Neutrophil degranulation involves a number of well-orchestrated structural and biochemical events. We have investigated the mechanism of intracellular signalling involved in neutrophil degranulation that was mediated by the high affinity leukotriene (LT)B[subscript 4] receptor, BLT1. The model systems used were consisted of Peripheral blood neutrophils as well as promyeloid PLB-985 cells, stably transfected with human BLT1 cDNA (PLB-BLT) or a substitution mutant (2L(304-305)/A) of the distal dileucine motif in helix VIII of BLT1, and differentiated into a neutrophil-like phenotype. The degranulation of these cells was measured in the presence and absence of factors that would affect the signaling pathway. The results show that Degranulation responses to LTB[subscript 4] were similar for differentiated PLB-BLT1 and neutrophils. However, the degranulation response of cells bearing the dileucine mutation in helix VIII of BLT1 was significantly reduced in response to LTB[subscript 4]. Pretreatment of differentiated PLB-BLT1 cells and neutrophils with Y-27632, a pharmacological inhibitor of p160-ROCK, the down-stream effector of the small GTPase RhoA, abrogated their degranulation in response to LTB[subscript 4]. The degranulation defect observed with the dileucine mutation was corrected by transient transfection of the cells bearing the mutation with a constitutively active form of RhoA. Taken together, our results suggest an essential role for the distal dileucine motif in helix VIII of BLT1 involving RhoA which allows normal neutrophil degranulation in response to LTB[subscript 4].

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BACKGROUND: A systems toxicology investigation comparing and integrating transcriptomic and proteomic results was conducted to develop holistic effects characterizations for the wildlife bird model, Northern bobwhite (Colinus virginianus) dosed with the explosives degradation product 2-amino-4,6-dinitrotoluene (2A-DNT). A subchronic 60d toxicology bioassay was leveraged where both sexes were dosed via daily gavage with 0, 3, 14, or 30 mg/kg-d 2A-DNT. Effects on global transcript expression were investigated in liver and kidney tissue using custom microarrays for C. virginianus in both sexes at all doses, while effects on proteome expression were investigated in liver for both sexes and kidney in males, at 30 mg/kg-d. RESULTS: As expected, transcript expression was not directly indicative of protein expression in response to 2A-DNT. However, a high degree of correspondence was observed among gene and protein expression when investigating higher-order functional responses including statistically enriched gene networks and canonical pathways, especially when connected to toxicological outcomes of 2A-DNT exposure. Analysis of networks statistically enriched for both transcripts and proteins demonstrated common responses including inhibition of programmed cell death and arrest of cell cycle in liver tissues at 2A-DNT doses that caused liver necrosis and death in females. Additionally, both transcript and protein expression in liver tissue was indicative of induced phase I and II xenobiotic metabolism potentially as a mechanism to detoxify and excrete 2A-DNT. Nuclear signaling assays, transcript expression and protein expression each implicated peroxisome proliferator-activated receptor (PPAR) nuclear signaling as a primary molecular target in the 2A-DNT exposure with significant downstream enrichment of PPAR-regulated pathways including lipid metabolic pathways and gluconeogenesis suggesting impaired bioenergetic potential. CONCLUSION: Although the differential expression of transcripts and proteins was largely unique, the consensus of functional pathways and gene networks enriched among transcriptomic and proteomic datasets provided the identification of many critical metabolic functions underlying 2A-DNT toxicity as well as impaired PPAR signaling, a key molecular initiating event known to be affected in di- and trinitrotoluene exposures.

International Telemetering Conference Proceedings / October 25-28, 1999 / Riviera Hotel and Convention Center, Las Vegas, Nevada / In this paper, we extended the matched filter bound (MFB) of time-discrete multipath Rayleigh fading channels derived in [1,2] for multiscale wavelet signaling communication.

