The electron microscope is an ideal tool to prepare an electron into a specified quantum state, entangle that state with states in a specimen of interest, and measure the electron final state to indirectly gain information about the specimen. There currently exist excellent technologies to prepare both momentum eigenstates (transmission electron microscopy) and position eigenstates (scanning transmission electron microscopy) in a narrow band of energy eigenstates. Similarly, measurement of the momentum and position final states is straightforward with post-specimen lenses and pixelated detectors. Measurement of final energy eigenstates is possible with magnetic electron energy loss spectrometers. In 2010 and 2011, several groups independently showed that it was straightforward to prepare electrons into orbital angular momentum eigenstates. This disseratation represents my contributions to the toolset we have to control these eigenstates: preparation, application (interaction with specimen states), and measurement. My collaborators and I showed that phase diffraction gratings efficiently produce electron orbital angular momentum eigenstates; that control of orbital angular momentum can be used to probe chirality and local magnetic fields; and that there are several routes toward efficient measurement.

Quando se utilizam os instrumentos rotatórios, oscilatórios e os recíprocos, de NiTi (Níquel-Titânio), cortando ou raspando as paredes do conduto radicular, estes também sofrem desgastes em suas lâminas de corte. Tal ocorrência determina atritos cada vez maiores, proporcionais à perda do corte de suas lâminas, diminuindo a eficiência do instrumento, exigindo mais torques e força de penetração, aumentando ainda mais o atrito e o esforço que este sofre para desgastar a dentina, predispondo acidentes. O presente estudo avaliou o desgaste nas lâminas de corte das limas Reciproc, após 3,6 e 9 usos em canais de dentes naturais. Foram utilizados 180 dentes pré-molares inferiores e 40 instrumentos R40. Após o preparo dos canais os instrumentos foram incluídos em corpos de prova e submetidos a cortes transversais a 3 mm e 6 mm, contados a partir da ponta do instrumento. Nas imagens dessas secções transversais obtidas no MEV foi mesurada a distância entre as extremidades das lâminas de corte. O desgaste dos instrumentos foi estabelecido pela diferença entre as medidas das distâncias dos instrumentos sem uso com a média de cada instrumento, nos diferentes grupos experimentais. Observou-se diferença significante, quando comparados os grupos de 6 e 9 usos com o grupo controle. Já o grupo com 3 utilizações não apresentou diferença estatisticamente significante em relação ao grupo controle. As diferenças percentuais de desgaste a 3 mm após 3, 6 e 9 utilizações foram de 5,96%, 8,01% e 18,09%, respectivamente, em relação ao Grupo controle. As diferenças percentuais de desgaste a 6 mm, após 3, 6 e 9 utilizações foram de 2,32%, 9,54% e 21,00%, respectivamente, em relação ao Grupo controle. Conclui-se, assim, que ocorre desgaste significante das lâminas de corte dos instrumentos Reciproc, já a partir do terceiro uso reforçando a recomendação do fabricante em utilizar cada instrumento em molar e, então, descartá-lo. Palavras-chave: Microscopia eletrônica de varredura. Sistema Rotatório. / The fracture of nickel-titanium instrument, whose consequences can range from the difficulty of its removal until the absolute inaccessibility to the apical portion of the canal, remains a situation to be avoided in the safest way possible. When using the rotary instruments, and reciprocal oscillatory, NiTi, cutting or scraping the walls of the root canal, they also suffer wear in its cutting blades. Such an occurrence determines friction increasing in proportion to the loss of cutting blades, reducing the efficiency of the instrument, requiring higher torque and penetration force, further increasing friction and effort that suffers when wearing the dentin. Different rotational speeds to the long axis of the active part of the instrument are thus provoked in areas of high friction, favoring fractures, regardless of the type of movement, even if it is rotating or reciprocating. The present study sought to assess whether there is wear on cutting blades System Reciproc, since this system works through reciprocal movement within the root canal. For that, we will instrumentation in selected teeth with the kinematic system recommended by the manufacturer at zero, three, six and nine uses, corresponding to a molar tooth, two molar teeth and three molar teeth after 10 uses in each experimental group (zero , three, six and nine) according to the number of uses. The instruments are inserted into a quadrangular centralized device in the same position, and fixed in resin, which have been cut into their cross initial 3 mm and 6 mm initial these selected samples with the cross section of the system is brought to the SEM, and thus wear measured by measuring the radius of your cutting blades, and thus statistically analyzed. The results showed statistically significant differences after successive instrumentations.

