Clonal analysis of stably transduced human epidermal stem cells in culture.

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population.

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.

Homozygous deletions have been central to the discovery of several tumor-suppressor genes, but their finding has often been either serendipitous or the result of a directed search. A recently described technique [Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, 946-951] held out the potential to efficiently discover such events in an unbiased manner. Here we present the application of the representational difference analysis (RDA) to the study of cancer. We cloned two DNA fragments that identified a homozygous deletion in a human pancreatic adenocarcinoma, mapping to a 1-centimorgan region at chromosome 13q12.3 flanked by the markers D13S171 and D13S260. Interestingly, this lies within the 6-centimorgan region recently identified as the BRCA2 locus of heritable breast cancer susceptibility. This suggests that the same gene may be involved in multiple tumor types and that its function is that of a tumor suppressor rather than that of a dominant oncogene.

Analysis of the colorimetric properties of goniochromatic colors using the MacAdam limits under different light sources

Technological innovation in all areas has led to the appearance in recent years of new metallic and pearlescent materials, yet no exhaustive studies have been conducted to assess their colorimetric capabilities. The chromatic variability of these special-effect pigments may largely be due to the three-dimensional effect of their curved shapes and orientations when they are directionally or diffusely illuminated. Our study examines goniochromatic colors using the optimal colors (MacAdam limits) associated with normal colors (photometric scale of relative spectral reflectance from 0 to 1) under certain conventional illuminants and other light sources. From a database of 91 metallic and interference samples and using a multi-gonio-spectrophotometer, we analyzed samples with lightness values of more than 100 and others with lightness values of less than 100, but with higher chromaticities than optimal colors, which places them beyond the MacAdam limits. Our study thus demonstrates the existence of chromatic perceptions beyond the normal solid color associated with these materials and independent of the light source. The challenge for future research, therefore, is to replicate and render these color appearances in current and future color reproduction technologies for computer graphics.

Nonlinear dynamics in discretionary accruals: An analysis of bank loan-loss provisions

Several studies have analyzed discretionary accruals to address earnings-smoothing behaviors in the banking industry. We argue that the characteristic link between accruals and earnings may be nonlinear, since both the incentives to manipulate income and the practical way to do so depend partially on the relative size of earnings. Given a sample of 15,268 US banks over the period 1996–2011, the main results in this paper suggest that, depending on the size of earnings, bank managers tend to engage in earnings-decreasing strategies when earnings are negative (“big-bath”), use earnings-increasing strategies when earnings are positive, and use provisions as a smoothing device when earnings are positive and substantial (“cookie-jar” accounting). This evidence, which cannot be explained by the earnings-smoothing hypothesis, is consistent with the compensation theory. Neglecting nonlinear patterns in the econometric modeling of these accruals may lead to misleading conclusions regarding the characteristic strategies used in earnings management.

Comparative analysis of the relationship between anterior and posterior corneal shape analyzed by Scheimpflug photography in normal and keratoconus eyes

Background To analyze and compare the relationship between anterior and posterior corneal shape evaluated by a tomographic system combining the Scheimpflug photography and Placido-disc in keratoconus and normal healthy eyes, as well as to evaluate its potential diagnostic value. Methods Comparative case series including a sample of 161 eyes of 161 subjects with ages ranging from 7 to 66 years and divided into two groups: normal group including 100 healthy eyes of 100 subjects, and keratoconus group including 61 keratoconus eyes of 61 patients. All eyes received a comprehensive ophthalmologic examination including an anterior segment analysis with the Sirius system (CSO). Antero-posterior ratios for corneal curvature (k ratio) and shape factor (p ratio) were calculated. Logistic regression analysis was used to evaluate if some antero–posterior ratios combined with other clinical parameters were predictors of the presence of keratoconus. Results No statistically significant differences between groups were found in the antero–posterior k ratios for 3-, 5- and 7-mm diameter corneal areas (p ≥ 0.09). The antero–posterior p ratio for 4.5- and 8-mm diameter corneal areas was significantly higher in the normal group than in the keratoconus group (p < 0.01). The k ratio for 3, 5, and 7 mm was significantly higher in the keratoconus grade IV subgroup than in the normal group (p < 0.01). Furthermore, significant differences were found in the p ratio between the normal group and the keratoconus grade II subgroup (p ≤ 0.01). Finally, the logistic regression analysis identified as significant independent predictors of the presence of keratoconus (p < 0.01) the 8-mm anterior shape factor, the anterior chamber depth, and the minimal corneal thickness. Conclusions The antero-posterior k and p ratios are parameters with poor prediction ability for keratoconus, in spite of the trend to the presence of more prolate posterior corneal surfaces compared to the anterior in keratoconus eyes.

