Transcriptional Regulation of a Human H4 Histone Gene is Mediated by Multiple Elements Interacting with Similar Transcription Factors: A Dissertation

Synthesis of histone proteins occurs largely during the S phase of the cell cycle and coincides with DNA replication to provide adequate amounts of histones necessary to properly package newly replicated DNA. Controlling transcription from cell cycle dependent and proliferation specific genes, including histone H4, is an important level of regulation in the overall governance of the cell growth process. Coordination of histone gene transcription results from the cumulative effects of cell signaling pathways, dynamic chromatin structure and multiple transcription factor interactions. The research of this dissertation focused on the characterization and identification of transcription factors interacting on the human histone H4 gene FO108. I also focused on the elucidation of regulatory elements within the histone coding region. Our results suggest a possible mechanism by which a transcription factor facilitates reorganization of histone gene chromatin structure. The histone promoter region between -418 nt and -215 nt, Site III, was previously identified as both a positive and negative cis-regulatory element for transcription. Results of in vitroanalyses presented in this dissertation identified multiple transcription factors interacting at Site III. These factors include H4UA-1/YY1, AP-2, AP-2 like factor and distal factor (NF-1 like factor). Transient transfection experiments show that Site III does not confer significant influence on transcription; however, there may exist a physiological role for Site III which would not be detected in these assay systems. We analyzed the histone H4 gene sequences for additional transcription factor binding motifs and identified several putative YY1 binding sites. Using electrophoretic mobility shift assays (EMSA), we found that Site IV, Site I and two elements within the histone H4 coding region are capable of interacting with YY1. In transient transfection experiments using reporter constructs containing either Site III or one of the coding region elements as potential promoter regulatory elements, and an expression vector encoding YY1, we observed levels of expression up to 2.7 fold higher than from the reporters lacking these elements. Therefore, YY1 appears to interact at multiple regulatory sites of the histone gene and can influence transcription through these elements. Prior to this study, the role of the coding region in histone gene expression was not known. To determine if the coding region is involved in regulating transcription, I constructed and tested a series of heterologous reporter constructs containing various sequences of the histone coding region. Results from these experiments demonstrated that the histone coding region contains three repressor elements. Extensive in vitro analysis indicated that the three repressor elements interact with the repressor CDP/cut. Further analysis showed that CDP/cut interactions with the repressor elements are cell cycle regulated and proliferation specific. CDP/cut interactions increase during the cell cycle when histone transcription decreases. These observations are consistent with the hypothesis that CDP/cutis a cell cycle regulated repressor factor which influences transcription of the histone H4 gene as such. The proximal promoter region of the histone H4 gene between -70 nt and +190 nt is devoid of normal nucleosome structure. This same region contains multiple CDP/cut binding sites. We hypothesized that CDP/cut is involved with chromatin remodeling of the histone gene. DNase I footprinting and EMSA results show purified recombinant CDP/cut interacts specifically with the histone promoter reconstituted into nucleosome cores. Thus, CDP/cutmay facilitate the organization of chromatin of the histone gene. In conclusion, the research presented in this dissertation supports the hypothesis that expression from the human histone H4 gene FO108 is regulated by multiple cis-regulatory elements which interact with several proteins. CDP/cut interacts with Site II, the three repressor elements in the histone coding region and at Distal Site I. YY1 interacts at Site IV, Site III, Site I, and twice in the coding region. ATF/CREB interacts with Site IV and Site I. Distal factor interacts with Site III and within the histone coding region. IRF 2 interacts with Site II and Distal Site I. Thus, histone gene expression is probably regulated by transcription factors CDP/cut, YY1, IRF 2 and ATF/CREB interacting with multiple regulatory elements dispersed throughout its promoter and the coding region. Cell cycle regulation of these transcription factors may contribute to cell cycle dependent expression of the histone gene

Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells

Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine-containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti-Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells

Concerted control of multiple histone promoter factors during cell density inhibition of proliferation in osteosarcoma cells: reciprocal regulation of cell cycle-controlled and bone-related genes

