Despite the significant progress in medicine, infectious diseases are life-threatening thanks to an increasing number of multiresistant strains of microorganisms and late detection of pathological agents. An opportunistic fungus Aspergillus fumigatus cause respiratory system diseases called aspergillosis. The invasive pulmonary aspergillosis affects immunocompromised patients after inhalation of ubiquitous conidia of A. fumigatus and results in 450,000 deaths per year. The biofilm formation in the infected tissue protects A. fumigatus against antimicrobial drugs. Late therapy may not be effective. Infection of immunocompromised patients and biofilm formation is characteristic also for gram negative bacteria Pseudomonas aeruginosa, which is due to the production of many factors of virulence and multiresistance a dreaded opportunistic pathogen. Scanning electron microscopy (SEM) provides detail information about morphology of microorganisms with the resolution in range of tens of nanometers that allows to observe microorganisms in the infected tissue and its pathological changes. Mass spectrometry allows to detect infection and its course based on identification of characteristic microbial molecules. The aim of this study was to optimize sample preparation of tissues infected with A. fumigatus or P....

In this work phase transformations in metastable β (primarily Ti-15Mo) alloys were studied utilizing electrical resistance, dilatometry, transmission electron microscopy and X-ray and neutron diffraction. The materials Ti-15Mo, Ti-6.8Mo-4.5Fe-1.5Al (LCB), Ti-5Al-5V-5Mo-3Cr (Ti-5553), Ti-29Nb-1Fe-0.5Si (TNFS), Ti-15Mo-3Nb-3Al-0.2Si (Timetal 21S) and Ti-13Cr-1Fe-3Al (TCFA) (in wt. %) - were subjected to a solution treatment at a temperature above β transus and quenched into water. In this condition, the microstructure of the investigated materials consists of β matrix and ω particles. Samples quenched from important temperatures determined from in-situ electrical resistance and dilatometry measurements were studied by post-mortem TEM. In-situ X-ray and neutron diffraction provided direct observations of microstructure of Ti-15Mo alloy during linear heating and confirmed statements based on results of indirect methods, such as: the decrease of volume fraction of ω phase during heating at low temperatures (up to 250 ◦ C), complete dissolution of ω phase at 560 ◦ C and precipitation of α phase without ω particles serving as its direct precursors. X-ray diffraction experiment allowed to determine relative evolution of the size of ω particles while phase fraction evolution was derived from neutron diffraction. The...

Tomographic electron holography combines tomography, the reconstruction of three-dimensionally resolved data from multiple measurements with different specimen orientations, with electron holography, an interferometrical method for measuring the complex wave function inside a transmission electron microscope (TEM). Due to multiple scattering and free wave propagation conventional, ray projection based, tomography does perform badly when approaching atomic resolution. This is remedied by incorporating propagation effects into the projection while maintaining linearity in the object potential. Using the Rytov approach an approximation is derived, where the logarithm of the complex wave is linear in the potential. The ray projection becomes a convolution with a Fresnel propagation kernel, which is considerably more computationally expensive. A framework for such calculations has been implemented in Python. So has a multislice electron scattering algorithm, optimised for large fields of view and high numbers of atoms for simulations of scattering at nanoparticles. The Rytov approximation gives a remarkable increase in resolution and signal quality over the conventional approach in the tested system of a tungsten disulfide nanotube. The response to noise seems to be similar as in conventional tomography, so rather benign. This comes at the downside of much longer calculation time per iteration. / Tomographische Elektronenholographie kombiniert Tomographie, die Rekonstruktion dreidimensional aufgelößter Daten aus einem Satz von mehreren Messungen bei verschiedenen Objektorientierungen, mit Elektronenholographie, eine interferrometrische Messung der komplexen Elektronenwelle im Transmissionselektronenmikroskop (TEM). Wegen Mehrfachstreuung und Propagationseffekten erzeugt konventionelle, auf einer Strahlprojektion basierende, Tomography ernste Probleme bei Hochauflösung hin zu atomarer Auflösung. Diese sollen durch ein Modell, welches Fresnel-Propagation beinhaltet, aber weiterhin linear im Potential des Objektes ist, vermindert werden. Mit dem Rytov-Ansatz wird eine Näherung abgeleitet, wobei der Logarithmus der komplexen Welle linear im Potential ist. Die Strahlen-Projektion ist dann eine Faltung mit dem Fresnel-Propagations-Faltungskernel welche rechentechnisch wesentlich aufwendiger ist. Ein Programm-Paket für solche Rechnungen wurde in Python implementiert. Weiterhin wurde ein Multislice Algorithmus für große Gesichtsfelder und Objekte mit vielen Atomen wie Nanopartikel optimiert. Die Rytov-Näherung verbessert sowohl die Auflösung als auch die Signalqualität immens gegenüber konventioneller Tomographie, zumindest in dem getesteten System eines Wolframdisulfid-Nanoröhrchens. Das Rauschverhalten scheint ähnlich der konventionallen Tomographie zu sein, also eher gutmütig. Im Gegenzug braucht die Tomographie basierend auf der Rytov-Näherung wesentlich mehr Rechenzeit pro Iteration.

