Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

The Cbl family of E3 ubiquitin ligases are important negative regulators of multiple receptor and cytoplasmic tyrosine kinases, and participate in a wide variety of cellular processes. Uncoupling of Cbl-mediated negative regulation allows activated receptor tyrosine kinases such as the Met receptor to escape degradation, enhancing their oncogenic potential in vitro and in vivo. Despite the consequences of loss of Cbl-mediated negative regulation for human disease, little is known about the mechanisms regulating Cbl protein levels themselves. / In this thesis work, I demonstrate a differential regulation of c-Cbl and Cbl-b downstream of the Met receptor tyrosine kinase. Cbl-b protein levels decrease in response to Met kinase activity, whereas c-Cbl levels remain stable. Cbl-b is partially degraded in a proteasome-dependant manner. This requires Cbl-b ubiquitin ligase activity and a carboxy terminal domain region located between the RING and UBA domains. I conclude that the regulation of c-Cbl and Cbl-b differs downstream of Met, and propose that negative regulation of Cbl-b by a dysregulated Met receptor may contribute to tumourigenesis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-104).

No description available.

Ph.D. / Molecular Microbiology and Immunology / Both poxviruses and γ2-herpesviruses share the K3-family of viral immune evasion proteins. These proteins are characterized by an amino-terminal RING-CH domain followed by two transmembrane domains. We analyzed several human homologues of the K3-family termed membrane-associated RING-CH (MARCH) proteins. All MARCH proteins localized to subcellular membranes while several reduced surface levels of known K3-family substrates. Thus, MARCH proteins appear to be structurally and functionally homologous to viral K3 proteins. One of the major challenges in determining the function of this family is the identification of their physiological substrates. To overcome this we created a quantitative proteomics approach which can be used to identify novel substrates for both the K3- and MARCH-families. Using stable isotope labeling by amino acids in cell culture, we compared the proteome of plasma membrane, golgi, and endoplasmic reticulum membranes in the presence and absence of K5 and MARCH-VIII. Quantitative mass spectrometric protein identification from these fractions revealed that CD316 (bone marrow stromal antigen 2), CD166 (activated leukocyte cell adhesion molecule) and syntaxin-4 were consistently underrepresented in the plasma membrane of K5 expressing cells, while CD44, CD81 (TAPA-1) and B-cell receptor-associated protein 31kDa (Bap31) were consistently underrepresented in the plasma membrane of MARCH-VIII expressing cells. Furthermore, downregulation of each of these proteins was independently confirmed. Our results both identify and characterize a novel family of human ubiquitin ligase enzymes and elucidate a novel technique which can analyze this family and be easily adapted to the analysis of other cellular enzymes viral immune modulators.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

In response to telomere shortening, oxidative stress, DNA damage or aberrant activation of oncogenes, normal somatic cells exit the cell cycle and enter an irreversible growth arrest termed senescence. The limited proliferative capacity imposed by senescence on cells impedes the accumulation of mutations necessary for tumorigenesis and prevents proliferation of cells at risk of neoplastic transformation. Opposite to the tumor suppressor function, accumulation of senescent cells in adult organisms is thought to contribute to aging by depleting the renewal capacity of tissues and stem/progenitor cells, and by interfering with tissue homeostasis and functions. The Antagonistic Pleiotropy Theory of senescence proposes that senescence is beneficial early in life by acting as a tumor suppressor, but harmful late in life by contributing to aging. Recent studies have provided evidence strongly supporting the tumor suppressor function of senescence, however, direct evidence supporting the role of senescence in aging remains largely elusive. In this thesis, I describe studies to test the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging. The approach that I have taken is to alter the senescence response in vivo by changing the expression of a senescence regulator in mice. The consequence of altered senescence response on tumorigenesis and stem cell self-renewal was investigated. The senescence regulator I studied is Smurf2, which has been shown previously to activate senescence in culture. I hypothesized that the senescence response will be impaired by Smurf2 deficiency in vivo. Consequently, Smurf2-deficient mice will develop tumors at an increased frequency, but also gain enhanced self-renewal capacity of stem/progenitor cells with age. I generated a Smurf2-deficient mouse model, and found that Smurf2 deficiency attenuated p16 expression and impaired the senescence response in primary cells and tissues. Smurf2-deficient mice exhibited an increased susceptibility to spontaneous tumorigenesis, indicating that Smurf2 is a tumor suppressor. At the premalignant stage of tumorigenesis, a defective senescence response was documented in the Smurf2-deficient mice, providing a mechanistic link between impaired senescence response and increased tumorigenesis. The majority of tumors developed in Smurf2-deficent mice were B-cell lymphomas with an origin in germinal centers of the spleen and a phenotype resembling human diffuse large B-cell lymphoma (DLBCL). I discovered that Smurf2 mediated ubiquitination of YY1, a master regulator of germinal centers. Stabilization of YY1 in the absence of Smurf2 was responsible for increased cell proliferation and drove lymphomagenesis in Smurf2-deficient mice. Consistently, a significant decrease of Smurf2 expression was observed in human primary DLBCL samples, and more importantly, a low level of Smurf2 expression in DLBCL correlated with poor survival prognosis. Moreover, I found that hematopoietic stem cells (HSCs) in Smurf2-deficient mice had enhanced function compared to wild-type controls. This enhanced stem cell function was associated with increased cell proliferation and decreased p16 expression, suggesting that defective senescence response in Smurf2-deficient mice leads to increased self-renewal capacity of HSCs. My study, for the first time, offers direct genetic evidence of an important tumor suppressor function for Smurf2 as well as its function in contributing to stem cell aging. Collectively, these findings provide strong evidence supporting the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging.

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin’s lymphoma. Patients who fail conventional therapy (~50%) have a poor prognosis and few treatment options. It is essential to understand the underlying biological processes, the progression of the disease, and utilize this information to develop new therapeutics. DLBCL patients with high C-MYC expression have a poor prognosis and new therapeutics for these patients are needed. This thesis describes work testing the hypothesis that JQ1, which can indirectly inhibit C-MYC in some tumors, can be used as an effective treatment for DLBCL. Some tumors have an unknown mechanism causing high C-MYC expression, leading me to investigate the underlying mechanisms. YY1 is a transcriptional regulator of c- Myc and has been implicated in DLBCL and as a potential regulator of the germinal center (GC) reaction. DLBCL arises from GC cells or post-GC cells. I tested the hypothesis that YY1 regulates the GC reaction. SMURF2 is an E3-ubiquitin ligase for YY1 and a tumor suppressor for DLBCL. I was interested in examining the mechanism underlying the suppression of DLBCL by SMURF2 leading to the hypothesis that SMURF2 regulates the GC. This thesis shows JQ1 leads to cell death and cellular senescence in human DLBCL cells. I conclude that BRD4 inhibition by JQ1 or derivatives could provide a new therapeutic avenue for DLBCL patients. I also show loss of YY1 perturbs the GC by decreasing the dark zone and increasing apoptosis. Finally I show modulation of SMURF2 does not affect the GC, suggesting SMURF2 utilizes a different mechanism to act as a tumor suppressor and may not modulate YY1 in the context of the GC.