Mutational Heterogeneity in Cancer

Thesis (Ph.D.)--University of Washington, 2014-06

Highly accurate RNA and DNA sequencing: Application to longstanding questions in aging and cancer

Thesis (Ph.D.)--University of Washington, 2016-06

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dual-axis electron microscope tomography to examine the architecture of the Golgi in three dimensions at approximate to6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all bypass interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion/fission of cisternal membranes and control the directionality and specificity of such events, and (it) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.

Columnar cell lesions of the breast: The missing link in breast cancer progression? A morphological and molecular analysis

Columnar cell lesions (CCLs) of the breast are a spectrum of lesions that have posed difficulties to pathologists for many years, prompting discussion concerning their biologic and clinical significance. We present a study of CCL in context with hyperplasia of usual type (HUT) and the more advanced lesions ductal carcinoma in situ (DCIS) and invasive ductal carcinoma. A total of 81 lesions from 18 patients were subjected to a comprehensive morphologic review based upon a modified version of Schnitt's classification system for CCL, immunophenotypic analysis (estrogen receptor [ER], progesterone receptor [PgR], Her2/neu, cytokeratin 5/6 [CK5/6], cytokeratin 14 [CK14], E-cadherin, p53) and for the first time, a whole genome molecular analysis by comparative genomic hybridization. Multiple CCLs from 3 patients were studied in particular detail, with topographic information and/or showing a morphologic spectrum of CCL within individual terminal duct lobular units. CCLs were ER an PgR positive, CK5/6 and CK14 negative, exhibit low numbers of genetic alterations and recurrent 16q loss, features that are similar to those of low grade in situ and invasive carcinoma. The molecular genetic profiles closely reflect the degree of proliferation and atypia in CCL, indicating some of these lesions represent both a morphologic and molecular continuum. In addition, overlapping chromosomal alterations between CCL and more advanced lesions within individual terminal duct lobular units suggest a commonality in molecular evolution. These data further support the hypothesis that CCLs are a nonobligate, intermediary step in the development of some forms of low grade in situ and invasive carcinoma. Copyright: © 2005 Lippincott Williams & Wilkins, Inc.

The importance of modelling heterogeneity in complex disease: application to NIMH Schizophrenia Genetics Initiative data

As for other complex diseases, linkage analyses of schizophrenia (SZ) have produced evidence for numerous chromosomal regions, with inconsistent results reported across studies. The presence of locus heterogeneity appears likely and may reduce the power of linkage analyses if homogeneity is assumed. In addition, when multiple heterogeneous datasets are pooled, intersample variation in the proportion of linked families ( a) may diminish the power of the pooled sample to detect susceptibility loci, in spite of the larger sample size obtained. We compare the significance of linkage. findings obtained using allele- sharing LOD scores ( LODexp) - which assume homogeneity - and heterogeneity LOD scores ( HLOD) in European American and African American NIMH SZ families. We also pool these two samples and evaluate the relative power of the LODexp and two different heterogeneity statistics. One of these ( HLOD- P) estimates the heterogeneity parameter a only in aggregate data, while the second ( HLOD- S) determines a separately for each sample. In separate and combined data, we show consistently improved performance of HLOD scores over LODexp. Notably, genome-wide significant evidence for linkage is obtained at chromosome 10p in the European American sample using a recessive HLOD score. When the two samples are combined, linkage at the 10p locus also achieves genome-wide significance under HLOD- S, but not HLOD- P. Using HLOD- S, improved evidence for linkage was also obtained for a previously reported region on chromosome 15q. In linkage analyses of complex disease, power may be maximised by routinely modelling locus heterogeneity within individual datasets, even when multiple datasets are combined to form larger samples.

Ena/VASP proteins can regulate distinct modes of actin organization at cadherin-adhesive contacts

Functional interactions between classical cadherins and the actin cytoskeleton involve diverse actin activities, including filament nucleation, cross-linking, and bundling. In this report, we explored the capacity of Ena/VASP proteins to regulate the actin cytoskeleton at cadherin-adhesive contacts. We extended the observation that Ena/vasodilator-stimulated phosphoprotein (VASP) proteins localize at cell-cell contacts to demonstrate that E-cadherin homophilic ligation is sufficient to recruit Mena to adhesion sites. Ena/VASP activity was necessary both for F-actin accumulation and assembly at cell-cell contacts. Moreover, we identified two distinct pools of Mena within individual homophilic adhesions that cells made when they adhered to cadherin-coated substrata. These Mena pools localized with Arp2/3-driven cellular protrusions as well as at the tips of cadherin-based actin bundles. Importantly, Ena/VASP activity was necessary for both modes of actin activity to be expressed. Moreover, selective depletion of Ena/VASP proteins from the tips of cadherin-based bundles perturbed the bundles without affecting the protrusive F-actin pool. We propose that Ena/VASP proteins may serve as higher order regulators of the cytoskeleton at cadherin contacts through their ability to modulate distinct modes of actin organization at those contacts.

