Membrane proteins live at the interface between a cell and its environment; hence, they play a variety of important physiological roles such as transmembrane transport, signal transduction, and cell adhesion. The importance of membrane proteins in biology and medicine requires that we understand their structure and function on the atomic level. In this thesis, I studied members of two different membrane protein families, namely the neuronal and keratinocyte TRPV ion channels that sense temperature changes and MP20, a member of the PMP22/EMP/MP20/claudin superfamily. Using a variety of biochemical, X-ray crystallographic and electrophysiological techniques, I addressed mechanistic questions pertaining to the regulation of thermosensitive TRPV channels by ATP and calmodulin in neurons and keratinocytes. For MP20, a protein specific for the lens of the mammalian eye, I used a vesicle assay in combination with electron microscopy (EM) to study its function, ruling out the possibility that MP20 is involved in the formation of membrane junctions. Furthermore, I made progress in expressing and crystallizing MP20 for X-ray diffraction studies. In a separate effort, I also worked on improving and expanding the use of monolayer purification and Affinity Grids, recently introduced techniques to prepare specimens for single-particle EM based on the recruitment of His-tagged proteins to nickel lipidcontaining lipid monolayers. I extended the use of these techniques by synthesizing a glutathione lipid that can be used to recruit GST-tagged proteins. A major hurdle in the use of monolayer purification techniques, however, is the extent of non-specific protein binding to the lipid monolayer. I found that incorporating PEG lipids in the monolayer appears to reduce the problem of non-specific protein binding. While it remains to be seen whether these techniques can be developed to a point at which it will be possible to recruit exclusively tagged proteins out of cell lysates, my goal is to continue to improve and expand the use of the monolayer purification and Affinity Grid techniques in hope to make single-particle EM more easily amenable to biochemists and cell biologists.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Laminated composites are materials with complex architecture made of continuous fibers embedded within a polymeric resin. The properties of the raw materials can vary from one point to another due to different local processing conditions or complex geometrical features for example. A first step towards the identification of these spatially varying material parameters is to image with precision the displacement fields in this complex microstructure when subjected to mechanical loading. This thesis is aimed to accurately measure the displacement and strain fields at the fiber-matrix scale in a cross-ply composite. First, the theories of both local subset-based digital image correlation (DIC) and global finite-element based DIC are outlined. Second, in-situ secondary electron tensile images obtained by scanning electron microscopy (SEM) are post-processed by both DIC techniques. Finally, it is shown that when global DIC is applied with a conformal mesh, it can capture more accurately sharp local variations in the strain fields as it takes into account the underlying microstructure. In comparison to subset-based local DIC, finite-element based global DIC is better suited for capturing gradients across the fiber-matrix interfaces.

Electron crystallography has evolved as a powerful method for structural characterization of a wide range of materials. It has two significant advantages over other methods for structure determination, e.g. X-ray diffraction. Electrons interact much more strongly with matter compared to X-rays and they can be focused by electromagnetic lenses to form images with atomic resolution. These advantages make electron crystallography a unique tool for characterization of crystalline materials suffering from small crystal size and complex or disordered structures.      Zeolites are a class of microporous materials with significance in several applications. They often possess complex and disordered structures, which demand large efforts in the structure determination.      Over the last years, two new electron crystallographic methods have been developed; the rotation electron diffraction (RED) and the structure projection reconstruction from a through-focus series of high resolution transmission electron microscopy (HRTEM) images. In this thesis, they will be applied for structure determination of four new zeolite structures, including EMM-25 and EMM-23 with two ordered structures, and ITQ-39 and ITQ-38 with disordered structures. Each of the structure solutions have different challenges to overcome. The high silica borosilicate EMM-25 was solved by the RED method. The aluminosilicate EMM-23 was solved by a combination of HRTEM and RED. The structure solution of two materials with disordered structures, ITQ-39 and ITQ-38, will be described. For materials containing disorders, structure projection images are of utmost importance.      Furthermore, the mesoporosity inside hierarchically porous ZSM-5 crystals was studied by a combination of focused ion beam (FIB) and HRTEM imaging. The last part of this thesis explores STEM imaging for use in structure determination from 3D reconstruction. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Papers 4 and 5: Manuscipts; Paper 10: Manuscript</p>

No description available.

