beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi.

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.

Magnetophoretic circuits for digital control of single particles and cells.

The ability to manipulate small fluid droplets, colloidal particles and single cells with the precision and parallelization of modern-day computer hardware has profound applications for biochemical detection, gene sequencing, chemical synthesis and highly parallel analysis of single cells. Drawing inspiration from general circuit theory and magnetic bubble technology, here we demonstrate a class of integrated circuits for executing sequential and parallel, timed operations on an ensemble of single particles and cells. The integrated circuits are constructed from lithographically defined, overlaid patterns of magnetic film and current lines. The magnetic patterns passively control particles similar to electrical conductors, diodes and capacitors. The current lines actively switch particles between different tracks similar to gated electrical transistors. When combined into arrays and driven by a rotating magnetic field clock, these integrated circuits have general multiplexing properties and enable the precise control of magnetizable objects.

Development of Stimulus-Responsive Ligands for the Modulation of Copper and Iron Coordination

The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.

Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N′-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.

Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.

Sensitivity to switching rates in stochastically switched ODEs

We consider a stochastic process driven by a linear ordinary differential equation whose right-hand side switches at exponential times between a collection of different matrices. We construct planar examples that switch between two matrices where the individual matrices and the average of the two matrices are all Hurwitz (all eigenvalues have strictly negative real part), but nonetheless the process goes to infinity at large time for certain values of the switching rate. We further construct examples in higher dimensions where again the two individual matrices and their averages are all Hurwitz, but the process has arbitrarily many transitions between going to zero and going to infinity at large time as the switching rate varies. In order to construct these examples, we first prove in general that if each of the individual matrices is Hurwitz, then the process goes to zero at large time for sufficiently slow switching rate and if the average matrix is Hurwitz, then the process goes to zero at large time for sufficiently fast switching rate. We also give simple conditions that ensure the process goes to zero at large time for all switching rates. © 2014 International Press.

The Innate Immune System in Acute and Chronic Wounds.

Significance: This review article provides an overview of the critical roles of the innate immune system to wound healing. It explores aspects of dysregulation of individual innate immune elements known to compromise wound repair and promote nonhealing wounds. Understanding the key mechanisms whereby wound healing fails will provide seed concepts for the development of new therapeutic approaches. Recent Advances: Our understanding of the complex interactions of the innate immune system in wound healing has significantly improved, particularly in our understanding of the role of antimicrobials and peptides and the nature of the switch from inflammatory to reparative processes. This takes place against an emerging understanding of the relationship between human cells and commensal bacteria in the skin. Critical Issues: It is well established and accepted that early local inflammatory mediators in the wound bed function as an immunological vehicle to facilitate immune cell infiltration and microbial clearance upon injury to the skin barrier. Both impaired and excessive innate immune responses can promote nonhealing wounds. It appears that the switch from the inflammatory to the proliferative phase is tightly regulated and mediated, at least in part, by a change in macrophages. Defining the factors that initiate the switch in such macrophage phenotypes and functions is the subject of multiple investigations. Future Directions: The review highlights processes that may be useful targets for further investigation, particularly the switch from M1 to M2 macrophages that appears to be critical as dysregulation of this switch occurs during defective wound healing.

Memory and learning for a novel written style.

Subjects read and recalled a series of five short stories in one of four plot and style combinations. The stories were written in one of two styles that consisted of opposing clause orders (i.e., independent-dependent vs. dependent-independent), tense forms (i.e., past vs. present), and descriptor forms (modifier modifier vs. modifier as a noun). The subjects incorporated both plot and style characteristics into their recalls. Other subjects, who, after five recalls, either generated a new story or listed the rules that had been followed by the stories read, included the marked forms of the characteristics they learned more often, except for tense. The subjects read and recalled four stories of the same plot and style and then read and recalled a fifth story of the same plot and style or of one of the other three plot/style combinations. Ability to switch style depended on both the characteristic and the markedness.