The microstructure, diffusion and phase transformations in aluminum-steel clad sheet were studied within the thesis by means of light optical microscopy, electron microscopy, resistometry and positron annihilation spectroscopy. Results of experimental methods were supplemented by finite element method employed for evaluation of electrical resistivity and simulation of diffusion between steel and aluminum. Several annealing experiments were carried out. The effective interdiffusion coefficient was evaluated by Boltzmann-Matano method from measured concentration profiles through the interface. Formation of an interfacial intermetallic phase was studied and orthorhombic phases Al13Fe4 and Al5Fe2 were identified present in the layer. Surprising results were obtained from the in-situ annealing in TEM (SEM) which go against the results found in a recent literature - the interfacial layer grows towards steel layer.

<div><div><div><p>Calcium-dependent protein kinase II alpha (CaMKIIα) is a highly abundant protein within the hippocampus, the region of the brain responsible for memory and learning. CaMKII has both structural and signaling roles in the regulation of the connective strength of synapses in excitatory neurons. It has a unique structure comprised of twelve subunits that form a dynamic assembly and is highly flexible. Its structural behavior has been shown to affect its activity, and a comprehensive mechanism of structure and function is still not fully understood. The determination of the quaternary structure of the CaMKII holoenzyme has been attempted for nearly 20 years by a variety of methods, with no one method giving a definitive structure. Problems in obtaining a structure originated with observation methods that estimated quaternary shape from low-resolution ensemble averages or required significant alteration of the protein to enforce a particular conformation. In this work, experiments were conducted to remove these limitations and provide a path towards the quaternary structure of CaMKIIα. Different expression and purification methods were evaluated to produce an optimal protocol for the generation of samples of concentrated, monodisperse, autoinhibited full-length wild-type CaMKIIα for study with cryo-electron microscopy. Strategies for microscopy sample preparation were investigated, including affinity girds, graphene-coated grids, and holey carbon grids. Lastly, experiments using negative stain electron microscopy, cryo-electron microscopy with single particle analysis, and cryo-electron tomography with subtomogram averaging were conducted to reveal the conditions required to produce an unambiguous three-dimensional structure. It was found that the assembly of the hexameric hub rings appeared to have flexible orientation, and superposition problems inherent in two-dimensional projection averaging requires the use of cryo-electron tomography to unravel the ambiguity in both hub orientation and catalytic module placement within the reconstructed volume. A subtomogram average of a limited number of particles revealed a hub domain that matched the morphology of prior reports, but the determination of catalytic module placement was not resolved. The cumulative result of this work establishes a strategy for the large-scale data collection needed to fully elucidate the structure of this challenging and fascinating protein.</p></div></div></div>