Este trabalho apresenta o estudo e caracterização de dois complexos moleculares, hRXRálfadeltaAB e hemocianina de Acanthoscurria gomesiana, através de técnicas estruturais e biofísicas. O uso da técnica de crio-microscopia eletrônica para o estudo da hemocianina de Acanthoscurria gomesiana, resultou em um modelo estrutural com resolução de 14 angstron- pelo métodode Fourier Shell Correlation com critério de 1/2 bit. Neste limite de resolução, já é possível observar detalhes estruturais que o mostram como sendo comptível com outros modelos de hemocianinas. Com relação ao estudo de hRXRalfadeltaAB, mostrou-se, através das técnicas de cromatografia analítica de exclusão por tamanho, eletroforese de gel de poliacrilamida e SAXS, que a proteína pode se apresentar no estado dimérico em solução, mesmo na ausência do seu ligante, 9-cis-RA. Também foi estudado a associação de hRXRalfadeltaAB a elementos responsivos: DR1, DR4, F2 e PAL. Suas constantes de dissociação foram calculadas através da técnica de espectroscopia por anisotropia de fluorescência. Os resultados obtidos mostram maior afinidade por DR1 e DR2 e indicam uma origem entrópica para o processo de associação / This work describes characterization of two biomolecular complexes: hRXR deltaAB and a hemocyanin from Acanthoscurria gomesiana using structural and biophysical techniques. Application of cryo-electron microscopy to studies of a hemocyanin from Acanthoscurria gomesiana resulted in its structural model to 14Å resolution, which was calculated by Fourier Shell Correlation with cut-off of 1/2 bit. At this resolution limit one can observe structural details of the complex which are compatible with other hemocyanin models. With respect to hRXR deltaAB, we showed using analytic size exclusion chromatography, SDS PAGE and SAXS, that the protein is dimeric in solution even at the absence of its ligand, 9-cis-RA. hRXR deltaAB binding to the responsive elements of DNA, DR1, DR4, F2 and PAL was investigated and the binding constants to these responsive elements have been determined using fluorescence anisotropy technique. Our results show higher affinity of the receptor to DR1 and DR4 and indicate entropic mechanism of DNA binding

Os crustáceos do gênero Aegla, endêmicos da América do Sul, são os únicos decápodes anomuros que vivem em ambientes de água doce. A descoberta de fósseis em sedimentos marinhos deixou poucas dúvidas sobre a origem do grupo. Diversos estudos taxonômicos, morfológicos e de distribuição geográfica têm sido realizados sobre os eglídeos. Entretanto, informações filogenéticas baseadas na morfologia do grupo limitam-se, basicamente, aos trabalhos sobre a posição da família Aeglidae na infraordem Anomura e a um trabalho pioneiro, no qual os autores propuseram um cladograma para sete espécies com ocorrência no Chile. A partir de revisões taxonômicas e de uma análise cladística, com base em caracteres morfológicos, das espécies de Aegla que ocorrem nas bacias hidrográficas do Alto Paraná e Alto Uruguai, obteve-se: 1. O não-monofiletismo do clado Alto Paraná-Alto Uruguai; 2. Aegla leptochela relacionada filogeneticamente com outras espécies do Alto Ribeira; 3. Aegla marginata é uma espécie parafilética e forma um complexo com Aegla quilombola n. sp.; 4. Aegla franca e A. perobae são espécies irmãs; 5. Aegla lata, entre outras espécies, aparecem relacionadas com populações de diferentes bacias hidrográficas demonstrando tanto a falta de caracteres derivados dentro de Aeglidae, bem como a possibilidade de existirem mais complexos de diferentes espécies nessa família; 6. A invasão ao ambiente subterrâneo ocorreu mais de uma vez ao longo do tempo e espécies estigobiontes não são irmãs recíprocas exclusivas; 7. Aegla paulensis trata-se de um complexo de sete espécies distintas: Aegla paulensis s. str., A. rosanae, A. lancinhas, A. japi n. sp., A. jaragua n. sp., A. jundiai n. sp. e A. vanini n. sp. Além disso, análises em microscopia eletrônica de varredura dos tubos sexuais de machos de 39 espécies de Aegla, revelaram dois principais tipos, o longo estreito e o curto largo e, ainda, que cada espécie possui um conjunto de características específicas para essa estrutura, podendo, então, ser utilizada como caráter diagnóstico em descrições taxonômicas / The South American endemic genus Aegla represents the only anomuran decapod crustaceans strictly living in freshwaters. Marine fossil records left no doubts regarding the origin