FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

NOVEL PHANTOMS AND POST-PROCESSING FOR DIFFUSION SPECTRUM IMAGING

High Angular Resolution Diffusion Imaging (HARDI) techniques, including Diffusion Spectrum Imaging (DSI), have been proposed to resolve crossing and other complex fiber architecture in the human brain white matter. In these methods, directional information of diffusion is inferred from the peaks in the orientation distribution function (ODF). Extensive studies using histology on macaque brain, cat cerebellum, rat hippocampus and optic tracts, and bovine tongue are qualitatively in agreement with the DSI-derived ODFs and tractography. However, there are only two studies in the literature which validated the DSI results using physical phantoms and both these studies were not performed on a clinical MRI scanner. Also, the limited studies which optimized DSI in a clinical setting, did not involve a comparison against physical phantoms. Finally, there is lack of consensus on the necessary pre- and post-processing steps in DSI; and ground truth diffusion fiber phantoms are not yet standardized. Therefore, the aims of this dissertation were to design and construct novel diffusion phantoms, employ post-processing techniques in order to systematically validate and optimize (DSI)-derived fiber ODFs in the crossing regions on a clinical 3T MR scanner, and develop user-friendly software for DSI data reconstruction and analysis. Phantoms with a fixed crossing fiber configuration of two crossing fibers at 90° and 45° respectively along with a phantom with three crossing fibers at 60°, using novel hollow plastic capillaries and novel placeholders, were constructed. T2-weighted MRI results on these phantoms demonstrated high SNR, homogeneous signal, and absence of air bubbles. Also, a technique to deconvolve the response function of an individual peak from the overall ODF was implemented, in addition to other DSI post-processing steps. This technique greatly improved the angular resolution of the otherwise unresolvable peaks in a crossing fiber ODF. The effects of DSI acquisition parameters and SNR on the resultant angular accuracy of DSI on the clinical scanner were studied and quantified using the developed phantoms. With a high angular direction sampling and reasonable levels of SNR, quantification of a crossing region in the 90°, 45° and 60° phantoms resulted in a successful detection of angular information with mean ± SD of 86.93°±2.65°, 44.61°±1.6° and 60.03°±2.21° respectively, while simultaneously enhancing the ODFs in regions containing single fibers. For the applicability of these validated methodologies in DSI, improvement in ODFs and fiber tracking from known crossing fiber regions in normal human subjects were demonstrated; and an in-house software package in MATLAB which streamlines the data reconstruction and post-processing for DSI, with easy to use graphical user interface was developed. In conclusion, the phantoms developed in this dissertation offer a means of providing ground truth for validation of reconstruction and tractography algorithms of various diffusion models (including DSI). Also, the deconvolution methodology (when applied as an additional DSI post-processing step) significantly improved the angular accuracy of the ODFs obtained from DSI, and should be applicable to ODFs obtained from the other high angular resolution diffusion imaging techniques.

BIOMEDICAL LANGUAGE UNDERSTANDING AND EXTRACTION (BLUE-TEXT): A MINIMAL SYNTACTIC, SEMANTIC METHOD

Clinical text understanding (CTU) is of interest to health informatics because critical clinical information frequently represented as unconstrained text in electronic health records are extensively used by human experts to guide clinical practice, decision making, and to document delivery of care, but are largely unusable by information systems for queries and computations. Recent initiatives advocating for translational research call for generation of technologies that can integrate structured clinical data with unstructured data, provide a unified interface to all data, and contextualize clinical information for reuse in multidisciplinary and collaborative environment envisioned by CTSA program. This implies that technologies for the processing and interpretation of clinical text should be evaluated not only in terms of their validity and reliability in their intended environment, but also in light of their interoperability, and ability to support information integration and contextualization in a distributed and dynamic environment. This vision adds a new layer of information representation requirements that needs to be accounted for when conceptualizing implementation or acquisition of clinical text processing tools and technologies for multidisciplinary research. On the other hand, electronic health records frequently contain unconstrained clinical text with high variability in use of terms and documentation practices, and without commitmentto grammatical or syntactic structure of the language (e.g. Triage notes, physician and nurse notes, chief complaints, etc). This hinders performance of natural language processing technologies which typically rely heavily on the syntax of language and grammatical structure of the text. This document introduces our method to transform unconstrained clinical text found in electronic health information systems to a formal (computationally understandable) representation that is suitable for querying, integration, contextualization and reuse, and is resilient to the grammatical and syntactic irregularities of the clinical text. We present our design rationale, method, and results of evaluation in processing chief complaints and triage notes from 8 different emergency departments in Houston Texas. At the end, we will discuss significance of our contribution in enabling use of clinical text in a practical bio-surveillance setting.

