The way in which the myocardium responds to its mechanical environment must be understood in order to develop reasonable treatments for congestive heart failure. The first step toward this understanding is to characterize and quantify the cardiac microstructure in healthy and diseased hearts. Myocardium has a laminar architecture made up of myolaminae, which are sheets of myocytes surrounded by a collagen weave. By enhancing the contrast between the myocytes and the surrounding collagen, the myocardium can be investigated and its laminar structure can be quantified. Many of the techniques that have been used to view the microstructure of the heart require the use of toxic or caustic chemicals for fixation or staining. An efficient imaging method that uses polarization microscopy and enhances the contrast between the collagen and myocytes while minimizing the use of harmful chemicals was developed in this research. Collagen is birefringent; therefore its visibility should be enhanced through polarization microscopy and image processing. The sheet angles were viewed directly by cutting slices of a rat septum perpendicular to the fiber angle. Images of different polarization combinations were taken and a region of interest was selected on the sample. Image processing techniques were used to reduce the intensity variation on the images and account for the variable gain of the camera. The contrast between the collagen and myocytes was enhanced by comparing adjusted images to the background and looking at a single image this comparison produced. Although the contrast was enhanced, the embedding media reduced the collagen signal and the enhancement was not as striking as expected.

Three-dimensional (3D) structural determination of biological macromolecules is not only critical to understanding their mechanisms, but also has practical applications. Combining the high resolution imaging of transmission electron microscopy (TEM) and efficient computer processing, protein structures in solution or in two-dimensional (2D) crystals can be determined. The lipid monolayer technique uses the high affinity binding of 6His-tagged proteins to a Ni-nitrilotriacetic (NTA) lipid to create high local protein concentrations, which facilitates 2D crystal formation. In this study, several proteins have been crystallized using this technique, including the SARS virus Nsp15 endonuclease and the human Toll-like receptor (TLR) 3 extracellular domain (ECD). Single particle analysis can determine protein structures in solution without the need for crystals. 3D structures of several protein complexes had been solved by the single particle method, including IniA from Mycobacterium tuberculosis, Nsp15 and TLR3 ECD. Determining the structures of these proteins is an important step toward understanding pathogenic microbes and our immune system.

The leading cause of death in patients who survive subarachnoid hemorrhage (SAH) is stroke as a result of cerebral arterial vasospasm1. Such vasospasms involve a vasoactive response, but they remain enigmatic and no clinical treatment has proven effective in prevention or reduction2. Arteries remodel in response to diverse mechanical loads and chemical factors. Following SAH, the surrounding vasculature is exposed to a radically altered chemo-mechanical environment. It is our hypothesis that chemical stimuli associated with the formation of an extravascular blood clot dominates the maladaptive growth and remodeling response early on, thus leading to important structural changes. However, it is not clear which of the many chemical factors are key players in the production of vasospasm. Before an accurate picture of the etiology of vasospasm can be produced, it is imperative to gain a better understanding of the non-pathogenic cerebral vasculature. In particular, the rabbit basilar artery is a well established model for vasospasm. However, surprisingly little is known about the mechanical properties of the rabbit basilar artery. Using an in vitro custom organ culture and mechanical testing device, acute and cultured basilar arteries from male White New Zealand specific pathogen free rabbits underwent cyclic pressurization tests at in vivo conditions and controlled levels of myogenic tone. Sections of basilar arteries were imaged for collagen fiber orientation at 0, 40 and 80 mmHg at in vivo stretch conditions using nonlinear optical microscopy. The nonlinear stress-strain curves provide baseline characteristics for acute and short-term culture basilar arteries. The active and passive testing creates a framework for interpreting the basal tone of arteries in our culture system. Nonlinear optical microscopy second harmonic generation provides unique microstructural information and allows imaging of live, intact arteries while maintaining in vivo geometries and conditions. Collagen fibers were found to be widely distributed about the axial direction in the adventitial layer and narrowly distributed about the circumferential direction in the adventitial layer. The quantified collagen fiber angles within the artery wall further support the development of accurate mathematical models.