Cell density-induced growth inhibition of osteosarcoma cells (ROS 17/2.8) results in the shutdown of proliferation-specific histone H4 and H2B genes and the concomitant up-regulation of several osteoblast-related genes. In several respects, this reciprocal regulatory relationship is analogous to the proliferation/differentiation transition stage during development of the bone cell phenotype in normal diploid osteoblasts. Here, we comprehensively analyzed the promoter binding activities interfacing with key regulatory elements in the cell cycle-dependent histone and bone-specific osteocalcin genes. Similarly, we examined factors interacting with a series of general transcription regulatory elements that are present in a broad spectrum of promoters. The results show that histone promoter binding activities HiNF-D, HiNF-P/H4TF-2, H4UA-1, and OCT-1, as well as AP-1 activity, are proliferation dependent. These factors decline coordinately during the cessation of proliferation in both ROS 17/2.8 bone tumor cells and normal diploid osteoblasts. Collective down-regulation of these trans-activating factors occurs in both cell types within the physiological context of constitutive regulation of ubiquitous transcription factors (Sp1, ATF, and CCAAT binding proteins). In addition, during growth inhibition of ROS 17/2.8 cells we observe a complex series of modifications in protein/DNA interactions of the osteocalcin gene. These modifications include both increased and decreased representation of promoter factor complexes occurring at steroid hormone response elements as well as tissue-specific basal promoter sequences. These results demonstrate cell growth regulation of the promoter factors binding to the proliferation-specific histone and tissue-specific osteocalcin genes during the cessation of proliferation

Pore-Water Extraction Scale-Up Study for the SX Tank Farm

The phenomena related to pore-water extraction from unsaturated sediments have been previously examined with limited laboratory experiments and numerical modeling. However, key scale-up issues have not yet been addressed. Laboratory experiments and numerical modeling were conducted to specifically examine pore-water extraction for sediment conditions relevant to the vadose zone beneath the SX Tank Farm at Hanford Site in southeastern Washington State. Available SX Tank Farm data were evaluated to generate a conceptual model of the subsurface for a targeted pore-water extraction application in areas with elevated moisture and Tc-99 concentration. The hydraulic properties of the types of porous media representative of the SX Tank Farm target application were determined using sediment mixtures prepared in the laboratory based on available borehole sediment particle size data. Numerical modeling was used as an evaluation tool for scale-up of pore-water extraction for targeted field applications

Psychosocial developmental milestones in men with classic galactosemia

Patients with classic galactosemia suffer from several long term effects of their disease. Research in a group of mainly female patients has shown that these patients may also have a developmental delay with regard to their social aptitude. To study if male galactosemia patients achieve psychosocial developmental milestones more slowly than male peers from the general Dutch population, we assessed their development with the Course of Life Questionnaire (CoLQ). A total of 18 male galactosemia patients participated in this study (response rate 69%): 11 Dutch patients and seven American patients. We found severe delays in the social and psychosexual scales of this questionnaire, but not on the autonomy axis. These results are comparable to an earlier study with a limited number of male patients. The observed delays could be secondary to less developed social skills, cognitive dysfunction, or disrupted language development. We strongly recommend screening of galactosemia patients for developmental delays, to ensure early intervention through social skills training

Bias magnification in ecologic studies: a methodological investigation

<p>Abstract</p> <p>Background</p> <p>As ecologic studies are often inexpensive to conduct, consideration of the magnitude and direction of ecologic biases may be useful in both study design and sensitivity analysis of results. This paper examines three types of ecologic bias: confounding by group, effect measure modification by group, and non-differential exposure misclassification.</p> <p>Methods</p> <p>Bias of the risk difference on the individual and ecologic levels are compared using two-by-two tables, simple equations, and risk diagrams. Risk diagrams provide a convenient way to simultaneously display information from both levels.</p> <p>Results</p> <p>Confounding by group and effect measure modification by group act in the same direction on the individual and group levels, but have larger impact on the latter. The reduction in exposure variance caused by aggregation magnifies the individual level bias due to ignoring groups. For some studies, the magnification factor can be calculated from the ecologic data alone. Small magnification factors indicate little bias beyond that occurring at the individual level. Aggregation is also responsible for the different impacts of non-differential exposure misclassification on individual and ecologic studies.</p> <p>Conclusion</p> <p>The analytical tools developed here are useful in analyzing ecologic bias. The concept of bias magnification may be helpful in designing ecologic studies and performing sensitivity analysis of their results.</p

Neonatal screening for congenital hypothyroidism in the Netherlands: Cognitive and motor outcome at 10 years of age

Contains fulltext : 35300.pdf (publisher's version ) (Open Access)CONTEXT: Patients with thyroidal congenital hypothyroidism (CH-T) born in The Netherlands in 1981-1982 showed persistent intellectual and motor deficits during childhood and adulthood, despite initiation of T(4) supplementation at a median age of 28 d after birth. OBJECTIVE: The present study examined whether advancement of treatment initiation to 20 d had resulted in improved cognitive and motor outcome. DESIGN/SETTING/PATIENTS: In 82 Dutch CH-T patients, born in 1992 to 1993 and treated at a median age of 20 d (mean, 22 d; range, 2-73 d), cognitive and motor outcome was assessed (mean age, 10.5 yr; range, 9.6-11.4 yr). Severity of CH-T was classified according to pretreatment free T(4) concentration. MAIN OUTCOME MEASURE: Cognitive and motor outcome of the 1992-1993 cohort in comparison to the 1981 to 1982 cohort was the main outcome measure. RESULTS: Patients with severe CH-T had lower full-scale (93.7), verbal (94.9), and performance (93.9) IQ scores than the normative population (P < 0.05), whereas IQ scores of patients with moderate and mild CH-T were comparable to those of the normative population. In all three severity subgroups, significant motor problems were observed, most pronounced in the severe CH-T group. No correlations were found between starting day of treatment and IQ or motor outcome. CONCLUSIONS: Essentially, findings from the 1992-1993 cohort were similar to those of the 1981-1982 cohort. Apparently, advancing initiation of T(4) supplementation from 28 to 20 d after birth did not result in improved cognitive or motor outcome in CH-T patients

