Amyloidosis is a group of diseases caused by proteins that have lost their correct three-dimensional conformation and instead assemble into insoluble fibrils in various tissues and organs. Today, more than 30 different proteins that can give rise to amyloid fibrils have been identified. Each protein that assembles into fibrils causes a specific disease. For clinical diagnosis of amyloid, antibodies are one of the most important tools. In this study, antibodies generated towards various amyloid-specific peptides were characterized and validated. This was assessed by immunohistochemistry, slot blot and SDS-PAGE and western blot. Congo red, an amyloid specific dye, was used for detection of amyloid. Immunohistochemical staining and slot blot analysis indicated that each antiserum used in this study was amyloid-specific. Antigen retrieval can facilitate staining by the techniques ability to break cross-linkages caused by fixation in formaldehyde. The results from the characterization of antisera in this study should be a great helpin clinical work on amyloid, and ensure correct diagnosis.

In 2012, more than 1.6 million new cases of breast cancer were diagnosed and about half a million women died of breast cancer. The incidence has increased in the developing world. The mortality, however, has decreased. This is thought to partly be the result of advances in diagnosis and treatment. Studying tissue samples from biopsies through a microscope is an important part of diagnosing breast cancer. Recent techniques include camera-equipped microscopes and whole slide scanning systems that allow for digital high-throughput scanning of tissue samples. The introduction of digital pathology has simplified parts of the analysis, but manual interpretation of tissue slides is still labor intensive and costly, and involves the risk for human errors and inconsistency. Digital image analysis has been proposed as an alternative approach that can assist the pathologist in making an accurate diagnosis by providing additional automatic, fast and reproducible analyses. This thesis addresses the automation of conventional analyses of tissue, stained for biomarkers specific for the diagnosis of breast cancer, with the purpose of complementing the role of the pathologist. In order to quantify biomarker expression, extraction and classification of sub-cellular structures are needed. This thesis presents a method that allows for robust and fast segmentation of cell nuclei meeting the need for methods that are accurate despite large biological variations and variations in staining. The method is inspired by sparse coding and is based on dictionaries of local image patches. It is implemented in a tool for quantifying biomarker expression of various sub-cellular structures in whole slide images. Also presented are two methods for classifying the sub-cellular localization of staining patterns, in an attempt to automate the validation of antibody specificity, an important task within the process of antibody generation.  In addition, this thesis explores methods for evaluation of multimodal data. Algorithms for registering consecutive tissue sections stained for different biomarkers are evaluated, both in terms of registration accuracy and deformation of local structures. A novel region-growing segmentation method for multimodal data is also presented. In conclusion, this thesis presents computerized image analysis methods and tools of potential value for digital pathology applications.

The sigma-1 receptor (σ-1R) is an endoplasmic reticulum (ER) protein that modulates a number of ion channels. It is hypothesized that σ-1Rs activated with agonist translocate to the plasma membrane. The σ-1R potentiates N-methyl-D-aspartate Receptors (NMDARs), important constituents of synaptic plasticity. NMDARs are anchored in the plasma membrane by Postsynaptic Density Protein-95 (PSD-95). The mechanism behind σ-1R modulation of NMDARs is not known. The results of my investigation confirm that σ-1Rs localize extrasomatically. Following σ-1R activation, σ-1R localization to dendrites and postsynaptic densities (PSDs) is upregulated. Unpublished work from our lab has shown that σ-1Rs associate with PSD-95 and NMDARs. Furthermore, immunocytochemistry (ICC) showed σ-1R colocalization with PSD-95 and NMDAR subunits. After σ-1R activation there was significantly increased colocalization between σ-1R, PSD-95, and GluN2B. Overall, this study may have provided insight into the molecular mechanism behind σ-1R modulation of NMDARs, which could have implications in the understanding of synaptic plasticity.

Angiogenesis has a crucial influence on tumour development and identification of microvessels in malignant breast cancer tissue is an indicator for worse prognosis. Angiogenesis is partially governed by the family of vascular endothelial growth factors (VEGF) and their receptors, by which the VEGF-A-protein seems to be the most important factor.     The aims of this work were to first establish a method for immunohistological (IH)-staining of the VEGF-A-protein for clinical use and then to label and evaluate the expression of this protein in 31 Nottingham Histology Graded (NHG I-III) breast carcinoma.      Formaldehyde-fixated tissues from invasive breast neoplasms and control tissues were labelled with monoclonal antibodies against VEGF-A and CD31-proteins using a semi-automated IH-system from Ventana BenchMark. Positively stained vessels were counted from digital copies of microscopic pictures related to mm2 tissue.     A method of IH-labelling with VEGF-A protein was successfully established before staining of the breast tissue and in 19 of the 31 breast cancers. Vessels were counted for both antibodies. The VEGF-A-antibody stained 2.7 ± 2.3 (mean ± SD) vessels/mm2 and the CD31-antibody stained 27.3 ± 19.3 in the breast carcinoma tissue. The percent of VEGF-A-stained vessels in relation to CD31-stained were 7.6% in the NHG-I- (n=3), 7.8% in the NHG-II- (n=10) and 15.0% in the NHG-III-group (n=6).     The results demonstrate that increased NHG-grade and lower differentiation can be associated with higher percent of vessels expressing VEGF-A-protein. The result was not statistically certified because of the small number of stained breast cancers and additional investigations are recommended before clinical use.