of the group. A number of studies on taxonomy, morphology and geographical distribution of aeglid have been carried out. Nevertheless, morphology based phylogenetic information about the group is limited to studies of the positioning of Aeglidae into the Anomuran infraorder and one pioneer work which presented a cladogram for seven species from Chile. Through cladistic analysis based on morphological characters of Aegla species occurring in the upper Paraná and upper Uruguay hydrographic basins the following results were obtained: 1. The non-monophyletic condition of the upper Paraná-upper Uruguay clade; 2. Aegla leptochela phylogenetically related to other species from Alto Ribeira; 3. Aegla marginata is paraphyletic, constituting a complex with Aegla quilombola n. sp.; 4. Aegla franca and A. perobae are sister species; 5. Aegla lata and other species were related with populations from different hydrographic basins, showing the lack of derived characters in Aeglidae and the possibility of existence of other species complexes in this family; 6. Invasion to the underground environments occurred more than once and the stygobiont species are not exclusive reciprocal sisters; 7. Aegla paulensis is a species complex encompassing seven species: Aegla paulensis s. str., A. rosanae, A. lancinhas, A. japi n. sp., A. jaragua n. sp., A. jundiai n. sp. e A. vanini n. sp.. In addition, scanning electron microscopy revealed two main types of sexual tubes (long and narrow; short and wide) in males from 39 Aegla species. Each species has a specific set of characters for this structure, hence indicating it can be used as diagnostic character in taxonomic descriptions

A realização do presente trabalho permitiu o estudo dos micromecanismos de deformação e fratura da liga de titânio Ti-6Al-4V, utilizada comercialmente para a fabricação de biomateriais metálicos. As técnicas empregadas para a análise do material em estudo foram: microscopia eletrônica de varredura (MEV), microscopia eletrônica de transmissão (MET) e a difração de raios X (DRX). Estudar a influência e comportamento das diversas fases existentes em ligas de titânio é importante para se avaliar o comportamento de trincas nas ligas de titânio com alta resistência mecânica, que possuem microestrutura fina, relacionando a presença das fases alfa (α), beta (β) e alfa+beta (α+β) com a resistência do material. A avaliação in situ dos micromecanismos de deformação e fratura foi realizada por MET e também foi feito o estudo das transformações de fase durante o resfriamento em ligas de titânio, por MET, utilizando-se as técnicas de campo claro, campo escuro e difração de elétrons (DEAS), em área selecionada. Após tratamento térmico foram observadas as diferenças entre a quantidade das fases α e β, em relação à microestrutura original do material para diferentes condições utilizadas no tratamento térmico aplicado à liga metálica. Observou-se a presença da microestrutura lamelar, formada durante o resfriamento no campo β, promovendo a transformação de parte da estrutura alfa secundária em beta, que se encontrava retida entre as lamelas de alfa. / This present work allowed the study of deformation and fracture micromechanisms of titanium alloy Ti-6Al-4V, used commercially for the manufacture of metallic biomaterials. The techniques employed for the analysis of the material under study were: scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD). The study of the influence and behavior of the phases present in titanium alloys is important to evaluate the behavior of cracks in titanium alloys with high mechanical strength, which have fine alpha (α), beta (β) and alpha+beta (α+β) microstructure, linking the presence of the phases with the strength of the material. The evaluation in situ of deformation and fracture micromechanisms were performed by TEM and was also a study of phase transformations during cooling in titanium alloys, using the techniques of bright field, dark field and diffraction of electrons in the selected area. After heat treatment differences were observed between the amount of in relation to the original microstructure of the β and α phases material for different conditions used in heat treatment applied to the alloy. The presence of lamellar microstructure formed during cooling in the β field was observed, promoting the conversion of part of the secondary alpha structure in β phase, which was trapped between the lamellar of alpha.