A systematic review of community-based participatory research interventions in pediatric and adolescent populations

Background. Community-based participatory research (CBPR) is a collaborative approach to research actively involving community members in all aspects of the research process. CBPR is not a new research method, but an approach that has gained increased attention in the field of public health over the last several years. Recognition of the inequalities in health status associated with social and environmental factors have led to calls for a renewed focus on ecological approaches to research. Ecological approaches acknowledge that the health of the community is dependent on an interaction between behavioral and environmental factors affecting the entire population. While many published studies document the benefits of CBPR in difficult-to-reach populations and describe successful implementation of this approach in adult populations, relatively few studies have been conducted in child and adolescent populations. Given that children and adolescents are particularly sensitive to the effects of their physical environments and may also be distrustful of outsiders, ecological approaches involving the community as partners, such as CBPR, may be especially useful in this population. ^ Objective. This thesis reviews published studies using a community-based participatory research approach in children and adolescents to assess the appropriateness of this approach in this population. ^ Method. Studies using CBPR in youth populations were identified using Medline and other Internet searches through both MeSH heading and text-word searches. ^ Results. A total of 16 studies were identified and analyzed for this review. Nine of the sixteen studies were experimental or quasi-experimental design, with Asthma being the most commonly studied disease. ^ Conclusions. While many studies using CBPR were not conducted with the level of scientific rigor typically found in clinical trial research, the studies reviewed each contributed to a greater understanding of the problems they investigated. Furthermore, interventional studies provided lasting benefits to communities under study above what would be found in studies using more traditional research approaches. While CBPR may not be appropriate for all research situations due to the time and resources required, we conclude that is a useful approach and should be considered when conducting community-based research for pediatric and adolescent populations.^

Syndromic surveillance at a long term shelter for persons displaced by hurricanes Katrina and Rita conducted by the San Antonio Metropolitan Health District

Background. Beginning September 2, 2005, San Antonio area shelters received approximately 12,700 evacuees from Hurricane Katrina. Two weeks later, another 12,000 evacuees from Hurricane Rita arrived. By mid-October, 2005, the in-shelter population was 1,000 people. There was concern regarding the potential for spread of infectious diseases in the shelter. San Antonio Metropolitan Health District (SAMHD) established a syndromic surveillance system with Comprehensive Health Services (CHS) who provided on-site health care. CHS was in daily contact with SAMHD to report symptoms of concern until the shelter closed December 23, 2005. ^ Study type. The objective of this study was to assess the methods used and describe the practical considerations involved in establishing and managing a syndromic surveillance system, as established by the SAMHD in the long-term shelter clinic maintained by CHS for the hurricane evacuees. ^ Methods. Information and descriptive data used in this study was collected from multiple sources, primarily from the San Antonio Metropolitan Health District’s 2006 Report on Syndromic Surveillance of a Long-Term Shelter by Hausler & Rohr-Allegrini. SAMHD and CHS staff ensured that each clinic visit was recorded by date, demographic information, chief complaint and medical disposition. Logs were obtained daily and subsequently entered into a Microsoft Access database and analyzed in Excel. ^ Results. During a nine week period, 4,913 clinic visits were recorded, reviewed and later analyzed. Repeat visits comprised 93.0% of encounters. Chronic illnesses contributed to 21.7% of the visits. Approximately 54.0% were acute care encounters. Of all encounters, 17.3% had infectious disease potential as primarily gastrointestinal and respiratory syndromes. Evacuees accounted for 86% and staff 14% of all visits to the shelter clinic. There were 782 unduplicated individuals who sought services at the clinic, comprised of 63% (496) evacuees and 36% (278) staff members. Staff were more likely to frequent the clinic but for fewer visits each. ^ Conclusion. The presence of health care services and syndromic surveillance provided the opportunity to recognize, document and intervene in any disease outbreak at this long-term shelter. Constant vigilance allowed SAMHD to inform and reassure concerned people living and working in the shelter and living outside the shelter.^

