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Thesis (Ph. D.)--Oklahoma State University, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oklahoma State University, 2004. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Oklahoma State University, 2007. / Vita. Includes bibliographical references.

Congenital Hyperinsulinism of Infancy (CHI) is a potentially lethal condition caused by excessive, unregulated insulin release from pancreatic β-cells. It is a complex clinical condition and the current understanding of this disease is still not completed. In this thesis, we investigated the disease islet pathobiology from 4 main perspectives; Using nucleomegaly as a novel diagnostic marker; Identifying the mosaic of immature delta-cells in atypical CHI (CHI-A); Assessing the insulin secretory profile at the ultrastructural level; Investigating endocrine cell turnover and the driving force/mechanism behind it. By quantifying the enlarged nuclei in the endocrine pancreas of patients with CHI, we discovered that the increased incidence of nucleomegaly is pathognomic for diffuse CHI (CHI-D). This finding potentially set a novel diagnostic hallmark for intraoperative diagnoses. A characteristic of CHI-A is a combination of active and quiescent islets. The maintained expression of NKX2.2 in somatostatin positive cells suggests an immature delta-cells phenotype in quiescent islets and this is potentially contributing to the pathobiology of CHI-A. By examining the insulin secretory profile at the ultrastructural level, as well as investigating the crucial exocytosis-related genes from both RNA and protein levels, our data suggested a greater secretory capacity in β-cells from focal CHI lesion compared to CHI-D. Despite seeing a maintained potential for proliferative (Ki67) in CHI samples, there was no significant increase in apoptosis rates (cleaved caspase-3) and whole cell mass compared to control samples. Alterations in the cellular localisation of cell cycle regulators are a plausible explanation for these abnormal disease dynamics. These data expanded our knowledge on understanding CHI, and provided us new clues for the phenotypical alterations and pathobiological mechanisms in patients with this disease. Meanwhile, they also provided new insights in the future management of CHI.

Synovial sarcoma is a soft tissue tumor defined by the presence of t(X;18)(p11.2;q11.2), fusing the SYT (SS18) gene on chromosome 18 and one of three SSX genes on chromosome X. T(X;18) results in production of a fusion protein (SYT-SSX) that is thought to underlie synovial sarcoma pathogenesis through aberrant targeting of both activating (trithorax, SWI/SNF) and repressing (Polycomb) transcription factors when expressed in a stem or progenitor-like cellular background. Clinically, synovial sarcomas present considerable diagnostic and therapeutic challenges. Whereas the classical biphasic histology is distinctive, the more common monophasic histology can be difficult to differentiate from other spindle cell tumors. In these situations, detection of t(X;18) is the gold standard for diagnosis, but it is a specialized and time-consuming process. Immunohistochemistry can be helpful, but no marker that is both highly sensitive and specific is available. Here I describe a fluorescence in situ hybridization based method employing an SYT break-apart probe set that can expedite detection of t(X;18). I also report that TLE1, which was identified in gene expression studies as a good discriminator of synovial sarcoma from other mesenchymal tumors, is a highly sensitive and specific immunohistochemical marker for synovial sarcoma. Both of these novel diagnostic techniques are applicable to small tissue samples such as core needle biopsies and are now being used clinically. The diagnosis of synovial sarcoma carries a poor prognosis and the 10-year overall survival rate is approximately 50%, most of whom are young adults. The addition of chemotherapy to surgical resection (the mainstay of treatment) does not appear to improve overall survival. Thus, there is a strong need for development of a clinically effective systemic therapy to improve patient outcome. I describe preclinical studies that demonstrate the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits proliferation of synovial sarcoma by inducing apoptosis and that this is associated with degradation of multiple receptor tyrosine kinases and disruption of the SYT-SSX-β-catenin interaction. I also identify a subset of synovial sarcoma cells, typified by expression of CD133, which exhibit stem-like properties and are relatively resistant to doxorubicin but susceptible to 17-AAG at clinically relevant concentrations.

