically Resolved Images 112 A.5.1 Au(111) 112 A.5.2 Ag(100) 112 Confirmation 113

No description available.

Solid-state nanopore sensors are an emerging platform for performing single-molecule characterization of biomolecules such as DNA. With the advent of Controlled Breakdown (CBD), creating a simple, tunable, ultra-low concentration sensing device in situ has enabled their direct integration with a host of platforms. The simplicity and sensitivity in performing measurements allows nanopore-based technologies to find uses which enhance existing methods. One such promising avenue for nanopore-based sensing is in the detection of infectious diseases, where early and accurate identification of the causative pathogen is essential for successful patient outcomes. Conventional assays, while effective, often have limitations in speed, cost, or target sensitivity, and may benefit from nanopore sensing approaches. However, solid-state nanopores currently lack the ability to discriminate between biomarkers sharing identical size and charge densities, such as sequentially-heterogeneous strands of DNA. Addressing the weakness of both conventional assays and nanopores could come from combining nanopore sensing with the polymerase chain reaction (PCR), a well-established and highly-selective nucleic acid amplification scheme. PCR is designed to produce large quantities of identical fragments of DNA, known as amplicons, if a user-defined parent copy is present. After the PCR process has finished, the signals produced by this population of amplicons on a nanopore sensor should therefore be indicative to the presence of a DNA biomarker in the starting sample. As PCR reactions can use a mix of different proteins, detergents, and other molecules, the challenge lies in ensuring the compatibility of these reagents with a nanopore, and determining whether the background signal they produce can be discriminated from an amplicon signal. To this end, this thesis investigates PCR-nanopore compatibility, and experimentally demonstrates a nanopore signal-classification technique to successfully identify the presence of chromosomal DNA from Group A Streptococcus (GAS), an infectious bacterium responsible for strep throat, in samples derived from clinical extracts.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23610号 / 医科博第133号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 松田 道行, 教授 林 康紀, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

La synthèse des protéines est un mécanisme central de la vie cellulaire dont la compré-hension est un enjeu pour la recherche biomédicale. Des phénomènes comme les erreurs programméesde la traduction eucaryote ou l’initiation par des structures IRES virales sont impliqués dans les processusde réplication de virus et de bactéries. Mieux appréhender ces processus est une étape essentiellepour aboutir au développement d’approches thérapeutiques innovantes. Les études en molécule uniquepermettent d’observer chaque système réactionnel individuellement et donnent accès à des évènementsasynchrones difficilement observables en mesure d’ensemble, tels la traduction de protéines.Ce manuscrit de thèse présente une approche d’étude de la traduction par un ribosome eucaryote(mammifère) en molécule unique. Nous observons les systèmes traductionnels grâce à des marqueursfluorescents liés à des oligonucléotides pouvant s’hybrider sur les séquences d’ARN messagers traduites.L’observation de ces marqueurs est faite par microscopie de fluorescence en réflexion totale (TIRFM),les ARNm étant accrochés à la surface de l’échantillon. En lisant l’ARNm, le ribosome détache lesmarqueurs, et leurs instants de départ nous permettent de remonter à la dynamique de traductionde ribosomes individuels. Cette méthode permet d’obtenir des données cinétiques statistiques surun grand nombre de systèmes traductionnels en parallèle pouvant alors être ajustées par des lois deprobabilité. Partant de ce principe, mes travaux de thèse ont eu pour objectif d’étendre nos expériencesà une nouvelle problématique biologique : l’étude des évènements non canoniques de la traductioneucaryote. Pour cela nous avons apporté les modifications et les optimisations nécessaires au dispositifet au protocole expérimental pour l’adapter à ces nouveaux enjeux.Nos mesures de la cinétique in vitro de l’élongation eucaryote ont mis à jour un délai dû à uneinitiation non-canonique. En effet, nous réalisons le recrutement du ribosome par l’ARNm grâce àune structure virale de type IRES. Dans nos conditions d’expérience, l’incorporation d’un acide aminéprend environ une seconde tandis que cette structure induit un retard à la traduction de plusieursdizaines de secondes. Nous avons réalisé une étude comparative de plusieurs de ces structures viraleset avons montré que le délai mesuré était une caractérisitique conservée dans le cadre de l’initiation noncanonique. Ce résultat ouvre des perspectives d’études cinétiques tant pour approfondir nos conclusionssur les IRES que pour aborder d’autres évènements non canoniques tel que le décalage de la phase delecture ou le franchissement du codon stop. / The synthesis of proteins is a central mechanism of cellular life whose understandingis an issue for biomedical research. Phenomena such as programmed errors of eukaryotic translation orinitiation by viral IRES structures are involved in virus and bacterial replication processes. A Betterunderstanding of these processes is an essential step towards the development of innovative therapeuticapproaches.Single molecule studies allow each reaction system to be observed individually and give accessto asynchronous events, such as protein translation, that are difficult to observe in overall measurements.This phD manuscript presents a single molecule approach to study translation by a eukaryotic(mammalian) ribosome.We observe the translational systems thanks to fluorescent primers linked to oligonucleotides thatare hybridized to the translated mRNA sequences. These markers are observed by Total InternalReflection Fuorescence Microscopy (TIRFM) ; with the mRNAs attached to the sample surface. Whilereading the mRNA, the ribosome detaches the primers, and their instants of departure give us access tothe translation dynamics of individual ribosomes. This method makes it possible to obtain statisticalkinetic data on a large number of parallel translational systems, which can then be fitted by probabilitylaws. On the basis of this principle, my phD work aimed at extending our experiments to a newbiological issue : the study of non-canonical events in eukaryotic translation. To this end, we havemade the modifications and optimizations necessary for the set-up and the experimental protocol toadapt them to these new challenges.Our measurements of the in vitro kinetics of eukaryotic elongation have revealed a delay due tonon-canonical initiation. Indeed, the ribosome are recruited on the mRNA thanks to a viral, IREStype structure. Under our experimental conditions, the incorporation of an amino acid takes aboutone second while this structure induces a translation delay of several tens of seconds. We carried outa comparative study of several of these viral structures and showed that the measured delay was acharacteristic preserved in the framework of the non-canonical initiation. This result opens up prospectsfor kinetic studies both to deepen our conclusions on IRES and to address other non-canonical eventssuch as programmed frameshifting or STOP codon readthrough.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19525号 / 理博第4185号 / 新制||理||1601(附属図書館) / 32561 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Authors are permitted to post the MBoC PDF of their articles (and/or supplemental material) on their personal websites or in an online institutional repository provided there appears always the proper citation of the manuscript in MBoC and a link to the original publication of the manuscript in MBoC (http://www.molbiolcell.org/site/misc/ifora.xhtml) / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19869号 / 生博第350号 / 新制||生||46(附属図書館) / 32905 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 河内 孝之, 教授 藤田 尚志, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20361号 / 工博第4298号 / 新制||工||1666(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 安達 泰治, 教授 小寺 秀俊, 教授 田畑 修 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