by Chan Lap-kong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 77-80). / ABSTRACT / ACKNOWLEDGEMENTS / LIST OF FIGURES / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.1 / Chapter 1.2 --- Problems in Testing VLSI Circuits --- p.3 / Chapter 1.2.1 --- Test-cost-per-gate --- p.3 / Chapter 1.2.2 --- Tester Complexity --- p.3 / Chapter 1.3 --- Tester Based on Terminals Characteristics -Automatic Testing Equipment(ATE) --- p.4 / Chapter 1.4 --- Tester Based on Terminal and Internal Characteristics --- p.6 / Chapter 1.4.1 --- Mechanical Probing Method --- p.6 / Chapter 1.4.2 --- E-beam Probing Method --- p.7 / Chapter 1.5 --- Movitation for this Research --- p.7 / Chapter 1.6 --- Outline of the Remaining Chapters --- p.9 / Chapter 2. --- E-BEAM TESTER --- p.10 / Chapter 2.1 --- State-of-art of E-Beam Tester --- p.10 / Chapter 2.2 --- An Electron-optical Column of a SEM --- p.12 / Chapter 2.3 --- Beam Rastering Methods --- p.13 / Chapter 2.4 --- Voltage Contrast Phenomenon --- p.14 / Chapter 2.5 --- Configuration of an E-Beam Test System --- p.18 / Chapter 2.6 --- Advantages of an E-beam Tester --- p.20 / Chapter 3. --- BASIC PRINCIPLES --- p.21 / Chapter 3.1 --- Single-Stuck-At Fault Model --- p.21 / Chapter 3.2 --- Observability and Controllability --- p.24 / Chapter 3.3 --- Netlist Format --- p.25 / Chapter 3.4 --- Level --- p.27 / Chapter 3.5 --- Reconvergent Fanout --- p.28 / Chapter 4. --- CONVENTIONAL TEST GENERATION --- p.29 / Chapter 4.1 --- Conventional Automatic Test Generation for ATEs --- p.29 / Chapter 4.3 --- Conventional E-Beam Test Generation --- p.31 / Chapter 5. --- TEST AND PROBE POINT GENERATION --- p.32 / Chapter 5.1 --- Wafer Stage E-beam Testing --- p.32 / Chapter 5.2 --- Critical Paths Generation --- p.33 / Chapter 5.3 --- Assumptions of the Test and Probe Point Generation Algorithm --- p.35 / Chapter 5.4 --- Rules of the Test and Probe Point Generation Algorithm --- p.36 / Chapter 5.5 --- Probe Points Selection and Reduction --- p.38 / Chapter 5.6 --- Test and Probe Point Generation Algorithm --- p.40 / Chapter 5.7 --- Propagation and Justification at Fanout Site --- p.42 / Chapter 6. --- EXAMPLES --- p.45 / Chapter 6.1 --- Example of Test and Probe Point Generation for Circuit sc2 --- p.45 / Chapter 6.2 --- Example of Test and Probe Point Generation for Circuit sfc4 --- p.53 / Chapter 7. --- CONCLUSIONS --- p.61 / Chapter 7.1 --- Summary of Results --- p.61 / Chapter 7.2 --- Further Research --- p.63 / APPENDIX / Appendix A: Algorithm to Find Reconvergent Fanouts / Appendix B: Results of Test Generation for Circuit sc1 / Appendix C: Results of Test Generation for Circuit sc3 / REFERENCES --- p.77

Diseases caused by pathogenic bacteria continue to be major health concerns. For example, it is estimated that in the year 2000 typhoid fever caused over 21,000,000 illnesses and ~200,000 deaths (Crump et al., 2004). The disease is caused by S. typhi, a closely-related serotype of S. typhiumurium, the salmonella strain in which BipA was first identified. The CDC estimated that in 2013, multidrug resistant bacteria caused over 2 million infections in the United States, ending in more than 23,000 deaths (CDC, 2013). This number is set to rise as more bacteria become resilient to the collection of conventional antibiotics. The increasing number of multidrug resistant bacterial strains necessitates the development of new antimicrobial drugs. BipA is an attractive target for drug research. As mentioned in Section 2.5.2, BipA is ubiquitous in eubacteria and lower eukaryotes such as protozoa, but is absent from higher-order eukaryotes such as humans. Because the protein is essential for bacterial survival, BipA presents a major vulnerability of pathogenic bacteria. A drug targeting the protein itself or its interactions to the ribosome will disable only the bacteria, but have no effect on the eukaryotic host. A comprehensive model of BipA bound to the 70S ribosome will provide unparalleled insight into BipA's binding site and its mechanism. Toward this goal, cryo-EM techniques were employed to visualize the binding site of BipA on the 70S ribosome, characterize its interactions with the ribosome, and elucidate its mechanism on the ribosome. An X-ray structure of isolated BipA-GMPPNP was elucidated, by