Synovial sarcoma is a soft tissue tumor defined by the presence of t(X;18)(p11.2;q11.2), fusing the SYT (SS18) gene on chromosome 18 and one of three SSX genes on chromosome X. T(X;18) results in production of a fusion protein (SYT-SSX) that is thought to underlie synovial sarcoma pathogenesis through aberrant targeting of both activating (trithorax, SWI/SNF) and repressing (Polycomb) transcription factors when expressed in a stem or progenitor-like cellular background. Clinically, synovial sarcomas present considerable diagnostic and therapeutic challenges. Whereas the classical biphasic histology is distinctive, the more common monophasic histology can be difficult to differentiate from other spindle cell tumors. In these situations, detection of t(X;18) is the gold standard for diagnosis, but it is a specialized and time-consuming process. Immunohistochemistry can be helpful, but no marker that is both highly sensitive and specific is available. Here I describe a fluorescence in situ hybridization based method employing an SYT break-apart probe set that can expedite detection of t(X;18). I also report that TLE1, which was identified in gene expression studies as a good discriminator of synovial sarcoma from other mesenchymal tumors, is a highly sensitive and specific immunohistochemical marker for synovial sarcoma. Both of these novel diagnostic techniques are applicable to small tissue samples such as core needle biopsies and are now being used clinically. The diagnosis of synovial sarcoma carries a poor prognosis and the 10-year overall survival rate is approximately 50%, most of whom are young adults. The addition of chemotherapy to surgical resection (the mainstay of treatment) does not appear to improve overall survival. Thus, there is a strong need for development of a clinically effective systemic therapy to improve patient outcome. I describe preclinical studies that demonstrate the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits proliferation of synovial sarcoma by inducing apoptosis and that this is associated with degradation of multiple receptor tyrosine kinases and disruption of the SYT-SSX-β-catenin interaction. I also identify a subset of synovial sarcoma cells, typified by expression of CD133, which exhibit stem-like properties and are relatively resistant to doxorubicin but susceptible to 17-AAG at clinically relevant concentrations.

Synovial sarcoma is a soft tissue tumor defined by the presence of t(X;18)(p11.2;q11.2), fusing the SYT (SS18) gene on chromosome 18 and one of three SSX genes on chromosome X. T(X;18) results in production of a fusion protein (SYT-SSX) that is thought to underlie synovial sarcoma pathogenesis through aberrant targeting of both activating (trithorax, SWI/SNF) and repressing (Polycomb) transcription factors when expressed in a stem or progenitor-like cellular background. Clinically, synovial sarcomas present considerable diagnostic and therapeutic challenges. Whereas the classical biphasic histology is distinctive, the more common monophasic histology can be difficult to differentiate from other spindle cell tumors. In these situations, detection of t(X;18) is the gold standard for diagnosis, but it is a specialized and time-consuming process. Immunohistochemistry can be helpful, but no marker that is both highly sensitive and specific is available. Here I describe a fluorescence in situ hybridization based method employing an SYT break-apart probe set that can expedite detection of t(X;18). I also report that TLE1, which was identified in gene expression studies as a good discriminator of synovial sarcoma from other mesenchymal tumors, is a highly sensitive and specific immunohistochemical marker for synovial sarcoma. Both of these novel diagnostic techniques are applicable to small tissue samples such as core needle biopsies and are now being used clinically. The diagnosis of synovial sarcoma carries a poor prognosis and the 10-year overall survival rate is approximately 50%, most of whom are young adults. The addition of chemotherapy to surgical resection (the mainstay of treatment) does not appear to improve overall survival. Thus, there is a strong need for development of a clinically effective systemic therapy to improve patient outcome. I describe preclinical studies that demonstrate the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits proliferation of synovial sarcoma by inducing apoptosis and that this is associated with degradation of multiple receptor tyrosine kinases and disruption of the SYT-SSX-β-catenin interaction. I also identify a subset of synovial sarcoma cells, typified by expression of CD133, which exhibit stem-like properties and are relatively resistant to doxorubicin but susceptible to 17-AAG at clinically relevant concentrations. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