Double stranded DNA bacteriophages achieve release of phage progeny by disrupting the cell envelope of the host cell. This is accomplished by two phage-encoded proteins, the holin and the endolysin. In bacteriophage lambda, the S holin is a small three TMD membrane protein that creates a lesion in the inner membrane of the host at a specific time, programmed in its primary structure. Lesion formation permits the cytoplasmic endolysin R access to the murein cell wall for degradation and cell lysis. Although it has been shown that S oligomerizes in the membrane, the structural nature of this complex has not been elucidated. In this study the S holin was purified using a mild non-ionic detergent and the structure of a ring complex formed by the holin was determined by electron microscopy and single particle analysis at a resolution of 2.6 nm. Biochemical characterization of the rings suggests that such a complex might represent the assembly formed by S in the membrane. Protein translocation in all organisms allows the export of proteins destined for localization outside the cytoplasm. In eubacteria, newly synthesized proteins are directed to the heterotrimeric membrane complex SecYEG by signals embedded in their sequence. The driving force through this complex is provided by the cytoplasmic ATPase SecA which combines ATP hydrolysis to mechanically insert proteins through the protein conducting channel. Using electron microscopy and single particle analysis we have obtained the structure of SecA from M. tuberculosis. The structure indicates that four SecA monomers assemble to form an elongated molecule with D2 symmetry. Docking of the EM map to the crystal structure of tb SecA confirms this arrangement of the subunits. This finding, that M. tuberculosis SecA forms a tetramer raises intriguing possibilities about SecA function.

Mesoporous silica crystals have a large variety of structures mainly due to the versatility of their structure template. The configuration and the chemical state of the templating micellar surfactants, together with the kinetic process of silica will determine the final outcome of the synthesis. Increasing the understanding of the complex formation processes involved will enable a possibilityto fine tune the material for specific uses, today focused into the fields of photoniccrystals, drug delivery, catalysis and separation technology. In this thesis emphasis is put on (1) increasing the understanding the formation mechanism yielding the different species of mesoporous silica crystals through an in depth study of quasicrystallinity (2) Characterization and description of the structural complexity through various characterization techniquesand also by studying the kinetic structural transformation phenomenon related to the minimal G- and D-surfaces. (3) The structural studies of the versatile surfactant liquid crystals for establishing a thermodynamically stable basis to evaluate the kinetic mesoporous silica growth processes. Furthermorethe thesis both enlightens the possibilities of and contributes to the developmentof electron microscopy characterization techniques. In these studies, electron microscopy is largely employed in the characterization to give a thorough picture of the mesoporous structures. This is combined with the sample preparation techniques cross-section polishing and ionslicing. Low voltage scanning electron microscopy is utilized for studying the surfaces and cross-sections of various materials at the limit of the resolution. Here, a deep understanding of the electron beam-material interaction is used for a better interpretation of the detected signals. Transmission electron microscopyis combined with electron crystallographic reconstruction to yield a three dimensional structural model. For determination of the quasicrystallinity level for a structure of dodecagonal tiling, revealed in the scope of this study,a phason strain analysis was made. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 7: Manuscript.</p>

The knowledge of the human genome sequence, as revealed in the HUGO project, has created exciting new possibilities for biomedical research. The Swedish Human Proteome Resource (HPR) program aims to make use of this information to gain further insight into the human proteome. Recombinant proteins are generated from coding sequences identified from the human genome sequence and used to produce specific antibodies to target proteins. Antibodies are subsequently utilized for functional analysis of the corresponding proteins using tissue micro arrays. The aim of my project was to investigate the possibility of distinguishing characteristic distribution patterns of intracellular proteins in the resolution capacity offered by light microscopy. A map of representative distribution patterns was created using immunohistological staining with commercially available antibodies toward well-characterised proteins in the cell. Such a map could then aid in interpreting the results of immunohistological staining of intracellular proteins using antibodies produced within the Human Proteome Resource program. Proteins manifested in nucleus, nuclear membrane and plasma membrane were clearly visible at the expected location. Proteins manifested in different organelles in the cytoplasm however, showed all a similar staining pattern, making determination of exact protein location uncertain. A possible explanation is the resolution of the light microscope not being sufficient to visualize certain proteins specific to organelles in the cytoplasm. Results may also have been influenced by the choice of secondary antibody, where the strenghtened signal generated by an enzyme labelled polymer may have a negative effect on depiction of details in the image generated.