collaborators, and used for further molecular modeling of the protein to reveal possible atomic interactions between BipA and 70S ribosome. Additional biochemical studies were performed to fully characterize the specific ribosomal complex that optimizes binding of the factor. Together, the cryo-EM reconstruction, the BipA X-ray structure, the subsequent molecular modeling, and the additional biochemical studies provide a comprehensive model for BipA binding. Over the last years, the introduction of new automated algorithms for particle selection (AutoPicker) and classification (RELION) for the cryo-EM technique has revolutionized the workflow of the entire imaging and reconstruction process. The BipA dataset was primed to be used as a test bed for these algorithms and classification technique, respectively. Using old and new techniques to process the dataset allows a discussion of how the single particle reconstruction process can be vastly improved, with greater automation and efficiency.

Translation is the process by which the cell produces new proteins on the ribosome, as directed by genetic instructions, in all living organisms. Structural studies of the ribosome have shed considerable lights on its mechanism and regulation. Cryogenic electron microscopy (cryo-EM) and single-particle reconstruction technique is one of the major approaches to studying ribosome structure. In this thesis, I report the use of cryo-EM and related new techniques to study the structure of ribosome complexes. This work is divided into three parts. First, in Chapter 3, I describe the development of a computational method in the classification of cryo-EM data. Recently developed classification methods have enabled resolving multiple structures/conformations of the molecules from cryo-EM data obtained on a heterogeneous biological sample. However, the classification methods all involve various amounts of arbitrary decisions made by researchers, which can limit the use of these methods by inexperienced users. As a step toward fully automated classification, I worked with colleagues to develop a "jumper analysis" to determine the number of distinguishable classes of 3D reconstruction, based on the statistics of cryo-EM particles. Second, in Chapter 4, I document the cryo-EM study of EttA-70S ribosome complex, which provided structural insights into the mechanism of EttA in translation regulation. Energy-dependent translation throttle A (EttA, previously named YjjK in Escherichia coli) is the most prevalent member of ATP-binding cassette F family proteins in eubacteria. Through a collaboration among the Hunt, Frank, and Gonzalez labs, we combined crystallography, biochemical, cryo-EM and single-molecule fluorescence energy transfer techniques to elucidate the function and mechanism of EttA. We demonstrated that EttA gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. We also showed that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, and restricts the ribosome and tRNA dynamics required for translation. Thirdly, in Chapter 5, I discuss the improvements to a new technique, time-resolved cryo-EM by mixing-spraying, and its application to ribosome studies. The mixing-spraying method can study processes involving two big biological molecules that are in the sub-second time scale. I worked with colleagues to apply this method to studying ribosome subunit association. By mixing the subunits and reacting for 60 ms and 140 ms, we were able to capture the association reaction in a pre-equilibrium state. We detected three 70S ribosome conformations in the system. Quantification of the proportions of particles assuming these conformations suggested that the 70S ribosome can undergo fast conformational changes upon formation, and reaches equilibrium among these conformations earlier than 60 ms. In addition, I present preliminary results of studying translation decoding using the mixing-spraying method. This study, performed before improving the mixing-spraying method, was inconclusive mainly due to the limited size of cryo-EM data. Now that we have demonstrated the capability of the mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, the translation decoding process can be revisited and many other processes, such as translation initiation, can be studied.