The lysosomal compartment is a major site for intracellular degradation. Lysosomal degradation of the cell’s own constituents, so-called autophagy, not only provides a cell with nutrients, but also removes damaged and potentially dangerous endogenous structures, thus securing intracellular homeostasis. On the other hand, lysosomes have been shown to be involved in the initial stages of apoptosis, and the protective effect of autophagy has been suggested to switch to cell death when excessive. Ageing-related changes of cellular structures result from damage caused by eactive oxygen species (ROS), which are an inevitable by-product of aerobic life. Intracellular turnover of compromised organelles and macromolecules, to which lysosomal degradation is a major contributor, does not function perfectly, even under favourable conditions. This inherent incompleteness of lysosomal degradation is responsible for the accumulation of a variety of nondegraded and functionally inefficient structures, which can be considered biological “garbage”. Biological “garbage” includes damaged non-degraded macromolecules and organelles, as well as intralysosomal non-degradable polymer-like structure called lipofuscin, or age pigment. Although accumulation of biological “garbage” has been suggested harmful, little is known about the mechanisms of its deleterious effects. To gain a better understanding of ageing-related changes of the lysosomal compartment and their influence on cell functions, we focused on studying: (1) the role of macroautophagy in the turnover of organelles and lipofuscin formation; (2) the role of biological “garbage” accumulation in the development of ageing-related changes and eventual death of growth-arrested, postmitotic-like cells; (3) the possible cell-protective effect of mitosis; (4) the influence of lipofuscin on cell survival during complete starvation; and (5) the effects of lipofuscin on lysosomal stability. As a model of induced biological “garbage” accumulation we used confluent human fibroblasts treated with the autophagy inhibitor 3-methyladenine (3MA). Alternatively, lysosomal degradation was suppressed by using the cysteine protease inhibitor leupeptin, or the cathepsin D inhibitor pepstatin A. As a cellular model of aged cells, we used lipofucsin-loaded human fibroblasts. Lipofuscin-loading was achieved by culturing confluent fibroblasts under hyperoxic conditions for 2-4 months. Using these in vitro models, the present study shows that: (1) inhibition of autophagy results in accumulation of lysosome-associated autofluorescent material and mitochondria with low membrane potential; (2) detrimental effect of biological “garbage” accumulation following inhibition of autophagy is prevented by continuous cell division; (3) lipofuscin-loaded cells are more resistant to starvation-induced cell death than control cells; (4) lysosomes of lipofuscinloaded fibroblasts are more resistant to the organelle-targeted stress then lysosomes of control cells. Based on the results of the present study we conclude that properly operating autophagic machinery plays a crucial role in preventing age-related changes associated with accumulation of biological “garbage”. We also suggest that continual proliferation is the natural mechanism by which cells cope with the accumulation of non-degradable material, employing mechanical dilution during the cell division. Finally, we introduce an idea of lipofuscin being a hormetic agent, and possibly possessing some lysosome-stabilising properties. Better understanding of the influence of the age-related accumulation of biological “garbage” on cellular functions may be helpful for future development of anti-ageing therapy and management of age-associated pathologies.

The intent of this research was to identify if the degenerative changes within arteries in the endometrium (endometrial angiopathies) correlate with degenerative changes in the uterine arteries and can be used as a predictor of increased risk for uterine artery rupture (UAR). With this objective specimens from 20 mares that died from uterine artery rupture and 21 control mares that died from unrelated causes were obtained from cases submitted to the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) over a two-year period. Postmortem specimens of each mare were collected from the left and right uterine arteries at the origin, bifurcation, and distal to the bifurcation as well as full thickness uterine wall sections at five different sites. An additional sample was taken from the uterine artery at the site of rupture in the affected mares. Tissue samples were immersed in 10% neutral buffered formalin, routinely processed, and stained with hematoxylin and eosin, Masson’s Trichrome, and Verhoeff´s Van Gieson histochemical stains as well as a smooth muscle-actin immunohistochemical marker. Elastosis, fibrosis, and vascular smooth muscle cell degeneration were identified in this study as potential contributors of vascular degeneration and a scoring system was developed to differentiate the degrees of severity of these specific degenerative changes within the intima and media of the vascular wall. Based on the scoring system, sections of uterine arteries and endometrial arterioles were blindly examined and the scored changes recorded for statistical analysis. Although the degenerative changes in endometrial and uterine arteries were similar within each group, the results could not not be used to predict an increased risk for UAR. Furthermore, we determined the major changes in vascular pathology of the affected uterine arteries and show there is a significant difference in degenerative changes between specific layers of the vascular wall.