Idiopathische Myositiden stellen die größte Gruppe der erworbenen Myopathien im Erwachsenenalter dar. Die Pathogenese dieser Erkrankungen ist sehr heterogen und nicht in allen Einzelheiten geklärt. Das Auftreten von mitochondrialen Veränderungen und mtDNADeletionen in idiopathischen Myositiden und deren pathophysiologische Bedeutung ist in der Literatur ein kontrovers diskutiertes Thema. Nach der Präsentation des bekannten Wissens über diese Erkrankungen wird in vorliegender Arbeit dieses Thema anhand lichtmikroskopischer Methoden unter Anwendung histologischer Spezialmethoden an Muskelbiopsien von 98 Patienten untersucht. Ziel der Arbeit ist es, mit verschiedenen histologischen Färbemethoden Hinweise für Mitochondrien-Alterationen und feinstrukturelle Charakteristika von primären Mitochondrialen Myopathien in idiopathischen Myositiden zu detektieren. Ein besonderer Schwerpunkt liegt auf der Anwendung einer neuen immunhistochemischen Methode unter Anwendung eines monoklonalen antimitochondrialen Antikörpers. Es wird der Fall eines Mädchens mit muskeldystrophischer Symptomatik dargestellt, dessen Muskelbiopsie im Alter von 7 Jahren die myohistologische Diagnose einer juvenilen Dermatomyositis und Hinweise auf eine mitochondriale Dysfunktion ergab. Die Ergebnisse der immunhistochemischen Methode korrelieren gut mit anderen bekannten mitochondrialen Färbungen, sind sensitiver und stellen möglicherweise eine gute Ergänzung zu den bekannten mitochondrialen Markern und Färbungen dar. Die beobachteten mitochondrialen Dysfunktionen sprechen für die gestörte Mitochondrienfunktion und eine früh im Krankheitsverlauf, am ehesten sekundäre, Beteiligung der Mitochondrien im Krankheitsprozess dieser primär nicht mitochondrialen Erkrankungen

Doctor of Philosophy (PhD) / GABAA receptors are implicated in the pathology of psychiatric disorders such as schizophrenia and depression. They are rapidly affected by stress in a sex-dependent fashion, suggesting that GABAA receptors may be relevant to understanding the association between stress and psychiatric disorders. Thus, this thesis examined how GABAA receptors are affected in both male and female mice exposed to stress in adulthood (Chapter 2), early-life (Chapter 3-5) and a combination of both early-life and adulthood stress (Chapter 6). 2. The effects of acute adulthood stress (3 minute warm swim stress) on GABAA receptor binding in the brains of male and female mice were examined using quantitative receptor autoradiography. The total number of GABAA receptor [3H]GABA binding sites was increased following swim stress in specific forebrain cortical regions of female mice swum individually or in a group, but decreased in male mice when swum in a group only. These findings confirm and extend previous studies, identifying the cortical regions involved in rapid stress-induced changes in GABAA receptors. 3. Post-natal handling models in rodents comparing control (brief handling sessions; EH) with no intervention stress conditions (NH), indicate that the NH condition results in an anxious adulthood phenotype and this was confirmed in the present thesis using the elevated plus-maze behavioural test. Using this model the effects of early-life stress on adulthood GABAA receptors were then examined. 4. Regional densities of GABAA receptor α1 and α2 subunit proteins were observed in the adult brain of male and female mice using immunoperoxidase histochemistry. NH males showed a loss of the α2 subunit from the thalamus and the lower layers (IV-VI) of the primary somatosensory cortex, whilst NH females showed a reduction of α2 but an increase in α1 protein in the lower layers of the primary somatosensory cortex only. These regionally specific alterations in the α1:α2 subunit ratio suggest that early-life stress disrupts the developmental α subunit switch, which occurs in a regionally-dependent fashion over the first two weeks of rodent life. 5. Double-labelling immunofluorescence and confocal microscopy were used to examine the effects of sex and early-life stress on GABAA receptor synaptic clustering. Regardless of sex, mice exposed to early-life stress (NH) showed reduced colocalisation of the GABAA receptor α2 subunit with the synaptic marker protein gephyrin relative to the control condition (EH). This suggests that early-life stress impairs adulthood inhibitory synaptic strength and is consistent with the increased anxiety of the stressed relative to control mice. 6. Finally, the effects of early-life stress on adulthood swim stress-induced changes in GABAA receptor binding were examined using quantitative receptor autoradiography in forebrain cortical regions. Findings showed that the effect of adulthood stress on the total number of GABAA receptor binding sites for [3H]GABA in forebrain cortical regions was altered by early-life stress in both male and female mice, suggesting that the rapid adulthood stress response of GABAA receptors is affected by early-life experience. 7. Together these results show that GABAA receptors are sensitive to subtle changes in the environment in both early-life and adulthood and that these neurochemical responses to stress in adulthood are sex-dependent. The short and long-term stress-sensitivity of the GABAergic system implicates GABAA receptors in the non-genetic aetiology of psychiatric illnesses in which sex and stress are important factors.

Thesis (Ph.D.)--University of Wisconsin--Madison, 2006. / Includes bibliographical references. Also available on the Internet.

Tez (Yüksek Lisans) - Süleyman Demirel Üniversitesi, Sağlık Bilimleri Enstitüsü, Histoloji ve Embriyoloji Anabilim Dalı, 2004. / Bibliyografya var.

Tez (Yüksek Lisans) - Süleyman Demirel Üniversitesi, Sağlık Bilimleri Enstitüsü, Histoloji ve Embriyoloji Anabilim Dalı, 2004. / Bibliyografya var.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.