Thesis advisor: Seth C. Kruckenberg / Quantitatively describing the deformational behavior (i.e. the rheology) of lower crustal materials has proven challenging due to the highly variable nature of structural and compositional fabrics in the lower crust. Further, many flow laws describing the rheology of monophase aggregates are experimentally derived and do not necessarily apply to polyphase materials, such as gabbro, that dominate the lower crust. Here, we present the results of integrated microstructural analysis and electron backscatter diffraction (EBSD) textural analysis from exhumed lower crustal granulites in the Mount Hay block of central Australia. The preservation of heterogeneous mafic and felsic granulites containing monophase and/or polyphase mixtures of anorthite, pyroxene, and quartz (interlayered on the mm- to m-scale) make this region uniquely suited for advancing our knowledge of the processes that affect deformation and the rheology of the lower crust. Forty-two samples from distinct structural and compositional domains were chosen to compare the microstructural record of deformation, the development of crystallographic textures, and to provide estimates of lower crustal rheology and deformation conditions. Full thin-section maps of crystallographic texture were produced using EBSD methods. The resultant orientation maps were processed to characterize crystallographic textures in all constituent phases, and all other quantifiable aspects of the rock microstructure (e.g., grain size, grain shape, misorientation axes). The EBSD analysis reveals the presence of strong crystallographic preferred orientations (CPO) in nearly all constituent phases, suggesting deformation dominated by dislocation creep. Differential stresses during deformation are calculated using grain size piezometry for all major phases, and range between 34-54 MPa in quartz within monophase layers. Two-pyroxene geothermometry was used to constrain deformation temperatures to ca. 780-810 C. Based on the estimated CPO patterns, stress, and temperature, we quantify strain rates and effective viscosities of all major phases through application of monophase flow laws. Monophase strain rates range from 2.10 x 10-12 s-1 to 1.56 x 10-11 s-1 for quartz, 4.68 x 10-15 s-1 to 2.48 x 10-13 s-1 for plagioclase feldspar, 1.56 x 10-18 s-1 to 1.64 x 10-16 s-1 for enstatite, and 5.66 x 10-16 s-1 to 1.00 x 10-14 s-1 for diopside. The determined flow law variables used for monophase calculations were subsequently applied to two different models – the Minimized Power Geometric model of Huet et al. (2014) and the Asymptotic Expansion Homogenization (AEH) method of Cook (2006) – in order to calculate a bulk aggregate viscosity of the polyphase material. At a strain rate of 10-14 s-1, polyphase effective viscosities for our samples range from 3.07 x 1020 to 2.74 x 1021 Pa·s. We find that the bulk viscosity of heterogeneous, gabbroic lower crust in the Mount Hay region lies between that of monophase plagioclase and monophase quartz, and varies as a function of composition. These results are consistent with past modeling studies and geophysical estimates. / Thesis (MS) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Earth and Environmental Sciences.

Electric and hybrid vehicles uses regenerative braking, where application of the brake triggers the electric motor to work as a generator to produce electricity, which in turn charges the battery. This results in much less use of the friction brake, changing the corrosion and wear behavior of the rotor surface. There is a need for research on this topic, since fully electric or hybrid vehicles are replacing combustion engines due to concerns about global warming and climate change. Here the corrosion behavior of coated cast iron vehicle rotors in 3.5wt% NaCl is studied. The corrosion study has been performed using electrochemical methods such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). All the coated samples were provided by Pure Forge Rotors. Based on the results from SEM and EDX, the coating is atomic forge proprietary coating, and the base material is gray cast iron. Our primary objective is to study the corrosion behavior of coated, non-coated and friction-tested samples. CV experiments indicate a shift in the corrosion potential and corrosion current density due to changes in the nature of the exposed surface. Cross-sectional SEM showed the thickness of the coating to be 16-23 µm. After friction testing, the friction layer created by rubbing the brake pad over the rotor plays a role in corrosion resistance, but this depends on the type of brake pad material (i.e. semi-metallic, non-asbestos organic and low metallic). Results showed that friction film that forms after testing against non-asbestos organic pads provides the highest corrosion resistance amongst the three brake pad materials.