The ribosome is a macromolecular machine, present in high copy number in the cell, that synthesizes proteins from information encoded in messenger RNA. It is a universal translator, found in all life forms and in all eras recent enough to bear life. The ribosome is structurally complex and its structure is highly evolutionarily conserved; that conservation reinforces the concept that its function in executing translation is essential. As a subject of study, the ribosome lends itself well to direct imaging, as it is large, asymmetric, dynamic, and it interacts with other heterogeneous agents throughout the translation process; if we are to infer function from structure, then the most certain way to observe the ribosome’s structure is to image it as directly as possible. Cryo-electron microscopy and single particle reconstruction are appropriate tools for this endeavor, as they can produce high-resolution three-dimensional structures of ribosomes or other macromolecular samples, and they can even reveal multiple biologically relevant states of a single sample. Although the ribosome is highly conserved in terms of its presence and core structure and functions, there is considerable variation among taxa, and the function of some of this variation is not yet understood. For example, the ribosome of the unicellular trypanosomatid parasite Leishmania major exhibits unusually large expansion segments of ribosomal RNA, as well as unusual cleavage sites in ribosomal RNA that is otherwise conserved. Here, we present a three-dimensional cryo-electron microscopy reconstruction of the 80S ribosome of Leishmania major and compare it to the available ribosome structures of closely related parasites. There is also structural variation related to the mechanism of translation: certain viruses with RNA genomes employ highly structured segments of RNA called internal ribosome entry sites to initiate translation of viral proteins on host cell ribosomes via noncanonical mechanisms. We explore one instance of this with a reconstruction of the encephalomyocarditis virus internal ribosome entry site bound with necessary protein factors to a eukaryotic 40S ribosomal subunit.

Graphene and other two-dimensional materials can be stacked together to form vander Waals heterostructures: synthetic crystals composed of different atomically thin layers with a bespoke electronic band structure. Structural characterisation of vander Waals heterostructures is difficult using conventional methods as the properties are almost entirely defined by the nature of the buried interfaces between dissimilar crystals. These methods also fall short of resolving the atomic structure of buried defects in van der Waals materials such as graphite. This work demonstrates the refinement and successful application of ion beam specimen preparation to produce cross sectional slices through these unique crystals so that they can be characterised by high resolution scanning transmission electron microscopy (STEM). Cross sectional specimen were prepared using in situ lift-out in a focused ion beam (FIB) dual-beam instrument. The fine polishing steps were optimised to prevent damage to the core of the specimen. High resolution STEM imaging of twin defects in graphene, hexagonal boron ni-tride and MoSe2 revealed that the boundaries are not atomically sharp but extended across many atoms. Advanced processing and analysis of these images uncovered fundamental mechanics which govern their geometry. This technique was further applied to complex transition metal dichalcogenide heterostructures to quantitatively determine the properties of buried interfaces between atomically thin crystals.