1 / Lucia Jie Chiao

p73 shares many structural and functional similarities with its homologue, the p53 tumour suppressor. Many p73 protein isoforms result from both alternative splicing and differential promoter utilization. Transcriptionally active (TA) isoforms are pro-apoptotic, while N-terminally truncated (dN) variants are anti-apoptotic and act as dominant-negative inhibitors of TAp73 and p53. p73 has been shown to activate the transcription of genes involved in cell cycle arrest and apoptosis in response to DNA damaging agents, including chemotherapies. We hypothesize that the activity of p73, like many transcription factors, may be modulated by binding to regulatory proteins. A novel p73-binding protein that we have identified is special AT-rich sequence binding protein 2 (SATB2), which is a nuclear matrix attachment region (MAR)-binding protein. It has important functions in the regulation of gene transcription, neuronal and osteoblast (OB) differentiation, and craniofacial patterning. The SATB2 family member, SATB1, has been implicated in breast tumour growth and metastasis. Here, I describe the initial characterization of the p73-SATB2 interaction. SATB2 binds to and stabilizes TAp73a, leading to the enhancement of TAp73a-mediated induction of p53-upregulated modulator of apoptosis (PUMA; apoptotic gene) and p21 (cell cycle gene). Conversely, SATB2 does not bind to but decreases TAp73b levels, resulting in inhibition of TAp73b-mediated transcription. Thus, our findings reveal a role for SATB2 in the regulation of p73 stability and function. Moreover, we demonstrate that SATB2 is overexpressed in osteosarcoma (OS), while it is undetectable in other sarcomas, thereby suggesting that SATB2 may be a potential biomarker that can distinguish OS from other sarcomas. Loss of SATB2 also inhibits the migration and invasion capabilities of OS cells, and suggests that SATB2 promotes both migration and invasion in OS. Therefore, I present work showing that SATB2 is a novel p73-binding partner that differentially regulates the stability and function of the C-terminal isoforms of p73, as well as the novel involvement of SATB2 in OS invasion.

p73 shares many structural and functional similarities with its homologue, the p53 tumour suppressor. Many p73 protein isoforms result from both alternative splicing and differential promoter utilization. Transcriptionally active (TA) isoforms are pro-apoptotic, while N-terminally truncated (dN) variants are anti-apoptotic and act as dominant-negative inhibitors of TAp73 and p53. p73 has been shown to activate the transcription of genes involved in cell cycle arrest and apoptosis in response to DNA damaging agents, including chemotherapies. We hypothesize that the activity of p73, like many transcription factors, may be modulated by binding to regulatory proteins. A novel p73-binding protein that we have identified is special AT-rich sequence binding protein 2 (SATB2), which is a nuclear matrix attachment region (MAR)-binding protein. It has important functions in the regulation of gene transcription, neuronal and osteoblast (OB) differentiation, and craniofacial patterning. The SATB2 family member, SATB1, has been implicated in breast tumour growth and metastasis. Here, I describe the initial characterization of the p73-SATB2 interaction. SATB2 binds to and stabilizes TAp73a, leading to the enhancement of TAp73a-mediated induction of p53-upregulated modulator of apoptosis (PUMA; apoptotic gene) and p21 (cell cycle gene). Conversely, SATB2 does not bind to but decreases TAp73b levels, resulting in inhibition of TAp73b-mediated transcription. Thus, our findings reveal a role for SATB2 in the regulation of p73 stability and function. Moreover, we demonstrate that SATB2 is overexpressed in osteosarcoma (OS), while it is undetectable in other sarcomas, thereby suggesting that SATB2 may be a potential biomarker that can distinguish OS from other sarcomas. Loss of SATB2 also inhibits the migration and invasion capabilities of OS cells, and suggests that SATB2 promotes both migration and invasion in OS. Therefore, I present work showing that SATB2 is a novel p73-binding partner that differentially regulates the stability and function of the C-terminal isoforms of p73, as well as the novel involvement of SATB2 in OS invasion.