The nuclear pore complex (NPC) is a proteinaceous gateway embedded in the nuclear envelope (NE) that regulates nucleocytoplasmic transport of molecules in eukaryotes. The NPC is formed by hundreds of proteins that are classified into approximately thirty different types of proteins called nucleoporin (Nup), each presents in multiples of eight copies. These nucleoporins are divided into two categories: the scaffold Nups forming the main structure of the NPC and the phenylalanine-glycine (FG) Nups that contain multiple repeats of intrinsically disordered and hydrophobic FG domains. These FG-Nups constitute the selective permeability barrier in the central channel of the NPC, which mediates the nuclear import of proteins into the nucleus, and the nuclear export of mRNA and pre-ribosomal subunits out of the nucleus. However, the precise copies of these Nups and their specific roles in the nucleocytoplasmic transport mechanism remain largely unknown. Moreover, the dysfunctional nuclear transport and the mutations of Nups have been closely associated with numerous human diseases, such as cancer, tumor and liver cirrhosis. We have developed and employed live-cell high-speed single-molecule microscopy to elucidate these critical questions remained in the nuclear transport and provide the fundamental knowledge for developing therapies. In this dissertation, I will present my major findings for the following three research projects: 1) determine the dynamic components of FG-Nups in native NPCs; 2) track the nucleocytoplasmic transport of transcription factor Smad proteins under ligand-activated conditions; and 3) elucidate the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells.Determination of the dynamic components for FG-Nups in native NPCs. Scaffold Nups have been intensively studied with electron microscopy to reveal their spatial positions and architecture in the past decades. However, the spatial organization of FG-Nups remains obscure due to the challenge of probing these disordered and dynamic polypeptides in live NPCs. By employing high-speed single-molecule microscopy and a live cell HaloTag labeling technique, I have mapped the spatial distribution for all eleven known mammalian FG-Nups within individual NPCs. Results show that all FG-Nups within NPCs are distinct in conformations and organized to form a ~300nm long hourglass shaped toroidal channel through the nuclear envelope. Exceptionally, the two remaining Nups (Nup98 and hCG1) almost extend through the entire NPC and largely overlap with all other FG-Nups in their spatial distributions. These results provide a complete map of FG-Nup organization within the NPC and also offer structural and functional insights into nucleocytoplasmic transport models. Tracking of the nucleocytoplasmic transport of Smad proteins under ligand-activated conditions. The inducement of transforming growth factor β1 (TGF-β1) was reported to cause the nuclear accumulation of Smad2/Smad4 heterocomplexes. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy technique (FRET), I tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complex in live cells. The FRET results have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, it was found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, these single-molecule tracking data elucidate the basic molecular mechanism to understand nuclear transport and accumulation of Smad protein. Elucidation of the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells. In addition to explore the dynamic organization of NPC, in vivo characterization of the exact copy number and the specific function of each nucleoporin in the nuclear pore complex (NPC) remains desirable and challenging. Using live-cell high-speed super-resolution single-molecule microscopy, we first quantify the native copies of nuclear basket FG-Nups (Nup153, Nup50 and Tpr). Second, with same imaging technique and the auxin-inducible degradation strategies, I track the nuclear export of mRNA through native NPCs in absence of these FG-Nups. I found that these FG-Nups proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs in live cells. Tpr’s absence in the NPC dominantly reduces nuclear mRNA’s probability of entering the NPC for export. Complete depletion of Nup153 causes mRNA’s successful nuclear export efficiency dropped approximately four folds. Remarkably, the relationship between mRNA’s successful export efficiency and the copy number of Nup153 is not linear but instead follows a sigmoid function, in which mRNA can gain its maximum successful export efficiency as Nup153 increased from zero to around half of their full copies in the NPC. Lastly, the absence of Tpr or Nup153 also alters mRNA’s export routes through the NPC, but the removal of only Nup50 has almost no impact upon mRNA export route and kinetics. / Biology