Molecular characterization of Thelastomatoidea (Nematoda : Oxyurida) from cockroaches in Australia

A molecular approach was used to genetically characterize 5 species (Aoruroides queenslandensis. Blattophila sphaerolaima, Cordonicola gibsoni, Desmicola ornato and Leidynemella fusiformis) belonging to the superfamily. Thelastomatoidea fi (Nematoda: Oxyurida), a group of pinworms that parasitizes terrestrial arthropods. The D3 domain of the large subunit Of nuclear ribosomal RNA (LSU) was sequenced for individual specimens, and the analysis of the sequence data allowed the genetic relationships of the 5 species to be studied dagger. The sequence variation in the D3 domain within individual species (0-1-8%) was significantly less than the differences among species (4(.)3-12(.)4%). Phylogenetic analyses, Using maximum parsimony, maximum likelihood, and neighbour-joining, tree-building methods, established relationships among the 5 species of Thelastomatoidea and Oxyuris equi (a species of the order Oxyurida). The molecular approach employed provides the prospect for developing DNA tools for the specific identification of the Thelastomatoidea, irrespective of developmental stage and sex, as a basis for systematic, ecological and/or population genetic investigations of members within this superfamily.

Physical activity and strength of the femoral neck during the adolescent growth spurt: A longitudinal analysis

Loading of the femoral neck (FN) is dominated by bending and compressive stresses. We hypothesize that adaptation of the FN to physical activity would be manifested in the cross-sectional area (CSA) and section modulus (Z) of bone, indices of axial and bending strength, respectively. We investigated the influence of physical activity on bone strength during adolescence using 7 years of longitudinal data from 109 boys and 121 girls from the Saskatchewan Paediatric Bone and Mineral Accrual Study (PBMAS). Physical activity data (PAC-Q physical activity inventory) and anthropometric measurements were taken every 6 months and DXA bone scans were measured annually (Hologic QDR2000, array mode). We applied hip structural analysis to derive strength and geometric indices of the femoral neck using DXA scans. To control for maturation, we determined a biological maturity age defined as years from age at peak height velocity (APHV). To account for the repeated measures within individual nature of longitudinal data, multilevel random effects regression analyses were used to analyze the data. When biological maturity age and body size (height and weight) were controlled, in both boys and girls, physical activity was a significant positive independent predictor of CSA and Z of the narrow region of the femoral neck (P < 0.05). There was no independent effect of physical activity on the subperiosteal width of the femoral neck. When leg length and leg lean mass were introduced into the random effects models to control for size and muscle mass of the leg (instead of height and weight), all significant effects of physical activity disappeared. Even among adolescents engaged in normal levels of physical activity, the statistically significant relationship between physical activity and indices of bone strength demonstrate that modifiable lifestyle factors like exercise play an important role in optimizing bone strength during the growing years. Physical activity differences were explained by the interdependence between activity and lean mass considerations. Physical activity is important for optimal development of bone strength. (c) 2005 Elsevier Inc. All rights reserved.

Velocity distributions in a plate heat exchanger channel

The development of a Laser Doppler Anemometer technique to measure the velocity distribution in a commercial plate heat exchanger is described. Detailed velocity profiles are presented and a preliminary investigation is reported on flow behaviour through a single cell in the channel matrix. The objective of the study was to extend previous investigations of plate heat exchanger flow patterns in the laminar range with the eventual aim of establishing the effect of flow patterns on heat transfer performance, thus leading to improved plate heat exchanger design and design methods. Accurate point velocities were obtained by Laser Anemometry in a perspex replica of the metal channel. Oil was used as a circulating liquid with a refractive index matched to that of the perspex so that the laser beams were not distorted. Cell-by-cell velocity measurements over a range of Reynolds number up to ten showed significant liquid mal-distribution. Local cell velocities were found to be as high as twenty seven times average velocity, contrary to the previously held belief of four times. The degree of mal-distribution varied across the channel as well as in the vertical direction, and depended on the upward or downward direction of flow. At Reynolds numbers less than one, flow zig-zagged from one side of the channel to the other in wave form, but increases in Reynolds number improved liquid distribution. A detailed examination of selected cells showed velocity variations in different directions, together with variation within individual cells. Experimental results are also reported on the flow split when passing through a single cell in a section of a channel . These observations were used to explain mal-distribution in the perspex channel itself.