Les anomalies de TP53, que sont la délétion génique associée ou non à des mutations somatiques, demeurent un facteur de résistance au traitement dans le Myélome Multiple (MM) malgré l’introduction de nouveaux agents thérapeutiques. Pour contourner les anomalies de TP53, nous avons étudié la possibilité d’activer p73, un membre de la famille de p53, qui n’est pas fréquemment muté dans les cancers. Nous avons étudié l’expression, la méthylation et la régulation de TP73 dans une collection de lignées de MM sauvages ou déficientes pour p53. Nous montrons que TP73 est rarement exprimé et surtout dans les lignées TP53 sauvage. Nous avons étudié la méthylation de l’ilot CpG situé en amont du gène par MS-PCR et montré que l’absence d’expression correspond à son hyperméthylation, qui peut néanmoins être réversée par la décitabine, un inhibiteur de la méthylation. Malgré l’augmentation d’expression de TP73, la décitabine ne permet pas une expression protéique significative de p73. Pour étudier la régulation de p73, nous avons utilisé des agents alkylants, des inhibiteurs de MDM2 et du protéasome. Nous montrons que les nutlin3a et MG132, ne stabilisent pas p73 mais diminuent son expression constitutive. Les agents alkylants induisent une augmentation de p73 mais uniquement dans les lignées TP53 sauvage et l’extinction de p53 par ARN interférence inhibe cette régulation. Dans les lignées déficientes pour p53, la décitabine augmente l’expression génique mais le melphalan ne permet pas de stabilisation de la protéine. L’ensemble de nos résultats montre que TP73 n’apparaît pas être un bon candidat pour contourner les anomalies de TP53. / TP53 deficiency remains a major adverse event in Multiple Myeloma despite therapeutic progresses. p73, a member of p53 family, is very rarely mutated and has been poorly studied in myeloma. Using human myeloma cell lines with different TP53 status, we assessed methylation, expression and regulation of TP73. We report that TP73 is silenced by methylation and that decitabine increases its expression, which remains however insufficient for significant protein expression. Alkylating drugs increase expression of TP73 only in TP53wt cells and fail to synergize with decitabine in p53 deficient cells. On the other hand, MG132 and nutlin-3a don’t stabilize p73 in response to in TP53wt p73 positive cell lines. TP73 does not appear as a promising target for bypassing p53 deficiency in Multiple Myeloma.

The purpose of this study was to evaluate the effects of pre and post visual (V) and verbal/visual (V/V) organizers on the learning of unfamiliar information at two rural community colleges in southwestern Virginia. A total of 153 students in five general English and speech classes in the two community colleges were used as subjects for the experiment. Pre and post V and V/V organizers (presented on video tape), each in the presence of objectives, were used in conjunction with a 20 minute lesson on the theory of communication written by the investigator. The control group did not receive any type of organizer. A 25 item multiple-choice test (criterion test) was administered to each treatment group following treatment. Total scores from the criterion test were subjected to a one-way analysis of variance using the Dunnett test. Results supported the first hypothesis; there was significant improvement in achievement in the presence of pre and post V and V/V organizers (p = < .05) as designed for this study and used with this particular population. Criterion test scores from the four experimental groups were analyzed further using a two-way factorial analysis of variance; one factor with two levels representing sequence of presentation (pre or post) and mode of presentation (V or V/V). Results did not support the second hypothesis: sequence or mode of presentation, or interaction of these two factors produced no significant effects (p = > .05) on achievement with this particular population. An evaluation form was administered to all five treatment groups to elicit student opinion about the lesson and the organizers. Results on items pertaining to the organizers were somewhat positive in nature. Recommendations for further research concerning V and V/V organizers included: replication of the present study using similar and more diverse populations, educational levels and subject matter content for the learning passage and organizer to test for consistency of results; investigation to isolate and define the as yet unidentified variable concerning organizers which facilitates learning; and, investigation of facilitative qualities of organizers in the achievement of affective objectives. / Ed. D.

The data gathered for this exploratory study on college students perceptions of hererosexual cohabitation support the thesis that college students do tend to view cohabitation as a courtship process. Of those who are currently cohabiting, females more than males tend to rate their own relationships as similar to the going steady phase of courtship. Background variables, such as sex, religious affiliation, and father's occupation were not found to be significantly related to how students perceive cohabitation. Degree of religiosity was found to be an indicator of how students view cohabitation. Overall, the basic perceptions of college students on this campus toward cohabitation agree with the major studies done on other college campuses across the country (Macklin, 1972; Croake, Keller and Catlin, 1974; Morrison and Anderson, 1973). That is, it is indeed a new stage in the courtship process, similar to going steady. It is not perceived as a substitute for marriage, a premarital or trial marriage, as was previously thought. / M. S.