Durability of antiretroviral therapy and predictors of virologic failure among perinatally HIV-infected children in Tanzania: a four-year follow-up.

BACKGROUND: In Tanzania, HIV-1 RNA testing is rarely available and not standard of care. Determining virologic failure is challenging and resistance mutations accumulate, thereby compromising second-line therapy. We evaluated durability of antiretroviral therapy (ART) and predictors of virologic failure among a pediatric cohort at four-year follow-up. METHODS: This was a prospective cross-sectional study with retrospective chart review evaluating a perinatally HIV-infected Tanzanian cohort enrolled in 2008-09 with repeat HIV-1 RNA in 2012-13. Demographic, clinical, and laboratory data were extracted from charts, resistance mutations from 2008-9 were analyzed, and prospective HIV RNA was obtained. RESULTS: 161 (78%) participants of the original cohort consented to repeat HIV RNA. The average age was 12.2 years (55% adolescents ≥12 years). Average time on ART was 6.4 years with 41% receiving second-line (protease inhibitor based) therapy. Among those originally suppressed on a first-line (non-nucleoside reverse transcriptase based regimen) 76% remained suppressed. Of those originally failing first-line, 88% were switched to second-line and 72% have suppressed virus. Increased level of viremia and duration of ART trended with an increased number of thymidine analogue mutations (TAMs). Increased TAMs increased the odds of virologic failure (p = 0.18), as did adolescent age (p < 0.01). CONCLUSIONS: After viral load testing in 2008-09 many participants switched to second-line therapy. The majority achieved virologic suppression despite multiple resistance mutations. Though virologic testing would likely hasten the switch to second-line among those failing, methods to improve adherence is critical to maximize durability of ART and improve virologic outcomes among youth in resource-limited settings.

Regulation of DLK-1 kinase activity by calcium-mediated dissociation from an inhibitory isoform.

MAPKKK dual leucine zipper-bearing kinases (DLKs) are regulators of synaptic development and axon regeneration. The mechanisms underlying their activation are not fully understood. Here, we show that C. elegans DLK-1 is activated by a Ca(2+)-dependent switch from inactive heteromeric to active homomeric protein complexes. We identify a DLK-1 isoform, DLK-1S, that shares identical kinase and leucine zipper domains with the previously described long isoform DLK-1L but acts to inhibit DLK-1 function by binding to DLK-1L. The switch between homo- or heteromeric DLK-1 complexes is influenced by Ca(2+) concentration. A conserved hexapeptide in the DLK-1L C terminus is essential for DLK-1 activity and is required for Ca(2+) regulation. The mammalian DLK-1 homolog MAP3K13 contains an identical C-terminal hexapeptide and can functionally complement dlk-1 mutants, suggesting that the DLK activation mechanism is conserved. The DLK activation mechanism is ideally suited for rapid and spatially controlled signal transduction in response to axonal injury and synaptic activity.

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast.

Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction delay caused by decreasing inducer concentrations across the population. Chromatin remodeling appears to be the basis for the dynamic GAL1 expression, because mutants with impaired histone dynamics show severely truncated dose-response profiles. In contrast, the GRE2 promoter operates like a rapid off/on switch in response to increasing osmotic stress, with almost constant expression rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 mitogen-activated protein (MAP) kinase seem to determine the different dose-response strategies at the two promoters. Accordingly, GAL1 becomes highly sensitive and dose independent if previously stimulated because of residual Gal3 levels, whereas GRE2 expression diminishes upon repeated stimulation due to acquired stress resistance. Our analysis reveals important differences in the way dynamic signals create dose-sensitive gene expression outputs.

Evaluation of an epithelial plasticity biomarker panel in men with localized prostate cancer.