Factors determining the growth curve of the foliose lichen Parmelia conspersa

Growth curves of the foliose lichen Parmelia conspersa (Ehrh. Ex Ach.)Ach. Were obtained by plotting radial growth (RGR, mm yr-1) of the fastest measured lobe, the slowest measured lobe, a randomly selected lobe, and by averaging a sample of lobes from each thallus against thallus diameter. Growth curves derived from the fastest-growing lobe and by averaging lobes were asymptotic and could be fitted by the growth model of Aplin and Hill. Mean lobe width increased with thallus size, reaching a maximum at approx. 4.5 cm thallus diameter. In four out of six thalli, radial growth of lobes over four months was positively correlated with initial lobe width or area. The RGR of isolated lobes was unaffected until the base of the lobe was removed to within 1-2 mm of the tip. The concentration (micrograms mg-1 biomass) of ribitol, arabitol and mannitol was greater in the marginal lobes of large than in small thalli. The results suggested that the growth curve of P. conspersa is determined by processes that occur within individual marginal lobes and can be explained by the Aplin and Hill model. Changes in lobe width and in the productive capacity of individual lobes with thallus size are likely to be more important factors than the degree of translocation within the lobe in determining the growth curve.

Immunoinformatic evaluation of multiple epitope ensembles as vaccine candidates:E coli 536

Epitope prediction is becoming a key tool for vaccine discovery. Prospective analysis of bacterial and viral genomes can identify antigenic epitopes encoded within individual genes that may act as effective vaccines against specific pathogens. Since B-cell epitope prediction remains unreliable, we concentrate on T-cell epitopes, peptides which bind with high affinity to Major Histacompatibility Complexes (MHC). In this report, we evaluate the veracity of identified T-cell epitope ensembles, as generated by a cascade of predictive algorithms (SignalP, Vaxijen, MHCPred, IDEB, EpiJen), as a candidate vaccine against the model pathogen uropathogenic gram negative bacteria Escherichia coli (E-coli) strain 536 (O6:K15:H31). An immunoinformatic approach was used to identify 23 epitopes within the E-coli proteome. These epitopes constitute the most promiscuous antigenic sequences that bind across more than one HLA allele with high affinity (IC50 <50nM). The reliability of software programmes used, polymorphic nature of genes encoding MHC and what this means for population coverage of this potential vaccine are discussed.

The response to and effects of microcomputers in the education sector:the introduction of an innovation in local authority secondary schools

The study addresses the introduction of an innovation of new technology into a bureaucratic profession. The organisational setting is that of local authority secondary schools at a time at which microcomputers were being introduced in both the organisational core (for teaching) and its periphery (school administration). The research studies innovation-adopting organisations within their sectoral context; key actors influencing the innovation are identified at the levels of central government, local government and schools.A review of the literature on new technology and innovation (including educational innovation), and on schools as organisations in a changing environment leads to the development of the conceptual framework of the study using a resource dependency model within a cycle of the acquisition, allocation and utilisation of financial, physical and intangible resources. The research methodology is longitudinal and draws from both positivist and interpretive traditions. lt includes an initial census of the two hundred secondary schools in four local education authorities, a final survey of the same population, and four case studies, using both interview methods and documentation. Two modes of innovation are discerned. In respect of administrative use a rationalising, controlling mode is identified, with local education authorities developing standardised computer-assisted administrative systems for use in schools. In respect of curricular use, in contrast, teachers have been able to maintain an indeterminate occupational knowledge base, derived from an ideology of professionalism in respect of the classroom use of the technology. The mode of innovation in respect of curricular use has been one of learning and enabling. The resourcing policies of central and local government agencies affect the extent of use of the technology for teaching purposes, but the way in which it is used is determined within individual schools, where staff with relevant technical expertise significantly affect the course of the innovation.

