Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Educational Technology-Benefits and Barriers

Statement of Problem: The second background paper for the Medical School Objective Project (MSOP), defined Educational Technology (ET) as the use of information technology to facilitate student’s learning.1 Medical schools as a group have made limited progress in accomplishing the recommended educational technology goals and there had been much greater use of such technology in basic sciences courses than in clinical clerkships. We will explore the positive and negative implications of incorporating ET into the educational experience of TMC schools. [See PDF for complete abstract]

First Trimester Screening and its Impact on Uptake of Diagnostic Testing

Introduction: First Trimester Screening (FTS) combines maternal age with fetal nuchal translucency (NT) and maternal analytes to identify pregnancies at an increased risk for Down syndrome and trisomy 18. Though the accuracy of this screening is high, it cannot replace the conclusive accuracy of prenatal diagnostic testing (PDT). Since FTS has been available, a decrease in the number of women who pursue PDT has been observed. This study sought to determine if there has been a significant change in the amount of PDT performed in our clinics, if the type of FTS result affects the patient’s decision regarding PDT, and what the patient’s intentions are regarding PDT. Material and Methods: A database review was performed for the two years prior and the two years after the January 2007 American College of Obstetricians and Gynecologists (ACOG) guidelines regarding FTS were issued. We compared the number of women who were AMA and the number of women who were AMA and had PDT between those time periods. We also determined the number of positive and negative FTS results, and determined how many of those patients had PDT. Finally, we surveyed our patients and referring physicians to determine: what the patient understands about FTS, what the patient’s intentions are regarding FTS, and how physicians present the option of FTS to their patients. Results: We determined that there was a 19.6% decrease in the amount of PDT performed when we compared the two time periods at our three specified clinics. Many of our patients were against having PDT prior to their genetic counseling session, but after they received genetic counseling, 76% of our population became open to the possibility of having PDT. Conclusion: Similar to previous studies, we determined that there has been a significant decrease in the number of PDT procedures performed at our clinics, which coincides with the release of the January 2007 ACOG statement regarding FTS. While our patients regarded FTS as a way to gain early information about their pregnancy in a non-invasive manner, they also stated they would use their results as a way to aid in their decision regarding PDT.

The performance of human papillomavirus high-risk DNA testing in the screening and diagnostic settings.

OBJECTIVE: We sought to evaluate the performance of the human papillomavirus high-risk DNA test in patients 30 years and older. MATERIALS AND METHODS: Screening (n=835) and diagnosis (n=518) groups were defined based on prior Papanicolaou smear results as part of a clinical trial for cervical cancer detection. We compared the Hybrid Capture II (HCII) test result with the worst histologic report. We used cervical intraepithelial neoplasia (CIN) 2/3 or worse as the reference of disease. We calculated sensitivities, specificities, positive and negative likelihood ratios (LR+ and LR-), receiver operating characteristic (ROC) curves, and areas under the ROC curves for the HCII test. We also considered alternative strategies, including Papanicolaou smear, a combination of Papanicolaou smear and the HCII test, a sequence of Papanicolaou smear followed by the HCII test, and a sequence of the HCII test followed by Papanicolaou smear. RESULTS: For the screening group, the sensitivity was 0.69 and the specificity was 0.93; the area under the ROC curve was 0.81. The LR+ and LR- were 10.24 and 0.34, respectively. For the diagnosis group, the sensitivity was 0.88 and the specificity was 0.78; the area under the ROC curve was 0.83. The LR+ and LR- were 4.06 and 0.14, respectively. Sequential testing showed little or no improvement over the combination testing. CONCLUSIONS: The HCII test in the screening group had a greater LR+ for the detection of CIN 2/3 or worse. HCII testing may be an additional screening tool for cervical cancer in women 30 years and older.

Comparison of OG1RF and an isogenic fsrB deletion mutant by transcriptional analysis: the Fsr system of Enterococcus faecalis is more than the activator of gelatinase and serine protease.