Cranial placodes are transient thickenings of the vertebrate embryonic head ectoderm that will give rise to sensory (olfactory, lens, and otic) and non-sensory (hypophyseal) components of the peripheral nervous system (PNS). In most vertebrate embryos, these four sensory placodes undergo invagination. Epithelial invagination is a morphological process in which flat cell sheets transform into three-dimensional structures, like an epithelial pit/cup. The process of invagination is crucial during development as it plays an important role for the formation of the lens, inner ear, nasal cavity, and adenohypophysis. Using the chick as the model system the following questions were addressed. What signals are involved in placode invagination? Is there any common regulatory molecular mechanism for all sensory placode invagination, or is it controlled by unique molecular codes for each individual placode? Are placode invagination and acquisition of placode-specific identities two independent developmental processes or coupled together? To address this we used in vivo assays like electroporation and whole embryo culture. Our in vivo results provide evidence that RhoA and F-actin rearrangements, apical constriction, cell elongation and epithelial invagination are regulated by a common BMP (Bone morphogenetic protein) dependent molecular mechanism. In addition, our results show that epithelial invagination and acquisition of placode-specific identities are two independent developmental processes. BMP signals have been shown to be essential for lens development and patterning of the retina. However, the spatial and temporal requirement of BMP activity during early events of lens development has remained elusive. Moreover, when and how retinal cells are specified, and whether the lens plays any role for the early development of the retina is not completely known. To address these questions, we have used gain- and loss-of-function analyses in chick explant and intact embryo assays. Here, we show that during lens development BMP activity is both required and sufficient to induce the lens specific marker, L-Maf. After the L-Maf upregulation the cells are no longer dependent on BMP signaling for the next step of fiber cell differentiation, which is characterized by up-regulation of δ-crystallin expression. Regarding the specification of retinal cells our results provide evidence that at blastula stages, BMP signals inhibit the acquisition of eye-field character. Furthermore, from optic vesicle stages, BMP signals emanating from the lens are essential for maintaining eye-field identity, inhibiting telencephalic character and inducing neural retina cells.

Context. Introduction of efficient multimedia technologies combined with the spreading of high-speed internet connection all over the world has led to the continuous increase in demand of multimedia services, particularly video and audio. One of the major demands are flexible, interoperable and cost-effective lightweight remote collaboration systems in companies. Web Real Time Communication (WebRTC) is an emerging peer to peer technology that is promising to be the solution to many digital real-time communication challenges. With its fantastic one-to-one communication capabilities, WebRTC supports fast and smooth audio calls, video calls, conferencing, data (media file, document and screen) sharing, gaming and all sorts of messages exchange, all being done straight out of the browser. However, as shown by investigations and interviews supported by Ericsson AB and Semcon AB as party of the MERCO (Mediated Effective Remote Collaboration) international project, many corporate use cases of remote collaboration involve applications beyond the conventional one to one communication. Present videoconferencing systems (telepresence) limits the collaboration flexibility due to their lack of the ability to adapt to system resource usage, hence tend to be too heavy for less powerful devices (laptops, tablets, phones). Moreover, their installation and maintenance costs are too expensive for small companies.  Therefore, new flexible, lightweight and less expensive solutions for remote collaboration need to be developed. Objectives. The main objective of this thesis is to identify technical solutions to address the challenges of resource usage flexibility in WebRTC multi-party remote collaboration systems. Despite concurrent developments of both commercial and free solutions that provide multi-party videoconferencing services using WebRTC, present solutions such as the conventional Multipoint Control Unit (MCU), Selective Forwarding Unit (SFU) and Fully Meshed architectures suffers from issues of excessive resource usage and cannot deliver the acceptable quality of experience in different use cases, particularly the mobile environment. The aim of this thesis is to investigate lightweight technical solutions that can be used to improve the system resource usage in WebRTC multiparty conferencing systems. Through understanding the architectural designs, benchmarking the performance of various technologies used in WebRTC and selecting the most suitable techniques a prototype is developed as a proof of concept. Methods. The first part of the thesis is dedicated to comprehensive study of fundamentals, background information and related works on WebRTC. This gives knowledge of technologies, techniques and performance evaluation metrics which help in making appropriate technical decisions during the experimental development of WebRTC solutions. The second part of the thesis is dedicated to experimental investigation in which two WebRTC signaling technologies (XSockets and NodeJs) are evaluated based on call setup time in WebRTC group call. Two lightweight technical solutions for improving resource usage flexibility (Switching video quality based on speech and using emotions and gestures instead of video) are evaluated based on system resources (CPU, memory, disk and network) and user experience. Results. Based on call setup time of WebRTC multi-party calls, the experimental results indicates that XSockets is a better signaling technology than NodeJs. The two proposed lightweight solutions have shown a remarkable improvement based on systems resource usage. A 15% reduction of CPU usage is observed when using speech controlled video quality switching and further 10% reduction is observed when video is replaced by emotions and gestures. Conclusions. Despite the minimal resource usage achieved by using emotions technique, this solution has usability issues as it cannot detect emotions in poor lighting environment. Consequently, the solution of switching video quality based on speech is chosen for further implementation. Though, this technique can be further improved through using machine learning techniques, the current implementation can significantly reduce the amount CPU, memory, disk and network usage to allow up to 6 participants to join a single conference call while maintain acceptable quality of experience.