Surfactants self-assemble into aggregates above a certain concentration. In this work mixtures of the cationic surfactant cetyltrimetylammonium bromide (CTAB) and the anionic surfactant sodium octyl sulfate (SOS) were investigated systematically. The measurements were accomplished by combining the two complimentary techniques static light scattering (SLS) and cryo-transmission electron microscopy (CRYO-TEM). It was found that CTAB-rich samples contain large threadlike micelles rather close to mole fractions where vesicles start to form. The mole fraction x of the surfactant in excess in the aggregates was calculated and it was found that it differs a lot from the mole fraction in the bulk, and the transition from micelles to vesicles occurs when x is about 0.7. In the SOS-rich samples small globular micelles were observed that transform into vesicles upon dilution. Some of the samples rich in SOS were found to contain open vesicles with CRYO-TEM and the reasons for this behavior have been discussed. One question that needs to be further investigated is whether or not these structures are the result of some kind of distortion of the equilibrium process during sample preparation in connection with CRYO-TEM measurements. In most cases the two methods showed consistent results and trends, but for some samples differences could be observed.

Many Proteins require the aid of molecular chaperones to achieve a stable folding state and avoid misfolding pathologies. A major eukaryotic chaperone is the cytosolic chaperonin CCT. While CCT is known to fold a significant portion of all cytosolic proteins, there is no general model for the mechanism CCT uses to fold substrate proteins. One class of proteins that CCT is known to fold are β-propeller containing proteins. Here, we present structural and biochemical data on the processes that CCT uses to fold three distinct β-propeller proteins: the G-protein Beta 5 (Gβ5) subunit of the Gβ5-RGS complex, mLST8 of the mTOR complexes, and BBS2, 7, and 9 of the BBSome. We also explore the mechanisms by which these proteins are assembled into their respective signaling complexes after being folded by CCT. We found that each CCT substrate follows a unique folding trajectory and posit that the major determinants underlying each trajectory are governed by interactions between the substrate and CCT and interactions with downstream binding partners.