The FsrABC system of Enterococcus faecalis controls the expression of gelatinase and a serine protease via a quorum-sensing mechanism, and recent studies suggest that the Fsr system may also regulate other genes important for virulence. To investigate the possibility that Fsr influences the expression of additional genes, we used transcriptional profiling, with microarrays based on the E. faecalis strain V583 sequence, to compare the E. faecalis strain OG1RF with its isogenic mutant, TX5266, an fsrB deletion mutant. We found that the presence of an intact fsrB influences expression of numerous genes throughout the growth phases tested, namely, late log to early stationary phase. In addition, the Fsr regulon is independent of the activity of the proteases, GelE and SprE, whose expression was confirmed to be activated at all three time points tested. While expression of some genes (i.e., ef1097 and ef0750 to -757, encoding hypothetical proteins) was activated in late log phase in OG1RF versus the fsrB deletion mutant, expression of ef1617 to -1634 (eut-pdu orthologues) was highly repressed by the presence of an intact Fsr at entry into stationary phase. This is the first time that Fsr has been characterized as a negative regulator. The newly recognized Fsr-regulated targets include other factors, besides gelatinase, described as important for biofilms (BopD), and genes predicted to encode the surface proteins EF0750 to -0757 and EF1097, along with proteins implicated in several metabolic pathways, indicating that the FsrABC system may be an important regulator in strain OG1RF, with both positive and negative effects.

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

Zoning and the built environment: A public health analysis of zoning in Austin, Texas

The built environment is part of the physical environment made by people and for people. Because the built environment is such a ubiquitous component of the environment, it acts as an important pathway in determining health outcomes. Zoning, a type of urban planning policy, is one of the most important mechanisms connecting the built environment to public health. This policy analysis research paper explores how zoning regulations in Austin, Texas promote or prohibit the development of a healthy built environment. A systematic literature review was obtained from Active Living Research, which contained literature published about the relationships between the built environment, physical activity, and health. The results of these studies identified the following four components of the built environment that were associated to health: access to recreational facilities, sprawl and residential density, land use mix, and sidewalks and their walkability. A hierarchy analysis was then performed to demonstrate the association between these aspects of the built environment and health outcomes such as obesity, cardiovascular disease, and general health. Once these associations had been established, the components of the built environment were adapted into the evaluation criteria used to conduct a public health analysis of Austin's zoning ordinance. A total of eighty-eight regulations were identified to be related to these components and their varying associations to human health. Eight regulations were projected to have a negative association to health, three would have both a positive and negative association simultaneously, and nine were indeterminable with the information obtained through the literature review. The remaining sixty-eight regulations were projected to be associated in a beneficial manner to human health. Therefore, it was concluded that Austin's zoning ordinance would have an overwhelmingly positive impact on the public's health based on identified associations between the built environment and health outcomes.^

Coping with advanced cancer: Caregivers and their patients

Background. Being diagnosed with advanced cancer may be one of the most difficult challenges a person faces. To help deal with advanced cancer, patients and caregivers lean towards the coping skills most familiar to them. Depending on the person, one’s coping may either help or perpetuate the problem. ^ Purpose. To identify predictors of negative coping skills among a group patients and caregivers, and to identify the predictors of prolonged grief among a group of providers, 6-months post loss. ^ Methods. Advanced cancer patients and caregivers were interviewed at the time of their enrollment (baseline) and caregivers were interviewed 6 months after the patient’s death (post-loss). Each participant was administered questionnaires regarding demographics, mental health disorders, mental health service use, coping methods, quality of life, and suicide attempts. Patients were asked about their treatment and diagnosis. Other patient-related information was obtained from medical charts. Moreover, caregivers were given an assessment regarding their alcohol and smoking history. ^ Results. Among White patients, positive coping skills were positively correlated with quality of life and negative coping skills were negatively correlated with quality of life. Gender, psychiatric disorders, suicidality, alcohol and smoking history, and treatment and diagnosis, were significantly related to negative coping skills; however this relationship only held for Hispanic patients. Gender and psychiatric disorders demonstrated predictive value for negative coping skills among all patients. On the other hand, psychiatric disorders, major depression, anxiety disorder, suicidality, and alcohol and smoking history, were not significant predictors of which caregivers experienced prolonged grief. ^ Conclusion. There was a significant relationship between quality of life and positive and negative coping skills of patients and negative coping skills for caregivers. Factors such as gender, psychiatric disorders, suicidality, alcohol and smoking history, and treatment and diagnosis demonstrated predictive value for negative coping skills in patients. ^