Motivational profiles Spanish students of Compulsory Secondary Education: Differential analysis of academic self-attributions

Academic goals and academic self-attributions are relevant variables in school settings. The objective of this study is to identify whether there are combinations of multiple goals that lead to different motivational profiles and to determine whether there are significant differences between the groups obtained regarding causal attributions of success and failure (ability, effort, or external causes) in Mathematics and Language and Literature, and in overall academic performance. The Goal Achievement Tendencies Questionnaire (AGTQ) and the Sydney Attribution Scale (SAS) were administered to a sample of 2022 students of compulsory secondary education, ranging in age from 12 to 16 years (M = 13.81, SD = 1.35). Cluster analysis identified four motivational profiles: a group of students with a high generalized motivation profile, a group of students with low generalized motivation profile, a group of students with predominance of learning goals and achievement goals, and a final group of students with predominance of social reinforcement goals. Results revealed statistically significant differences between the profiles obtained in academic self-attributions.

Multivariate statistical analysis of highway accident and highway conditions. Final report.

Transportation Department, Office of University Research, Washington, D.C.

An analysis of ultimate performance measures to determine total project impact. (A supplementary report to analytic studies no. 1 and no. 3 for project year 1973.).

National Highway Traffic Safety Administration, Washington, D.C.

Analysis of the Hydrogeological Conditions and the Effects of Development on the Hydraulic Head at Redmond Ridge East

Redmond Ridge East (RRE) is a large-scale master plan community in East King County, WA. In this report, I evaluate the spatial variability of the Quaternary Advance Outwash (Qva) at RRE and the time-series data for 16 water wells with the intent to better understand groundwater below the RRE area. I investigate changes between pre- and post-development conditions through the determination of temporal changes in annual water level, annual water level fluctuations, hydraulic head response to precipitation, and ambient drainage of the aquifer. I also perform a basic analysis of the annual aquifer recharge and a determination for the storage through the implementation of the water table fluctuation (WTF) method. Associated Earth Sciences (AESI) was tasked with monitoring the geological and environmental impacts during the development of RRE and collected the data I use in this report. AESI involvement in monitoring began in 1998 and extends to the present. Sixteen wells were identified in the RRE area with adequate temporal data to conduct the analysis. A comparison of the well logs and aquifer testing data allowed local variations in the Qva to be mapped. The WTF was used to determine a range of reasonable specific yield values for locations where the Qva was unconfined. Yearly average of the seasonal water level high and lows, and the fluctuations were quantified. Temporal relationships were established through linear regression. The average water level was found to be increasing in some locations, and the corresponding fluctuations were found to decrease. However, no clear change between pre- and post-development was observed. The response of hydraulic head to precipitation was investigated through an analysis of hydrographs for ten wells. Periods of consistent response and the corresponding precipitation during each period were delineated. A linear relationship between precipitation and water level change was determined. The threshold precipitation under which there is a positive response in the hydraulic head was established. No observable changes were apparent between pre- and post-development conditions. The ambient drainage for the Qva was calculated using recessional periods on the hydrograph. The transmissivity of Qva varies with thickness of the overlying lodgment till and thickness of the Qva, itself. Water level fluctuations observed in the Qva are consistent with regional observations. Localized areas in the Qva display the large 10 foot fluctuations and these anomalies are likely due to a combination of the local variability in the storativity as well as the concentration and channeling of water due to geographical variations in the Qva and the overlying topography. All trends seen in the RRE area remained relatively constant through time. There was no evidence showing an effect of development on the hydraulic head at RRE. This implies that the style and distribution of infiltration has not changed as a result of development, and that any measures in place are properly mitigating the effects of development on the RRE region.

A combined analysis of two randomized controlled trials evaluating the effect of Mindfulness-Based Stress Reduction on self-reported emotional experience and physiological symptoms among Veterans with Posttraumatic Stress Disorder

Thesis (Master's)--University of Washington, 2016-06

Pregnancy Intention and Postpartum Depressive Symptoms: An Analysis of PRAMS Data

Thesis (Master's)--University of Washington, 2016-06