HPV knowledge, HPV vaccine awareness and initiation among Hispanics: The role of language, information seeking, and information scanning

Background. Among Hispanics, the HPV vaccine has the potential to eliminate disparities in cervical cancer incidence and mortality but only if optimal rates of vaccination are achieved. Media can be an important information source for increasing HPV knowledge and awareness of the vaccine. Very little is known about how media use among Hispanics affects their HPV knowledge and vaccine awareness. Even less is known about what differences exist in media use and information processing among English- and Spanish-speaking Hispanics.^ Aims. Examine the relationships between three health communication variables (media exposure, HPV-specific information scanning and seeking) and three HPV outcomes (knowledge, vaccine awareness and initiation) among English- and Spanish-speaking Hispanics.^ Methods. Cross-sectional data from a survey administered to Hispanic mothers in Dallas, Texas was used for univariate and multivariate logistic regression analyses. Sample used for analysis included 288 mothers of females aged 8-22 recruited from clinics and community events. Dependent variables of interest were HPV knowledge, HPV vaccine awareness and initiation. Independent variables were media exposure, HPV-specific information scanning and seeking. Language was tested as an effect modifier on the relationship between health communication variables and HPV outcomes.^ Results. English-speaking mothers reported more media exposure, HPV-specific information scanning and seeking than Spanish-speakers. Scanning for HPV information was associated with more HPV knowledge (OR = 4.26, 95% CI = 2.41 - 7.51), vaccine awareness (OR = 10.01, 95% CI = 5.43 - 18.47) and vaccine initiation (OR = 2.54, 95% CI = 1.09 - 5.91). Seeking HPV-specific information was associated with more knowledge (OR = 2.27, 95% CI = 1.23 - 4.16), awareness (OR = 6.60, 95% CI = 2.74 - 15.91) and initiation (OR = 4.93, 95% CI = 2.64 - 9.20). Language moderated the effect of information scanning and seeking on vaccine awareness.^ Discussion. Differences in information scanning and seeking behaviors among Hispanic subgroups have the potential to lead to disparities in vaccine awareness.^ Conclusion. Findings from this study underscore health communication differences among Hispanics and emphasize the need to target Spanish language media as well as English language media aimed at Hispanics to improve knowledge and awareness.^

Functional Analysis of Drosophila Integrator Complex in snRNA 3' End Processing

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex. In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression in response to a deficiency in snRNA processing. Besides scoring the known Integrator subunits, we identified Asunder and CG4785 as additional core members of the Integrator Complex. Additionally, we also found a conserved requirement for Cyclin C and Cdk8 in both fly and human snRNA 3’ end processing. We have further demonstrated that the kinase activity of Cdk8 is critical for snRNA 3’ end processing and is likely to function independent of its well-documented function within the Mediator Cdk8 module. Taken together, this work functionally defines the Drosophila Integrator Complex and demonstrates a novel function for Cyclin C/Cdk8 in snRNA 3’ end formation. This thesis work has also characterized an important functional interaction mediated by a microdomain within Integrator subunit 12 (IntS12) and IntS1 that is required for the activity of the Integrator Complex in processing the snRNA 3’ end. Through the development of a reporter-based functional RNAi-rescue assay in Drosophila S2 cells, we analyzed domains within IntS12 required for snRNA 3’ end formation. This analysis unexpectedly revealed that an N-terminal 30 amino acid region and not the highly conserved central PHD finger domain, is required for snRNA 3’ end cleavage. The IntS12 microdomain (1-45) functions autonomously, and is sufficient to interact and stabilize the putative scaffold protein IntS1. Our findings provide more details of the Integrator Complex for understanding the molecular mechanism of snRNA 3’ end processing. Moreover, these results lay the foundation for future studies of the complex through the identification of a novel functional domain within one subunit and the identification of additional subunits.