Evaluation of Soil Flushing for Application to the Deep Vadose Zone in the Hanford Central Plateau

Soil flushing was included in the Deep Vadose Zone Treatability Test Plan for the Hanford Central Plateau as a technology with the potential to remove contaminants from the vadose zone. Soil flushing operates through the addition of water, and if necessary an appropriate mobilizing agent, to mobilize contaminants and flush them from the vadose zone and into the groundwater where they are subsequently captured by a pump-and-treat system. There are uncertainties associated with applying soil flushing technology to contaminants in the deep vadose zone at the Hanford Central Plateau. The modeling and laboratory efforts reported herein are intended to provide a quantitative assessment of factors that impact water infiltration and contaminant flushing through the vadose zone and into the underlying groundwater. Once in the groundwater, capture of the contaminants would be necessary, but this aspect of implementing soil flushing was not evaluated in this effort. Soil flushing was evaluated primarily with respect to applications for technetium and uranium contaminants in the deep vadose zone of the Hanford Central Plateau

TABADO: "Evaluation of a smoking cessation program among Adolescents in Vocational Training Centers": Study protocol

<p>Abstract</p> <p>Background</p> <p>Most of the efforts to reduce teenagers' tobacco addiction have focused on smoking prevention and little on smoking cessation. A smoking cessation program (TABADO study), associating pharmacologic and cognitive-behavioural strategy, on a particularly vulnerable population (vocational trainees), was developed. This study aims to evaluate the efficacy of the program which was offered to all smokers in a population aged 15 to 20 years in Vocational Training Centers (VTC). This paper presents the TABADO study protocol.</p> <p>Methods</p> <p>The study is quasi-experimental, prospective, evaluative and comparative and takes place during the 2 years of vocational training. The final population will be composed of 2000 trainees entering a VTC in Lorraine, France, during the 2008-2009 period. The intervention group (1000 trainees) benefited from the TABADO program while no specific intervention took place in the "control" group (1000 trainees) other than the treatment and education services usually available. Our primary outcome will be the tobacco abstinence rate at 12 months.</p> <p>Discussion</p> <p>If the program proves effective, it will be a new tool in the action against smoking in populations that have been seldom targeted until now. In addition, the approach could be expanded to other young subjects from socially disadvantaged backgrounds in the context of a public health policy against smoking among adolescents.</p> <p>Trial registration</p> <p>Clinical trial identification number is NTC00973570.</p

How Well Do Randomized Trials Inform Decision Making: Systematic Review Using Comparative Effectiveness Research Measures on Acupuncture for Back Pain

Background: For Comparative Effectiveness Research (CER) there is a need to develop scales for appraisal of available clinical research. Aims were to 1) test the feasibility of applying the pragmatic-explanatory continuum indicator summary tool and the six CER defining characteristics of the Institute of Medicine to RCTs of acupuncture for treatment of low back pain, and 2) evaluate the extent to which the evidence from these RCTs is relevant to clinical and health policy decision making. Methods: We searched Medline, the AcuTrials™ Database to February 2011 and reference lists and included full-report randomized trials in English that compared needle acupuncture with a conventional treatment in adults with non-specific acute and/or chronic low back pain and restricted to those with ≥30 patients in the acupuncture group. Papers were evaluated by 5 raters. Principal Findings: From 119 abstracts, 44 full-text publications were screened and 10 trials (4,901 patients) were evaluated. Due to missing information and initial difficulties in operationalizing the scoring items, the first scoring revealed inter-rater and inter-item variance (intraclass correlations 0.02-0.60), which improved after consensus discussions to 0.20-1.00. The 10 trials were found to cover the efficacy-effectiveness continuum; those with more flexible acupuncture and no placebo control scored closer to effectiveness. Conclusion: Both instruments proved useful, but need further development. In addition, CONSORT guidelines for reporting pragmatic trials should be expanded. Most studies in this review already reflect the movement towards CER and similar approaches can be taken to evaluate comparative effectiveness relevance of RCTs for other treatments. © 2012 Witt et al.published_or_final_versio