Amelogenesis imperfecta (AI) is a genetically determined enamel defect characterized by genetic and clinical heterogeneity . The prevalence and incidence of AI were established in the county of Västerbotten, northern Sweden, in 3-19-yr-olds born 1963-79, as were the mode of inheritance and clinical manifestation of AI. The distribution of the inorganic component in the enamel of AI teeth was studied as well as the surface morphology and other morphological details, and the findings were correlated to genetic and clinical data. AI was diagnosed in 79 children and adolescents (index cases). The prevalence in the study population was 1.4: 1 000. The mean yearly incidence 1963-79 was 1.3:1 000. The inheritance patterns for AI were established in 78 index cases from 51 families. Pedigree and segregation analyses suggested autosomal dominant (AD) inheritance in 3 3 families, autosomal recessive (AR) in six families, and X- linked recessive in two families; in ten families only sporadic cases were found. In one of the families with an AD inheritance pattern, X-linked dominant was a possible alternative. Examination of the families of the 78 index cases revealed 107 new cases of AI. The hypoplastic form was seen in 72% of all diagnosed cases and the hypomineralization form in 28% of the cases. A further classification of the clinical manifestations led to the identification of eight clinical variants. In 3 3 of the 51 families the same clinical variant was found in all affected members. In eight families affected members were assigned to different clinical variants. In three families with an X-linked inheritance pattern for AI, the clinical manifestation differed between women and men due to lyo- nization. Among the remaining five families, with an AD inheritance pattern for AI, variants clinically characterized by hypoplasia as well as variants characterized by hypomineralization were found in three families; in the other two families the clinical manifestation varied within the same main form of AI, i.e. hypoplasia or hypomineralization. Hypoplasia as well as hypomineralization were observed microradiographically in the enamel of most of the examined teeth. These findings were supported by scanning electron microscopy (SEM). Both clinically and microradiographically as well as by SEM, similar variants of AI were found as AD and AR traits and/or among the sporadic cases. In the families with AI as an X-linked trait the genetic hypothesis was confirmed by the clinical, microradiographic and scanning electron microscopic findings. / <p>S. 1-46: sammanfattning, s. 47-134: 5 uppsatser</p> / digitalisering@umu

Although molybdenum blue solutions have been known for more than twocenturies, an understanding of their chemical nature is only beginning to emerge.This dissertation aimed at elucidating the structural nature of the polydisperse,nanoscopic components in the solution phases and the solid states of partiallyreduced polyoxomolybdate (Mo-POM). The study offered at least fourcontributions to the area: (1) a rational protocol for the molecular recognition ofMo-POM with de novo organic hosts. (2) demonstration of kinetic precipitation ofa dynamic mixture of polyoxomolybdates and application of the technique to thestudy of the dynamic mixture by TEM (3) characterization of the Mo-POMnanostructures by an unusual combination of complementary analyticaltechniques. (4) a general approach for the synthesis of crown-ethers-containingtripodal molecules.The molecular recognition of Mo-POM with designer tripodal hexaminetris-crown ethers opened a window to the solution phase structures of Mo-POMnanoscopic components. Studies with a series of structurally analogous hostsprobed the relationship between the structure of the molecular host and theformation of nanostructures.An unusual combination of complementary analytical protocols: flow fieldflowfractionation, electron microscopy (transmission and scanning), andinductively coupled plasma – emission spectroscopy, was used to monitor thesolution-phase evolution of Mo-POM nanostructures. The crystallization – drivenformation of keplerate Mo-POM and solution-phase evolution of structurallyrelated nanoscopic species were apparent in the self-assembling process ofpartially reduced Mo-POM.