The goal of this dissertation is to investigate the feasibility of using a bivariate Chebyshev polynomial to approximate solutions to the two-dimensional neutron diffusion equation. The two-dimensional two-group neutron diffusion equations are solved by expanding neutron fluxes in a finite series of Chebyshev polynomials over large regions of a fission reactor. All the equations for the expansion coefficients necessary to satisfy the appropriate boundary conditions for the flux and current for a typical region are developed. The resulting system of algebraic equations is solved, using the power iteration method. Since the system of equations is overdetermined, the Gram-Schmidt method of orthogonalization is used. Calculations are done with the aid of a computer code, CDP, developed as part of this dissertation. Two different test problems are solved using a first order finite difference computer code, PDQ-7 as a standard for comparison, and CDP. The first problem is a water-reflected square core with homogeneous material properties in each region. This problem is selected to provide a rather severe test of the CDP method in the calculation of large thermal neutron flux peaking at the core- reflector interface. The second problem is an actual problem solved by a utility for on-line-fuel management in a Pressurized Water Reactor. The reactor core consists of four-different fuel assemblies arranged in a checkerboard pattern in the interior of the core. For the first problem, both PDQ-7 and CDP give about the same fast neutron flux distributions. The eigenvalues calculated by both methods are identical to one part in 4800. Except for a small region near the core-reflector interface, the thermal neutron fluxes within the core differ by less than about 4%. At the core-reflector interface, the difference in thermal fluxes is about 20%. A significant reduction in computer time required for the solution is achieved (0.45 sec for CDP vs 32.6 sec for PDQ-7). It is important to note, however, that the time required to obtain the "standard" solution for comparison, using PDQ-7 is larger than would be needed for a large mesh spacing. Thus, the savings in computer time required to achieve a solution using a PDQ-7 type code giving results comparable to those obtained with the CDP code cannot be inferred directly from these results. In any event, note that the CDP method is indeed. economical in both preparation of input data and computer time. In the second problem, four very large regions are used in the CDP method for the entire reactor. These regions are much larger than those used in a typical finite difference solution for the same problem. Both methods give about the same fast neutron flux distribution. The eigenvalues calculated by the two methods are identical to one part in 2140. Although the CDP method does not show the assembly-to-assembly variation in thermal neutron flux, it gives the Same average thermal neutron flux as calculated with PDQ-7 to within 2%, except near the core boundary. Again, a large reduction in computer time is achieved (0.69 sec vs 101 sec). The feasibility of using Chebyshev polynomial expansions for two-dimensional multi-group diffusion calculations has been demonstrated for these two problems. The method gives, not only very accurate eigenvalues, but also reasonably accurate neutron flux distributions within the reactor core. / Ph. D.

Tumour suppressor 73 (Trp73) gene shares structural and functional homology with p53. Trp73 encodes multiple isoforms with opposing effects. The TAp73 isoform is a transcription factor and is classified as a tumour suppressor where as the DNp73 isoform is a putative oncogene. Imbalance of the two opposing isoforms has been reported in human malignancies including ovarian cancer. TAp73 deficiency results in a dramatic increase in the DNp73 isoform, mimicking the altered isoform balance observed in human ovarian cancer. The effects of TAp73 deficiency in mouse OSE cells were assessed. TAp73 deficiency is not sufficient to induce ovarian cancer in mice. However, TAp73 deficiency compromises cellular proliferation in OSE cell lines. Furthermore, expression of p73 isoforms and epithelial markers is altered in TAp73 deficient cell line. Further studies are needed to determine if TAp73 deficiency in conjunction with other molecular alterations can mediate transformation of OSE cells leading to ovarian cancer.

Tumour suppressor 73 (Trp73) gene shares structural and functional homology with p53. Trp73 encodes multiple isoforms with opposing effects. The TAp73 isoform is a transcription factor and is classified as a tumour suppressor where as the DNp73 isoform is a putative oncogene. Imbalance of the two opposing isoforms has been reported in human malignancies including ovarian cancer. TAp73 deficiency results in a dramatic increase in the DNp73 isoform, mimicking the altered isoform balance observed in human ovarian cancer. The effects of TAp73 deficiency in mouse OSE cells were assessed. TAp73 deficiency is not sufficient to induce ovarian cancer in mice. However, TAp73 deficiency compromises cellular proliferation in OSE cell lines. Furthermore, expression of p73 isoforms and epithelial markers is altered in TAp73 deficient cell line. Further studies are needed to determine if TAp73 deficiency in conjunction with other molecular alterations can mediate transformation of OSE cells leading to ovarian cancer.

Würzburg, Univ., Diss., 2009. / Zsfassung in engl. Sprache.