Latino perceptions of and barriers to preventive healthcare maintenance in the southwest United States

Between the 1990 and 2000 Censuses, the Latino population accounted for 40% of the increase in the nation’s total population. The growing population of Latinos underscores the importance for understanding factors that influence whether and how Latinos take care of their health. According to the U.S. Department of Human Health Service’s Office of Minority Health (OMH), Latinos are at greater risk for health disparities (2003). Factors such as lack of health insurance and access to preventive care play a major role in limiting Latino use of primary health care (Institute of Medicine, 2005). Other significant barriers to preventive health care maintenance behaviors have been identified in current literature such as primary care physician interaction, self-perceived health status, and socio-cultural beliefs and traditions (Rojas-Guyler, King, Montieth and 2008; Meir, Medina, and Ory, 2007; Black, 1999). Despite these studies, there remains less information regarding interpersonal perceptions, environmental dynamics and individual and cultural attitudes relevant to utilization of healthcare (Rojas-Guyler, King, Montieth and 2008; Aguirre-Molina, Molina and Zambrana, 2001). Understanding the perceptions of Latinos and the barriers to health care could directly affect healthcare delivery. Improved healthcare utilization among Latinos could reduce the long term health consequences of many preventable and manageable diseases. The purpose of this study was to explore Latino perceptions of U.S. health care and desired changes by Latinos in the U.S. healthcare system. The study had several objectives, including to explore perceived barriers to healthcare utilization and the resulting effects on health among Latinos, to describe culturally influenced attitudes about health care and use of health care services among Latinos, and to make recommendations for reducing disparities by improving healthcare and its utilization. The current study utilized data that were collected as part of a larger study to examine multidimensional, cross-cultural issues relevant to interactions between healthcare consumers and providers. Qualitative methods were used to analyze four Spanish-language focus group transcripts to interpret cultural influences on perceptions and beliefs among Latinos. Direct coding of transcript content was carried out by two reviewers, who conducted independent reviews of each transcript. Team members developed and refined thematic categories, positive and negative cases, and example text segments for each theme and sub-theme. Incongruities of interpretations were resolved through extensive discussion. Study participants included 44 self-identified Latino adults (16 male, 28 female) between age 18 and 64 years. Thirty seven (84.1%) of the participants were immigrants. The study population comprised eight ethnic subgroups. While 31% of the participants reported being employed on a full-time basis, only 18.4% had medical insurance that was private or employee sponsored. Five major themes regarding the perceptions and healthcare utilization behaviors of Latinos were consistent across all focus groups and were identified during the analysis. These were: (1) healthcare utilization, experience, and access; (2) organizational and institutional systems; (3) communication and interpersonal interactions between healthcare provider, staff, and patient; (4) Latinos’ perception of their own health status; (5) cultural influences on healthcare utilization, which included an innovation termed culturally-bound locus of control. Healthcare utilization was directly influenced by healthcare experience, access, current health status, and cultural factors and indirectly influenced by organizational systems. There was a strong interdependence among the main themes. The ability to communicate and interact effectively with healthcare providers and navigate healthcare systems (organizational and institutional access) significantly influenced the participant’s health care experience, most often (indirectly) impacting utilization negatively. ^ Research such as this can help to identify those perceptions and attitudes held by Latinos concerning utilization or underutilization of healthcare systems. These data suggest that for healthcare utilization to improve among Latinos, healthcare systems must create more culturally competent environments by providing better language services at the organizational level and more culturally sensitive providers at the interpersonal level. Better understanding of the complex interactions between these impediments can aid intervention developments, and help health providers and researchers in determining appropriate, adequate, and effective measurers of care to better increase overall health of Latinos.^

Sex, drugs, and STIs: Syphilis infection and hepatitis B vaccine compliance among illicit drug users in Houston