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

Diffusion tensor imaging of rat spinal cord in vivo

Magnetic resonance imaging, with its exquisite soft tissue contrast, is an ideal modality for investigating spinal cord pathology. While conventional MRI techniques are very sensitive for spinal cord pathology, their specificity is somewhat limited. Diffusion MRI is an advanced technique which is a very sensitive and specific indicator of the integrity of white matter tracts. Diffusion imaging has been shown to detect early ischemic changes in white matter, while conventional imaging demonstrates no change. By acquiring the complete apparent diffusion tensor (ADT), tissue diffusion properties can be expressed in terms of quantitative and rotationally invariant parameters. ^ Systematic study of SCI in vivo requires controlled animal models such as the popular rat model. To date, studies of spinal cord using ADT imaging have been performed exclusively in fixed, excised spinal cords, introducing inevitable artifacts and losing the benefits of MRI's noninvasive nature. In vivo imaging reflects the actual in vivo tissue properties, and allows each animal to be imaged at multiple time points, greatly reducing the number of animals required to achieve statistical significance. Because the spinal cord is very small, the available signal-to-noise ratio (SNR) is very low. Prior spin-echo based ADT studies of rat spinal cord have relied on high magnetic field strengths and long imaging times—on the order of 10 hours—for adequate SNR. Such long imaging times are incompatible with in vivo imaging, and are not relevant for imaging the early phases following SCI. Echo planar imaging (EPI) is one of the fastest imaging methods, and is popular for diffusion imaging. However, EPI further lowers the image SNR, and is very sensitive to small imperfections in the magnetic field, such as those introduced by the bony spine. Additionally, The small field-of-view (FOV) needed for spinal cord imaging requires large imaging gradients which generate EPI artifacts. The addition of diffusion gradients introduces yet further artifacts. ^ This work develops a method for rapid EPI-based in vivo diffusion imaging of rat spinal cord. The method involves improving the SNR using an implantable coil; reducing magnetic field inhomogeneities by means of an autoshim, and correcting EPI artifacts by post-processing. New EPI artifacts due to diffusion gradients described, and post-processing correction techniques are developed. ^ These techniques were used to obtain rotationally invariant diffusion parameters from 9 animals in vivo, and were validated using the gold-standard, but slow, spinecho based diffusion sequence. These are the first reported measurements of the ADT in spinal cord in vivo . ^ Many of the techniques described are equally applicable toward imaging of human spinal cord. We anticipate that these techniques will aid in evaluating and optimizing potential therapies, and will lead to improved patient care. ^

An ethnographic study of home remedy use for African-American children

Background and significance. The use of herbs and other remedies by adult and elderly African-Americans has been documented. However, little is understood regarding the use of herbs for African-American children. The purpose of this study was to document and describe the historical and present day uses of herbal and other remedies, specifically for the health and illness of African-American children. This information will provide health care providers with a better understanding of their African-American patients. This information may also contribute to the emerging appreciation of indigenous uses of phytotherapeutics. ^ Methods. A focused ethnographic approach was used to describe the cultural context, including the beliefs, values, customs, and behaviors of a particular culture. The use of intensive fieldwork, including participant observation, audiotaped formal interviews, photographs, and specimen collection of plants helped to describe herb use in this population. Information on the growing, harvesting, preparation, and storage of these plant remedies, as well as the amount and dosage of these compounds was collected in a typology. Detailed information was gathered to discern how, when, and under what conditions these remedies were used and their expected results. Further data collection focused on the history of herbal use, and explanations for how and why informants thought the herbs work. ^ Setting and participants. The setting for this study was in East Texas and field work extended over the period of one year. Thirty African-Americans, age 38 to 98, were interviewed for the study. The African-American population in this area has been relatively stable, with roots dating back prior to the reconstruction period, which allowed excellent historical information. Informants were chosen by a nominated sampling technique starting with two key informants knowledgeable about the use of home remedies for children. ^ Findings. The findings of this study suggest that African-American children in East Texas have a long history of receiving herbs and home remedies for health promotion and illness. Data further suggests that there is a strong connection between spirituality and the health beliefs and practices of this community. This spiritual component underlies the accuracy of oral recall for remedies that have been used over many generations and the use of natural folk remedies. A typology of the herbal remedies was developed with folk and Latin name, herb place of origin, known scientific properties, and informant folk usage and dosage information. ^