The study of the three-dimensional structures of biological macromolecules has given us significant insight into life and its mechanisms. Understanding these structures in their native contexts, a challenging but important goal, came closer to reality with the development of electron microscopy. After many years of technological development, we are now starting to understand previously intractable biological phenomena at an unprecedented resolution. One such phenomenon is how neighboring cells interact, both to communicate and send signals, and to adhere and form complex tissue structures. While the molecules that mediate such processes have long been studied in isolation, electron microscopy allows us to examine them in a more native biophysical environment; as hydrated, dynamic molecules tethered to opposed cellular membranes.Imaging unadulterated biological material using electron microscopy requires that the sample be embedded in a thin layer of vitreous ice to immobilize the molecules and protect them from the vacuum of the microscope, and thus is generally referred to as cryogenic electron microscopy (cryo-EM). Samples can be imaged using two common cryo-EM modalities: single particle analysis (SPA), where many two-dimensional projection images of molecules in solution are collected, and cryo-electron tomography (cryo-ET), where the sample is tilted as it is imaged at multiple angles to reconstruct a three-dimensional volume. In this work, I will describe how I have used both SPA and cryo-ET to understand cell surface interactions involving a variety of proteins. The first chapter will look at the cell surface molecules known as the Toll receptors, a family of molecules found in Drosophila melanogaster, with orthologs in mammals known as the Toll-like receptors (TLRs). I will focus on their role in the development of the Drosophila embryo during germ band extension, a kind of convergent extension that is a conserved process through all metazoans. Biophysical assays of the three implicated Toll receptors, Toll-2, -6, and -8, revealed both homophilic and heterophilic interactions. SPA was used to determine the structure of monomeric Toll-2 which closely resembles the overall fold of Toll, whose structure was previously solved by x-ray crystallography. Surface plasmon resonance (SPR) spectroscopy and analytical ultracentrifugation (AUC) showed Toll-6 is a dimer in solution, which I visualized using cryo-EM. The Toll-6 homodimer is a novel dimer interface for Tolls and TLRs, where molecules on the same cell surface have been shown to dimerize in the presence of a wide variety of ligands. In contrast, the Toll-6 dimer is formed in the absence of any ligand and exists in an antiparallel arrangement that could be formed by molecules on opposing cell surfaces. Together, these results provide a biochemical basis for germ band extension which may be further explored through the study of structure-based mutations. While cryo-EM SPA is a powerful tool, cryo-ET allows one to reconstruct three dimensional volumes of highly heterogeneous samples, such as the interior of cells, where molecules of interest may not exist in enough copies to facilitate averaging. This technique, where the sample is imaged multiple times as it is tilted to obtain three-dimensional information of a region of interest, was used to study cell adhesion of a different type: that mediated by the classical cadherins. These calcium-dependent adhesion molecules cluster into adherens junctions, spot-like protein densities found in a wide variety of tissues. In the second chapter, these junctions are recapitulated between synthetic liposome membranes by tethering the adherent cadherin molecules to chemically functionalized lipids. They are then imaged using cryo-ET to reveal higher-order structural details. First, this method is applied to the clustered protocadherins, a family of cadherins that mediate neuronal self-avoidance in mammals. Cryo-ET in combination with x-ray crystallography revealed that clustered protocadherins form extended one-dimensional zippers between membranes, which are a combination of strictly homophilic trans interactions coupled with promiscuous cis interactions. Neurons express unique subsets of the ~50-60 possible isoforms, and when two neuronal processes express identical subsets, which happens only when those processes are a part of the same cell, these linear chains grow and initiate a repulsive signal. If the subsets are different, the chains terminate and no repulsive signal is generated. The same technique has been used previously to study the type I classical cadherins, perhaps the most well-studied members of the cadherin superfamily. In the second half of this chapter, we extend our analysis to include the type II classical cadherins, which possess more complex expression patterns and binding specificities. Cryo-ET of type II cadherin ectodomains tethered to synthetic liposomes revealed that several representative members of this family form only moderately ordered arrays between liposomes, a finding in agreement with their role in cell sorting and migration. However, VE-cadherin, an outlier type II expressed in vascular endothelial cells where it withstands blood pressure, forms extraordinarily ordered junctions. Subtomogram averaging reveals the regularity of this two-dimensional array. In the final chapter, I describe my work on a membrane surface molecule of a different kind, one not involved in cell adhesion but viral infection. The global COVID-19 pandemic gave me the opportunity to contribute to our understanding of SARS-CoV-2 by studying the structure of neutralizing antibodies bound to the viral spike protein, perhaps the most infamous membrane surface protein. The first subchapter describes the initial isolation, neutralization, and structural analysis of antibodies isolated from convalescent COVID-19 patients. This work revealed that patients with severe COVID-19 produce potently neutralizing antibodies that target two spike protein domains: the receptor binding domain (RBD) and the N-terminal domain (NTD). RBD-directed antibodies occlude binding to ACE2, the human receptor that mediates viral fusion, but the neutralization mechanism of NTD-directed antibodies is unknown. The following two subchapters are more detailed structural studies of two specific types of antibodies. The first looks at a class of RBD-directed antibodies derived from the VH1-2 gene, which are some of the most potent and common antibodies against SARS-CoV-2. The heavy chains of these antibodies recognize almost identical epitopes, but the antibodies employ a modular approach to recognize the RBD in either of its possible conformations. The second class are antibodies that target the NTD, which our work revealed all bind to a single antigenic supersite. The final subchapter focuses on emerging SARS-CoV-2 variants and includes the structures of two antibodies that are still capable of neutralizing these new variants. They are also infrequent in the human antibody response to SARS-CoV-2, meaning they put little selective pressure on the virus to produce escape mutations, making them good candidates for monoclonal antibody therapies. Though Drosophila embryogenesis, adherens junction formation, and SARS-CoV-2 neutralization are seemingly unrelated systems, they are united by the incredible flexibility of cryo-EM to visualize biological molecules in more native environments. Whether it is the ability to study multiprotein complexes or assemblies formed between membranes, cryo-EM is a powerful technique that promises to help bridge the divide between structure and function.