Although the association between syphilis infection status and compliance with the hepatitis B virus vaccine has been the focus of investigation, there is a lack of data regarding the association between syphilis infection and HBV vaccine compliance. The author investigated the association between the exposure of syphilis infection and the outcome of HBV vaccine completion, defined as degree of constancy and accuracy with which a patient follows a prescribed regimen. A cohort design was employed using interview and serological data from the Drugs, AIDS, STDs, Hepatitis (DASH) Research Project; analysis was restricted to HIV and HBV seronegative (at baseline), illicit drug users residing in Harris County. Syphilis negative and syphilis positive infection status was determined from the serological data while covariates and outcome information were determined from the DASH Project Questionnaire; enrolled subjects (n=1160) were selected from the data. Association between exposure and outcome was assessed with logistic regression adjusted for data-based confounders. ^ A prevalence of 7% and 71% was found for syphilis and HBV vaccine compliance, respectively. When measuring the actual association between syphilis infection status and HBV vaccine compliance, an odds ratio of 1.49 (95% CI: 0.86, 2.72) was obtained. There was a non-significant association between these two variables. 78% of the study population was syphilis positive and completed the vaccine series compared to 70% of the population that was syphilis negative and received all three doses. This finding confirms that there is a difference between syphilis positive and negative drug users with respect to HBV vaccine compliance. The fact that differences were found in these drug users with respect to vaccine schedule supports the idea that sub-group differences may exist and thus merits further investigation. If these differences are confirmed, it is recommended that STI interventions identify community characteristics of their samples and target populations based on practices specific to that community. ^

A screening for anti-West Nile immunoglobulin-G and immunoglobulin-M in the Cameron County Hispanic cohort

This study is designed to be a cross-sectional, retrospective analysis of the seroprevalence of anti-WNV antibodies in 1,006 plasma samples collected from February 2, 2006 to June 18, 2007 originally for The Cameron County Hispanic Cohort: Extreme obesity and uncontrolled diabetes on the U.S.-Mexico border, major concerns for populations with health disparities. The aim of this thesis research is to give a more up-to-date picture of Flavivirus activity in south Texas, which can potentially contribute to the surveillance objective of arboviral control in this area. A West Nile virus (WNV) seroprevalence study in humans in this particular area has never before been completed. Plasma samples were tested using immunoglobulin-G (IgG) and immunoglobulin-M (IgM) WNV enzyme linked immunosorbent assays (ELISA). Estimated seroprevalence for this particular population was 0.98% or 9.8 cases of West Nile disease per 1,000 citizens. After IgG testing, seroprevalence in the study population was found to be 15.4%. Specimens tested for WNV IgG were compared with a subset of specimens (N=803) tested for history of primary dengue virus (DENV) infection. Of the 803 specimens tested for IgG to DENV, 308 were positive. Of the 132 positive WNV IgG specimens in the subset, 131 (99.2%) tested also tested positive for DENV IgG. It would be helpful to use standard plaque reduction neutralization testing to determine if the seroprevalence is in fact lower because of cross-reaction to DENV on testing. Regardless of the possibility of other Flavivirus activity occurring prior to the introduction of WNV into the United States and the potential for cross-reactivity, Texas has ranked in the top 5 states with the highest, laboratory confirmed incidence of infection with WNV since 2003. Indicating that climate factors and the presence of suitable vectors makes Texas a hotspot for WNV activity. ^ A description of the study population by age, gender, and income was done indicating a statistically significant income difference with a mean household income per year being $13,413.55 for a case and $20,268.80 for non-cases (p=0.001). Lower income neighborhoods should be targeted for education and prevention of vector-borne diseases during the summer months in Cameron County. With respect to gender, being male has been noted in the literature to be a risk factor for infection with WNV (25). In this study, females comprised approximately 68% of the study population, they also made up 66.5% of the positive IgG specimens. An odds ratio of 0.91 indicates that women are less likely to be IgG positive for WNV as compared to men; however, this was not found to be significant based on the 95% confidence interval, but is consistent with the literature. When looking at age difference between positive and negative/equivocal cases, there was no statistical difference found between the two groups. ^ We concluded that this study will enable us to understand the epidemiology of WNV transmission since its introduction into the United States and hopefully to maintain or improve the current measures we have in place to prevent infections that are seen annually with WNV.^

