Raman Spectroscopic Modeling Of T- Lymphocyte Activation And Detection Of Acute Renal Allograft Rejection

ABSTRACT RAMAN SPECTROSCOPIC MODELING OF T-LYMPHOCYTE ACTIVATION AND DETECTION OF ACUTE RENAL ALLOGRAFT REJECTION By KRISTIAN L. BROWN 2010 Advisor: Gregory Auner, PhD Major: Biomedical Engineering Degree: Doctor of Philosophy Despite the advances made in the area of kidney transplantation, the disparity between the demand and available donated organs remains a dominant and unresolved issue. Given the paucity of available renal allografts the preservation of existing grafts is vital. One factor that has negatively impacted renal allograft survival is acute rejection (AR). Traditionally, kidney transplant centers have used elevations in serum creatinine as a screening tool for detecting AR. However, with its diagnostic delay, low sensitivity, and low specificity, serum creatinine has proven to be an unreliable and problematic bio-marker. Acute rejection is an activated T lymphocyte driven process that leads to graft dysfunction and possible loss. The activation state of T lymphocytes is determined by the specific cell surface receptor composition present. A technologic tool that could resolve these receptor differences could detect T lymphocyte activation and thus provide a diagnostic modality for AR. Raman spectroscopy (RS), a laser-based technology that is able to characterize substances based on molecular vibrational signatures, represents this modality. Using T lymphocytes isolated from human peripheral blood and clean-catch urine we investigated three aspects of T lymphocyte activation using a modified RS system. First, we explored the sensitivity (the ability to detect activation) of a RS-based system by analyzing mixed lymphocyte reacted (MLR), Mitomycin C inactivated, and resting T lymphocytes at 785nm and 514.5nm wavelengths. Second, the specificity (the ability to distinguish T cells activated by different stimuli) of the system was determined by comparing the signatures of MLR and CD3/CD28-activated T lymphocytes. Third, we analyzed the biomolecular events that conveyed the spectral changes detected by RS. This was carried out by coupling RS analysis of Mitogen-activated T lymphocytes with antigen expression kinetic studies designed to quantify the intensity and timing of cell surface receptor up-regulation. We found that there were significant RS signature differences between the MLR and non-activated (inactivated and resting) T lymphocytes while there was only a trend toward difference seen between the resting and inactivated cellular populations. When analyzing MLR versus CD3/CD28-activated cells, both samples differed from the inactivated and resting groups and demonstrated differences in Raman shifts at multiple foci when compared to one another. Receptor expression kinetics of Mitogen-activated T lymphocytes analyzed at the early and late phases of activation showed differential antibody immuno-fluorescent intensity. This correlated to spectral differences at defined peaks. Moreover, when analyzing all forms of activation (i.e. MLR, CD3/CD28, or Mitogen) there were conserved and reproducible signature changes regardless of the mode of activation which supports the notion that there are receptor and receptor moiety changes that are required in all forms of T lymphocyte activation. This dissertation outlines the use of RS in the resolution and modeling of cell surface receptor differences that define T lymphocyte activation. The accurate detection of T lymphocyte activation within a biomatrix is the foundational step toward the development of a noninvasive tool capable of accurately detecting AR in real-time within the clinical setting

Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray

BACKGROUND: We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested. RESULTS: We have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA. CONCLUSION: We validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content

The developmental dynamics of terrorist organizations

We identify robust statistical patterns in the frequency and severity of violent attacks by terrorist organizations as they grow and age. Using group-level static and dynamic analyses of terrorist events worldwide from 1968-2008 and a simulation model of organizational dynamics, we show that the production of violent events tends to accelerate with increasing size and experience. This coupling of frequency, experience and size arises from a fundamental positive feedback loop in which attacks lead to growth which leads to increased production of new attacks. In contrast, event severity is independent of both size and experience. Thus larger, more experienced organizations are more deadly because they attack more frequently, not because their attacks are more deadly, and large events are equally likely to come from large and small organizations. These results hold across political ideologies and time, suggesting that the frequency and severity of terrorism may be constrained by fundamental processes.Comment: 28 pages, 8 figures, 4 tables, supplementary materia