The goal of fully decoding how the brain works requires a detailed wiring diagram of the brain network that reveals the complete connectivity matrix. Recent advances in high-throughput 3D electron microscopy (EM) image acquisition techniques have made it possible to obtain high-resolution 3D imaging data that allows researchers to follow axons and dendrites and to identify pre-synaptic and post-synaptic sites, enabling the reconstruction of detailed neural circuits of the nervous system at the level of synapses. However, these massive data sets pose unique challenges to structural reconstruction because the inevitable staining noise, incomplete boundaries, and inhomogeneous staining intensities increase difficulty of 3D reconstruction and visualization. In this dissertation, a new set of algorithms are provided for reconstruction of neuronal morphology from stacks of serial EM images. These algorithms include (1) segmentation algorithms for obtaining the full geometry of neural circuits, (2) interactive segmentation tools for manual correction of erroneous segmentations, and (3) a validation method for obtaining a topologically correct segmentation when a set of segmentation alternatives are available. Experimental results obtained by using EM images containing densely packed cells demonstrate that (1) the proposed segmentation methods can successfully reconstruct full anatomical structures from EM images, (2) the editing tools provide a way for the user to easily and quickly refine incorrect segmentations, (3) and the validation method is effective in combining multiple segmentation results. The algorithms presented in this dissertation are expected to contribute to the reconstruction of the connectome and to open new directions in the development of reconstruction methods.

A number of diseases are linked to protein folding problems which lead to the deposition of insoluble protein plaques in the brain or other organs. These diseases include prion diseases such as Creutzfeld-Jakob disease, Alzheimer's disease, Parkinson's disease and type II (non-insulin dependent) diabetes. The protein plaques are found to consist of amyloid fibrils - cross-beta-sheet polymers with the beta-strands arranged perpendicular to the long axis of the fibre. Studies of ex vivo fibrils and fibrils produced in vitro showed that amyloid structures possess similar tinctorial and morphological properties. These suggest that the ability to form amyloid fibrils is an inherent property of polypeptide chains. The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the involved mechanisms. The model proteins used in the studies were the equine and hen lysozymes and de novo designed protein albebetin. Lysozymes are naturally ubiquitous proteins. Equine lysozyme belongs to an extended family of structurally related lysozymes and α-lactalbumins and can be considered as an evolutional bridge between them. Hen lysozyme is one of the most characterized protein and its amyloidogenic properties were described earlier. De novo protein albebetin and its constructs are designed to perform the function of grafted polypeptide sequence. Fibrils of equine lysozyme are formed at acidic pH and elevated temperatures where a partially folded molten globule state is populated. We have shown that lysozyme assembles into annular and linear protofilaments in a calcium-dependent manner. We showed that albebetin and its constructs are inherently highly amyloidogenic under physiological conditions. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils, among which two distinct types of oligomeric intermediates were characterized. Pivotal oligomers comprise of 10-12 monomers and on-pathway amyloid-prone oligomers constitute of 26-30 molecules. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in albebetin fibrillation. Cytotoxic studies of albebetin amyloid species have revealed that initial, pivotal oligomers do not effect on cell viability while amyloid-prone ones induce cell death. We suggest that oligomeric size is important for the stabilizing cross-beta-sheet core which is crucial for cell toxicity. Cytotoxic studies of both oligomers and fibrils of hen lysozyme have revealed that both species induce cell death. The amyloid sample containing cross-β-sheet oligomers induces an apoptosis-like cell death. The oligomers without cross-β-sheet appeared to be non-toxic, indicating that the stabilization of this structural pattern is critical for the induced toxicity. In contrast, the fibrils induce more rapid, necrosis-like death. These studies gained insights into a structure–function relationship of different forms of amyloid and general pathways of cell death. This is an important step in understanding the mechanisms of amyloid-associated degeneration and defining specific therapeutic targets.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.