No description available.

Abstract The properties of starch nanoparticles (SNPs) labeled with the fluorescent dye pyrene (Py-SNPs) were probed by using fluorescence quenching, pyrene excimer formation, and transmission electron microscopy (TEM). Pyrene labeling of the SNPs was achieved by reacting 1-pyrenebutyric acid with the hydroxyl groups of the SNPs under basic conditions and in the presence of diisopropylcarbodiimide. This procedure did not degrade the SNPs as confirmed by dynamic light scattering (DLS) and afforded a means to generate a pyrene labeling level ranging from 0.5 to 5.0 mol% of the glucose units making up the SNPs. A polymeric quencher was also synthesized to probe the accessibility of the interior of the Py-SNPs by using fluorescence quenching measurements. The polymeric quencher was a 2K poly(ethylene glycol) terminated at one end with a methyl group and a nitropropane group at the other. Unfortunately these quenching experiments were abandoned when it was found that the polymeric quencher synthesized for these experiments absorbed too strongly where pyrene absorbs. Intramolecular pyrene excimer formation in the Py-SNPs was investigated by steady-state and time-resolved fluorescence. These experiments demonstrated that the Py-SNPs contract but do not overlap like linear polymers do in the semi-dilute regime. They also showed that despite the inherent rigidity of starch, the Py-SNPs deformed in water to allow their hydrophobic pyrene labels to cluster toward the center of the SNPs to minimize pyrene-solvent contacts. This segregation of the hydrophobic pyrene labels led to a distinct core-shell structure for the Py-SNPs which was illustrated in TEM images acquired on films prepared with the Py-SNPs. In summary, this thesis has uncovered some unexpected properties of the SNPs. Their branched structure makes their interpenetration difficult in the semi-dilute regime which forces them to contract. SNPs are thus deformable and their deformation can be probed quantitatively by using fluorescence and TEM.

Neocortical circuits can adapt to changes in sensory input by modifying the strength or number of synapses. These changes have been well-characterized electrophysiologically in primary somatosensory (barrel) cortex of rodents across different ages and with different patterns of whisker stimulation. Previous work from our lab has identified layer-specific critical periods for synaptic potentiation after selective whisker experience (SWE), where all but one row of facial whiskers has been removed. Although whole-cell patch-clamp recording methods enable a mechanistic understanding of how synaptic plasticity can occur in vivo, they are painstakingly slow, typically focus on a small number of observed events, and are focused on a single pathway or restricted anatomical area. For example, most studies of plasticity in barrel cortex have focused on analyses of experience-dependent synaptic changes in layer 4 and layer 2/3, at a single time point, but it is unclear whether such changes are limited to these layers, or whether they persist over long time periods. Here we employ an established electron-microscopic technique that selectively intensifies synaptic contacts, in combination with unbiased, automated synapse detection, to broadly explore experience-dependent changes in synaptic size and density across many neocortical layers, regions, and time periods in a high-throughput fashion. To validate the method, we focused on imaging synaptic contacts at time points surrounding the critical period for strengthening of excitatory synapses in mouse barrel cortex, and compared these to electrophysiological analyses that show a doubling of synaptic events targeting layer 2/3 pyramidal neurons following SWE. We found that the pattern of occurrence of synapses across the cortical layers is significantly different following SWE. Also, an increase in length was observed specifically in layer 3 synapses. Furthermore, we uncovered potential bidirectional plasticity in L6 synapses depending on the developmental state of circuit and a potential critical period onset for L5A synapse at PND 18. The high resolution imaging and unbiased synapse detection has enabled us to potentially tease apart synaptic changes that occur in a laminar specific fashion. This high-throughput method will facilitate analysis of experience-dependent changes in synaptic density by age, sensory experience, genotype, pharmacological treatments or behavioral training, and will enable classification of synaptic structure to identify key parameters that can be changed by these variables.