Null dust in canonical gravity

We present the Lagrangian and Hamiltonian framework which incorporates null dust as a source into canonical gravity. Null dust is a generalized Lagrangian system which is described by six Clebsch potentials of its four-velocity Pfaff form. The Dirac--ADM decomposition splits these into three canonical coordinates (the comoving coordinates of the dust) and their conjugate momenta (appropriate projections of four-velocity). Unlike ordinary dust of massive particles, null dust therefore has three rather than four degrees of freedom per space point. These are evolved by a Hamiltonian which is a linear combination of energy and momentum densities of the dust. The energy density is the norm of the momentum density with respect to the spatial metric. The coupling to geometry is achieved by adding these densities to the gravitational super-Hamiltonian and supermomentum. This leads to appropriate Hamiltonian and momentum constraints in the phase space of the system. The constraints can be rewritten in two alternative forms in which they generate a true Lie algebra. The Dirac constraint quantization of the system is formally accomplished by imposing the new constraints as quantum operator restrictions on state functionals. We compare the canonical schemes for null and ordinary dust and emhasize their differences.Comment: 25 pages, REVTEX, no figure

Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 20132016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP\u27s roles in protein structure, virion budding, and transcription and replication

Kepler eclipsing binary stars. VII. the catalogue of eclipsing binaries found in the entire Kepler data set

The primary Kepler Mission provided nearly continuous monitoring of ~200,000 objects with unprecedented photometric precision. We present the final catalog of eclipsing binary systems within the 105 deg2 Kepler field of view. This release incorporates the full extent of the data from the primary mission (Q0-Q17 Data Release). As a result, new systems have been added, additional false positives have been removed, ephemerides and principal parameters have been recomputed, classifications have been revised to rely on analytical models, and eclipse timing variations have been computed for each system. We identify several classes of systems including those that exhibit tertiary eclipse events, systems that show clear evidence of additional bodies, heartbeat systems, systems with changing eclipse depths, and systems exhibiting only one eclipse event over the duration of the mission. We have updated the period and galactic latitude distribution diagrams and included a catalog completeness evaluation. The total number of identified eclipsing and ellipsoidal binary systems in the Kepler field of view has increased to 2878, 1.3% of all observed Kepler targets

Nuclear charge radii of silicon isotopes

The nuclear charge radius of $^{32}$Si was determined using collinear laser spectroscopy. The experimental result was confronted with ab initio nuclear lattice effective field theory, valence-space in-medium similarity renormalization group, and mean field calculations, highlighting important achievements and challenges of modern many-body methods. The charge radius of $^{32}$Si completes the radii of the mirror pair $^{32}$Ar - $^{32}$Si, whose difference was correlated to the slope $L$ of the symmetry energy in the nuclear equation of state. Our result suggests $L \leq 60$\,MeV, which agrees with complementary observables

Orientation of Galaxies in the Local Supercluster: A Review

The progress of the studies on the orientation of galaxies in the Local Supercluster (LSC) is reviewed and a summary of recent results is given. Following a brief introduction of the LSC, we describe the results of early studies based on two-dimensional analysis, which were mostly not conclusive. We describe next the three-dimensional analysis, which is used widely today. Difficulties and systematic effects are explained and the importance of selection effects is described. Then, results based on the new method and modern databases are given, which are summarized as follows. When the LSC is seen as a whole, galaxy planes tend to align perpendicular to the LSC plane with lenticulars showing the most pronounced tendency. Projections onto the LSC plane of the spin vectors of Virgo cluster member galaxies, and to some extent, those of the total LSC galaxies, tend to point to the Virgo cluster center. This tendency is more pronounced for lenticulars than for spirals. It is suggested that 'field' galaxies, i.e., those which do not belong to groups with more than three members, may be better objects than other galaxies to probe the information at the early epoch of the LSC formation through the analysis of galaxy orientations. Field lenticulars show a pronounced anisotropic distribution of spin vectors in the sense that they lay their spin vectors parallel to the LSC plane while field spirals show an isotropic spin-vector distribution.Comment: 21 pages, 10 figures; Accepted for publication in Astrophysics and Space Scienc