BACKGROUND: Given the potential importance of epithelial plasticity (EP) to cancer metastasis, we sought to investigate biomarkers related to EP in men with localized prostate cancer (PC) for the association with time to PSA recurrence and other clinical outcomes after surgery. METHODS: Men with localized PC treated with radical prostatectomy at the Durham VA Medical Center and whose prostatectomy tissues were included in a tissue microarray (TMA) linked to long-term outcomes. We performed immunohistochemical studies using validated antibodies against E-cadherin and Ki-67 and mesenchymal biomarkers including N-cadherin, vimentin, SNAIL, ZEB1 and TWIST. Association studies were conducted for each biomarker with baseline clinical/pathologic characteristics an risk of PSA recurrence over time. RESULTS: Two hundred and five men contributed TMA tissue and had long-term follow-up (median 11 years). Forty-three percent had PSA recurrence; three died of PC. The majority had high E-cadherin expression (86%); 14% had low/absent E-cadherin expression. N-cadherin was rarely expressed (<4%) and we were unable to identify an E-to-N-cadherin switch as independently prognostic. No associations with clinical risk group, PSA recurrence or Gleason sum were noted for SNAIL, ZEB1, vimentin or TWIST, despite heterogeneous expression between patients. We observed an association of higher Ki-67 expression with Gleason sum (P=0.043), National Comprehensive Cancer Network risk (P=0.013) and PSA recurrence (hazard ratio 1.07, P=0.016). CONCLUSIONS: The expression of EP biomarkers in this cohort of men with a low risk of PC-specific mortality was not associated with aggressive features or PSA relapse after surgery.

Sea Urchin Body Plan Development and Evolution: An Integrative Transcriptomic Approach

My dissertation work integrates comparative transcriptomics and functional analyses to investigate gene expression changes underlying two significant aspects of sea urchin evolution and development: the dramatic developmental changes associated with an ecologically significant shift in life history strategy and the development of the unusual radial body plan of adult sea urchins.

In Chapter 2, I investigate evolutionary changes in gene expression underlying the switch from feeding (planktotrophic) to nonfeeding (lecithotrophic) development in sea urchins. In order to identify these changes, I used Illumina RNA-seq to measure expression dynamics across 7 developmental stages in three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and an outgroup planktotroph Lytechinus variegatus. My analyses draw on a well-characterized developmental gene regulatory network (GRN) in sea urchins to understand how the ancestral planktotrophic developmental program was altered during the evolution of lecithotrophic development. My results suggest that changes in gene expression profiles occurred more frequently across the transcriptome during the evolution of lecithotrophy than during the persistence of planktotrophy. These changes were even more pronounced within the GRN than across the transcriptome as a whole, and occurred in each network territory (skeletogenic, endomesoderm and ectoderm). I found evidence for both conservation and divergence of regulatory interactions in the network, as well as significant changes in the expression of genes with known roles in larval skeletogenesis, which is dramatically altered in lecithotrophs. I further explored network dynamics between species using coexpression analyses, which allowed me to identify novel players likely involved in sea urchin neurogenesis and endoderm patterning.

In Chapter 3, I investigate developmental changes in gene expression underlying radial body plan development and metamorphosis in H. erythrogramma. Using Illumina RNA-seq, I measured gene expression profiles across larval, metamorphic, and post-metamorphic life cycle phases. My results present a high-resolution view of gene expression dynamics during the complex transition from pre- to post-metamorphic development and suggest that distinct sets of regulatory and effector proteins are used during different life history phases.

Collectively, my investigations provide an important foundation for future, empirical studies to investigate the functional role of gene expression change in the evolution of developmental differences between species and also for the generation of the unusual radial body plan of sea urchins.