Mechanisms of heterosynaptic modulation of transmission in Aplysia

Heterosynaptic plasticity has received considerable attention as a means to induce and maintain cell-wide, as opposed to synapse-specific, learning-related modifications. Modulatory neurotransmitters are thought to provide the attentional and motivational state for memory formation. However, the cellular and molecular mechanisms mediating the effects of most of these modulators on synaptic plasticity and learning remain unclear. A well established system for the study of heterosynaptic plasticity is the Aplysia sensorimotor synapse, which is subject regulation by at least two neuromodulators, serotonin (5-HT) and FMRFa. ^ 5-HT engages multiple second messenger cascades to induce short- and long-term facilitation (STF and LTF, respectively) of synaptic transmission. One mechanism proposed to be involved in STF is mobilization of synaptic vesicles from a storage pool to a releasable pool. To investigate this hypothesis, we examined the involvement of the protein synapsin, a central element in the regulation of the storage pool of vesicles in nerve terminals, in STF. 5-HT induced phosphorylation of synapsin and modified its subcellular distribution via PKA and p42/44 MAPK. Electrophysiological experiments and computer simulations suggested that synapsin can support heterosynaptic plasticity by regulating vesicle mobilization. ^ FMRFa induce short- and long-term synaptic depression in Aplysia . Long-term depression (LTD) correlates with morphological changes, the mechanisms of which remain elusive. LTD is also transcription- and translation-dependent, but little is known about the genes expressed and their regulation. We investigated the role of protein degradation via the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by the transcription factor CREB2, which is generally regarded as a transcription repressor. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions. ^ These and additional studies on the interaction of the 5-HT and FMRFa-activated pathways suggest that different neuromodulators, by activating several and sometimes overlapping signaling cascades, can exercise bidirectional control on synaptic gain and information processing.^

The social construction of infant mortality: From grassroots to medicalization

Infant mortality as a problematic situation has been recognized for some 130 years in one form or another. It has undergone various changes in its empirical dimensions relative to whom we study within the population, what we study--low birth-weight vs. pre-term births--and how we study it--whether demographically or medically. An analysis of the process by which the condition was raised by claims makers as an intolerable situation among America's urban residents reveals that demographic and medical data were sparse. Nonetheless, a judgement about the meaning and significance of the condition was made, and that interpretation led to the promulgation of systems to both document and address the condition as it has come to be defined.^ This investigation depicts the historical context and natural history of infant mortality as one of a number of social problems that came to be defined through the interplay among groups and individuals making claims and how their claims came to the public policy agenda as worthy of collective resources--who won, who lost and why. The process of social definition focuses attention on the claims makers and the ways they contrast the meaning, origins and remedies for this troubling condition. The historical context becomes the frame of reference for understanding the actions of the claims makers and the meaning and significance they attached to the problem.^ We purport that "context" provides a closer reality than disjoined "value free" accounts. Context provides the evidence for the definition, who participated in the process, why and by what means.^ The role of women in the definitional process reveals the differences in approaches utilized by the women of the settlement house reform movement and African-American women working at the grass-roots. Much of the work done by these two groups provided options to the problem's remedy; however, their differences paved the way to our current (principally medically-oriented) definition and its inherent limitations. ^