Clinical Illness and Outcomes in Patients with Ebola in Sierra Leone

Background Limited clinical and laboratory data are available on patients with Ebola virus disease (EVD). The Kenema Government Hospital in Sierra Leone, which had an existing infrastructure for research regarding viral hemorrhagic fever, has received and cared for patients with EVD since the beginning of the outbreak in Sierra Leone in May 2014. Methods We reviewed available epidemiologic, clinical, and laboratory records of patients in whom EVD was diagnosed between May 25 and June 18, 2014. We used quantitative reverse-transcriptase–polymerase-chain-reaction assays to assess the load of Ebola virus (EBOV, Zaire species) in a subgroup of patients. Results Of 106 patients in whom EVD was diagnosed, 87 had a known outcome, and 44 had detailed clinical information available. The incubation period was estimated to be 6 to 12 days, and the case fatality rate was 74%. Common findings at presentation included fever (in 89% of the patients), headache (in 80%), weakness (in 66%), dizziness (in 60%), diarrhea (in 51%), abdominal pain (in 40%), and vomiting (in 34%). Clinical and laboratory factors at presentation that were associated with a fatal outcome included fever, weakness, dizziness, diarrhea, and elevated levels of blood urea nitrogen, aspartate aminotransferase, and creatinine. Exploratory analyses indicated that patients under the age of 21 years had a lower case fatality rate than those over the age of 45 years (57% vs. 94%, P=0.03), and patients presenting with fewer than 100,000 EBOV copies per milliliter had a lower case fatality rate than those with 10 million EBOV copies per milliliter or more (33% vs. 94%, P=0.003). Bleeding occurred in only 1 patient. Conclusions The incubation period and case fatality rate among patients with EVD in Sierra Leone are similar to those observed elsewhere in the 2014 outbreak and in previous outbreaks. Although bleeding was an infrequent finding, diarrhea and other gastrointestinal manifestations were common. (Funded by the National Institutes of Health and others.

Antenatal HIV testing in rural eastern Uganda in 2003: incomplete rollout of the prevention of mother-to-child transmission of HIV programme?

BACKGROUND: Uganda began to implement the prevention of mother-to-child transmission (PMTCT) of HIV programme in 2000, and by the end of 2003 it had expanded to cover 38 of the 56 districts including Mbale District. However, reports from Mbale Hospital showed that less than 10% of pregnant women accepted antenatal HIV testing. We therefore conducted a study to determine the proportion of pregnant women who tested for HIV and the gaps and barriers in PMTCT implementation. METHODS: The study was a cross sectional household survey of women aged 18 years or more, with children aged one year or less, who resided in Mbale Town or in the surrounding Bungokho County. We also conducted in-depth interviews with six health workers in Mbale Hospital. RESULTS: In 2003, we interviewed 457 women with a median age of 24 years. The prevalence of antenatal HIV testing was 10 percent. The barriers to antenatal HIV testing were unavailability of voluntary counselling and testing services (44%), lack of HIV counselling (42%) and perceived lack of benefits for HIV infected women and their infants. Primipara (OR 2.6, 95% CI 1.2–5.8), urban dwellers (OR 2.7, 95% CI 1.3–5.8), women having been counselled on HIV (OR 6.2, 95% CI 2.9–13.2), and women with husbands being their primary confidant (OR 2.3, 95% CI 1.0–5.5) were independently associated with HIV testing. CONCLUSION: The major barriers to PMTCT implementation were unavailability of PMTCT services, particularly in rural clinics, and poor antenatal counselling and HIV testing services. We recommend that the focus of the prevention of mother-to-child transmission of HIV programme should shift to the district and sub-district levels, strengthen community mobilization, improve the quality of antenatal voluntary counselling and HIV testing services, use professional and peer counsellors to augment HIV counselling, and ensure follow-up care and support for HIV positive women and their infants