Adenovirus-36 infection and obesity related hormones in children

Obesity has a complex, multi-factorial etiology. Infectious agents have recently emerged as a possible contributor to the current obesity epidemic. Seven viruses have demonstrated an association with obesity in animals; however, Adenovirus-36 (Ad-36) is the only known virus associated with obesity in humans. The primary aim of this research was to determine the association between Ad-36 infection and the expression of obesity related hormones in children. Additionally, this study proposed to compare the mean three year change in the level of obesity related hormones between Ad-36 positive and negative children. This study utilized pilot data collected from 98 children at baseline and year three of the Project Heartbeat! cohort. Fasting serum samples were analyzed for the concentration of adiponectin, insulin and leptin. The crude analysis uncovered Ad-36 positive children had significantly lower mean concentrations of insulin (p=0.039) and leptin (p=0.038) at baseline compared to Ad-36 negative children. The results of the adjusted analysis indicated the leptin association with Ad-36 infection at baseline was statistically significant even after controlling for age, sex, ethnicity, and BMI percentile. The longitudinal evaluation revealed individuals with a history of Ad-36 infection experienced a larger mean decrease in adiponectin, a larger mean increase in leptin, and a smaller mean increase in insulin levels over a three year period compared to individuals without a history of infection. These results suggest Ad-36 infection may produce changes in hormone expression. The only statistically significant findings in the crude and adjusted longitudinal analysis occurred at baseline when the children were younger, suggesting physical changes that occur during sexual maturation may mask or enhance Ad-36 induced changes in hormone expression. Furthermore, the longitudinal analysis revealed the duration and course of Ad-36 infection may influence changes in the expression of obesity-related hormones. Taken together, the results of this pilot study are suggestive of an association between Ad-36 infection and the expression of obesity-related hormones.^

Targeting AKT/mTOR Signaling Pathways During Murine Skin Tumor Promotion and the Impact of Dietary Energy Balance Manipulation

The prevalence of obesity has continued to rise over the last several decades in the United States lending to overall increases in risk for chronic diseases including many types of cancer. In contrast, reduction in energy consumption via calorie restriction (CR) has been shown to be a potent inhibitor of carcinogenesis across a broad range of species and tumor types. Previous data has demonstrated differential signaling through Akt and mTOR via the IGF-1R and other growth factor receptors across the diet-induced obesity (DIO)/CR spectrum. Furthermore, mTORC1 is known to be regulated directly via nutrient availability, supporting its role in the link between epithelial carcinogenesis and diet-induced obesity. In an effort to better understand the importance of mTORC1 in the context of both positive and negative energy balance during epithelial carcinogenesis, we have employed the use of specific pharmacological inhibitors, rapamycin (mTORC1 inhibitor) and metformin (AMPK activator) to target mTORC1 or various components of this pathway during skin tumor promotion. Two-stage skin carcinogenesis studies demonstrated that mTORC1 inhibition via rapamycin, metformin or combination treatments greatly inhibited skin tumor development in normal, overweight and obese mice. Furthermore, mechanisms by which these chemopreventive agents may be exerting their anti-tumor effects were explored. In addition, the effect of these compounds on the epidermal proliferative response was analyzed and drastic decreases in epidermal hyperproliferation and hyperplasia were found. Rapamycin also inhibited dermal inflammatory cell infiltration in a dose-dependent manner. Both compounds also blocked or attenuated TPA-induced signaling through epidermal mTORC1 as well as several downstream targets. In addition, inhibition of this pathway by metformin appeared to be, at least in part, dependent on AMPK activation in the skin. Overall, the data indicate that pharmacological strategies targeting this pathway offset the tumor-enhancing effects of DIO and may serve as possible CR mimetics. They suggest that mTORC1 contributes significantly to the process of skin tumor promotion, specifically during dietary energy balance effects. Exploiting the mechanistic information underlying dietary energy balance responsive pathways will help translate decades of research into effective strategies for prevention of epithelial carcinogenesis.

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^