Predictors of safer sex behaviors of midlife and older women

Heterosexual transmission of the human immunodeficiency virus (HIV) in midlife and older women is increasing, yet little is known about the safer sex practices of these women. Identification of HIV risk and protective factors necessitates understanding of the influences of individual, interpersonal, and socio-environmental factors on the sexual behaviors of this understudied, at-risk population. The purpose of this study was to determine the influence of self esteem, sensation seeking, self silencing, sexual assertiveness, and HIV-stigma on the safer sex behaviors of women aged 50 and older. ^ This study was guided by the ecological perspective which emphasizes the multilevel factors affecting health behaviors within individual, interpersonal, and socio-environmental contexts. A community-based, ethnically diverse sample of 572 women aged 50 to 93 (M = 63.6 years, SD = 10.5) completed a 128-item anonymous questionnaire. This study used a cross-sectional, correlational research design. The data were analyzed using Pearson correlation coefficients and multiple regression analysis. ^ Results from the regression analysis with the predictors (i.e., ethnicity, education, self esteem, sensation seeking, self silencing, sexual assertiveness, and HIV stigma) indicated the model significantly predicted safer sex behaviors (p < .001). Self silencing (β = -.115, p < .05) was a significant predictor. The lower the self silencing scores, the higher the safer sex behavior scores. Further exploration of the data revealed that the greater the age of the woman, the more likely she is to engage in safer sex behaviors (β = .173, p < .001). While the data showed this model was statistically significant, its practical significance may be limited due to the low proportion of variance explained by age and self silencing. HIV/AIDS prevention interventions that are socially anchored, age-appropriate, and gender-specific are discussed and recommendations for socially meaningful strategies to reduce the number of new cases of HIV/AIDS in midlife and older women are presented. ^

Hispanic adolescents with severe substance abuse problems: Parental involvement, acculturation-related factors, and attachment

The main objective of the study was to investigate the relationship between parent-related, acculturation-related, and substance use-related variables found within individual, familial/parental, peer and school adolescent ecological domains, in a clinical sample (i.e. adolescents who met criteria for a Diagnostic Statistical Manual-IV [DSMIV] clinical diagnosis of substance abuse/dependence) of Hispanic adolescents from Miami, Florida. ^ The sample for this study consisted of 94 adolescent-mother pairs. The adolescent sample was 65% male, and 35% female, with a mean age of 15 years. More than half of the adolescents were born in the United States (60%) and had resided in the U.S. for an average of 12 years; 80% of the caregivers (primarily mothers) were foreign-born and lived in the U.S. for an average of 21 years. ^ Correlation and hierarchical regression were used to answer the research questions. The findings indicate that the hypothesized model and corresponding anticipated effect of the relationship between parental school and peer involvement on adolescents’ frequency of alcohol, marijuana and cocaine use was not supported by the data. Parental “acculturation-related” variables did not explain any of the variance in adolescent substance use frequency in this sample. Mediation and moderation models were not supported either. However, some interesting relationships were found: ^ The larger the acculturation gap, the lower the parental involvement in school tended to be (r = -.21, p < .05). Adolescents who experienced a greater acculturation gap with their parents (-.81, p >.01) had an earlier onset of marijuana (-.33, p < .01) and cocaine use (r = -.24, p <.01). The less acculturated parents experienced more parenting stress (r = -.31, p = < .01). Attachment was positively associated with parental peer involvement (r = .24, p < .05) and inversely associated with parenting acculturative stress (r = -.24, p < .05). Attachment was also positively associated with marijuana (r = .39, p < .01) and cocaine use (r = .33, p < .01). Adolescent males reported being more attached to their mothers when compared to adolescent females (r = .22, p >.05), they also reported using marijuana more frequently than females (.21, p >.05). ^

Hispanic Adolescents With Severe Substance Abuse Issues: Parental Involvement, Acculturation-Related Factors and Attachment

The main objective of the study was to investigate the relationship between parent-related, acculturation-related, and substance use-related variables found within individual, familial/parental, peer and school adolescent ecological domains, in a clinical sample (i.e. adolescents who met criteria for a Diagnostic Statistical Manual-IV [DSM-IV] clinical diagnosis of substance abuse/dependence) of Hispanic adolescents from Miami, Florida. The sample for this study consisted of 94 adolescent-mother pairs. The adolescent sample was 65% male, and 35% female, with a mean age of 15 years. More than half of the adolescents were born in the United States (60%) and had resided in the U.S. for an average of 12 years; 80% of the caregivers (primarily mothers) were foreign-born and lived in the U.S. for an average of 21 years. Correlation and hierarchical regression were used to answer the research questions. The findings indicate that the hypothesized model and corresponding anticipated effect of the relationship between parental school and peer involvement on adolescents’ frequency of alcohol, marijuana and cocaine use was not supported by the data. Parental “acculturation-related” variables did not explain any of the variance in adolescent substance use frequency in this sample. Mediation and moderation models were not supported either. However, some interesting relationships were found: The larger the acculturation gap, the lower the parental involvement in school tended to be (r = -.21, p < .05). Adolescents who experienced a greater acculturation gap with their parents (-.81, p >.01) had an earlier onset of marijuana (-.33, p < .01) and cocaine use (r = -.24, p .05), they also reported using marijuana more frequently than females (.21, p >.05).