Violence Exposure and Welfare over Time: Evidence from the Burundi Civil War

We investigate the relationship between exposure to conflict and poverty dynamics over time, using original three-waves panel data for Burundi which tracked individuals and reported local-level violence exposure from 1998 to 2012. Firstly, the data reveal that headcount poverty has not changed since 1998 while we observe multiple transitions into and out of poverty. Moreover, households exposed to the war exhibit a lower level of welfare than non-exposed households, with the difference between the two groups predicted to remain significant at least until 2017, i.e. twelve years after the conflict termination. The correlation between violence exposure and deprivation over time is confirmed in a household-level panel setting. Secondly, our empirical investigation shows how violence exposure over different time spans interacts with households' subsequent welfare. Our analysis of the determinants of households' likelihood to switch poverty status (i.e. to fall into poverty or escape poverty) combined with quantile regressions suggest that, (i) exposure during the first phase of the conflict has affected the entire distribution, and (ii) exposure during the second phase of the conflict has mostly affected the upper tail of the distribution: initially non-poor households have a higher propensity to fall into poverty while initially poor households see their propensity to pull through only slightly decrease with recent exposure to violence. Although not directly testable with the data at hand, these results are consistent with the changing nature of violence in the course of the Burundi civil war, from relatively more labour-destructive to relatively more capital-destructive.

Modeling the effect of lead-free soldering on flexible substrates

Flexible Circuit Boards (FPCs) are now being widely used in the electronic industries especially in the areas of electronic packages. Due to European lead-free legislation which has been implemented since July 2006, electronic packaging industries have to switch to use in the lead-free soldering technology. This change has posed a number of challenges in terms of development of lead-free solders and compatible substrates. An increase of at least 20-50 degrees in the reflow temperature is a concern and substantial research is required to investigate a sustainable design of flexible circuit boards as carrier substrates. This paper investigates a number of design variables such as copper conductor width, type of substrate materials, effect of insulating materials, etc. Computer modeling has been used to investigate thermo-mechanical behavior, and reliability, of flexible substrates after they have been subjected to a lead- free solder processing. Results will show particular designs that behave better for a particular rise in peak reflow temperature. Also presented will be the types of failures that can occur in these substrates and what particular materials are more reliable.

"System in package technology" - design for manufacture challenges

Purpose – To present key challenges associated with the evolution of system-in-package technologies and present technical work in reliability modeling and embedded test that contributes to these challenges. Design/methodology/approach – Key challenges have been identified from the electronics and integrated MEMS industrial sectors. Solutions to optimising the reliability of a typical assembly process and reducing the cost of production test have been studied through simulation and modelling studies based on technology data released by NXP and in collaboration with EDA tool vendors Coventor and Flomerics. Findings – Characterised models that deliver special and material dependent reliability data that can be used to optimize robustness of SiP assemblies together with results that indicate relative contributions of various structural variables. An initial analytical model for solder ball reliability and a solution for embedding a low cost test for a capacitive RF-MEMS switch identified as an SiP component presenting a key test challenge. Research limitations/implications – Results will contribute to the further development of NXP wafer level system-in-package technology. Limitations are that feedback on the implementation of recommendations and the physical characterisation of the embedded test solution. Originality/value – Both the methodology and associated studies on the structural reliability of an industrial SiP technology are unique. The analytical model for solder ball life is new as is the embedded test solution for the RF-MEMS switch.

Multiple attractors of host-parasitoid models with integrated pest management strategies: eradication, persistence and outbreak

Host-parasitoid models including integrated pest management (IPM) interventions with impulsive effects at both fixed and unfixed times were analyzed with regard to host-eradication, host-parasitoid persistence and host-outbreak solutions. The host-eradication periodic solution with fixed moments is globally stable if the host's intrinsic growth rate is less than the summation of the mean host-killing rate and the mean parasitization rate during the impulsive period. Solutions for all three categories can coexist, with switch-like transitions among their attractors showing that varying dosages and frequencies of insecticide applications and the numbers of parasitoids released are crucial. Periodic solutions also exist for models with unfixed moments for which the maximum amplitude of the host is less than the economic threshold. The dosages and frequencies of IPM interventions for these solutions are much reduced in comparison with the pest-eradication periodic solution. Our results, which are robust to inclusion of stochastic effects and with a wide range of parameter values, confirm that IPM is more effective than any single control tactic.