Skeletal muscle protein synthesis is regulated by the mammalian target of rapamycin (mTOR) signaling pathway. The first objective was to optimize the methodological procedures for assessing mTOR signaling in horses. The response of mTOR signaling (P-Akt Ser473, P-S6K1 Thr389, P-rpS6 Ser235/26 & 240/244, and P-4EBP1 Thr37/46 by Western blotting techniques) to meal consumption was determined at three gluteal muscle biopsy depths (6, 8, and 10 cm), and the repeatability of the contralateral side at 8 cm during 5 days of repeated biopsies. There was no effect (P > 0.05) of sampling side or biopsy depth on mTOR signaling in mature horses. During repeated biopsies there was an increase (P < 0.05) in downstream (P-S6K1 Thr389, P-rpS6 Ser235/236 & 240/244 and P-4EBP1 Thr389) mTOR signaling in response to feeding. The second objective was to characterize alterations in mTOR signaling throughout the equid lifespan. Adolescent horses (yearlings and two year olds) studied in the postprandial had a lowered (P < 0.05) activation of downstream mTOR signaling with aging. There was a lower (P < 0.05) abundance of P-S6K1 Thr389 in aged horses (23.5 years old) than in mature horses (11 years old) during the post-absorptive state. The final objective was to assess mTOR signaling during acute and chronic inflammation. Acute inflammation occurred during 5 days of repeated biopsies, and chronic inflammation is characteristic of the aged. During acute inflammation, characterized by increased muscle mRNA expression of inflammatory cytokines, there was an increase (P < 0.05) in downstream mTOR signaling. Chronic inflammation resulted in a decrease (P < 0.05) in the abundance of P-S6K1 Thr389. Phenylbutazone was administered to reduce (P < 0.05) acute and chronic inflammation in muscle. Phenylbutazone administration during acute inflammation reduced (P < 0.05) the activation of downstream mTOR signaling and trended to increase (P = 0.09) P-S6K1 Thr389 abundance during chronic inflammation. Whole-body protein synthesis determined using isotope infusion techniques increased (P < 0.05) when chronic inflammation was reduced due to phenylbutazone administration. This research provides new standards for muscle biopsy collection when examining mTOR signaling, and insight into management and feeding practices for adolescent and aging horses.

Parthenolide (PN), a major active component of the traditional herbal medicine feverfew, has been shown to have anti-inflammatory and anti-tumor properties. More remarkably, the cytotoxicity of PN seems selective to tumor cells but not their normal cell counterparts. In the present study, we investigate whether and how PN selectively enhances tumor sensitivity to radiation therapy by using prostate cancer cells LNCaP, DU145 and PC3, as well as normal prostate epithelial cells PrEC. Our study demonstrates that inhibition of NF-κB pathway and suppression of its downstream target MnSOD are common mechanisms for the radiosensitization effect of PN in prostate cancer cells. The differential susceptibility to PN in two radioresistant cancer cells, DU145 and PC3, is due, in part, to the fact that in addition to NF-κB inhibition, PN activates the PI3K/Akt pro-survival pathway in both cell lines. The presence of PTEN in DU145 cells enhances the radiosensitization effect of PN by suppression of the steady state level of activated p-Akt. We also demonstrate that PN selectively exhibits a radiosensitization effect on prostate cancer PC3 cells but not on normal prostate epithelial PrEC cells. PN causes oxidative stress in PC3 cells but not in PrEC cells, as determined by the oxidation of the ROS-sensitive probe H2DCFDA and intracellular reduced thiol and disulfide levels. In PC3 but not PrEC cells, PN activates NADPH oxidase leading to a decrease in the level of reduced thioredoxin, activation of PI3K/Akt and consequent FOXO3a phosphorylation, which results in the downregulation of FOXO3a targets, antioxidant enzyme MnSOD and catalase. Importantly, when combined with radiation, PN further increases ROS levels in PC3 cells, while it decreases radiation-induced oxidative stress in PrEC cells, possibly by increasing GSH level. Overall, our data support the concept that increasing oxidative stress in cancer cells, which are already under high constitutive oxidative stress, will lead to cell death, while the same stress may allow normal cells to maintain redox homeostasis through adaptive response. Thus, modulating cell redox status may be a novel approach to efficiently and selectively kill cancer cells.

The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multistep phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): the type-B ARRs (response activators) and type-A ARRs (negative-feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the related to ubiquitin (RUB) modification pathway, leads to decreased cytokinin sensitivity. This research shows that the cytokinin resistance of axr1 seedlings is suppressed by loss-of-function of type-A ARRs and that the cytokinin resistance caused by ectopic expression of ARR5, a type-A ARR family member, is enhanced in axr1 background. Based on the established role of the RUB pathway in ubiquitin-dependent proteolysis, these data suggested that AXR1 promotes the cytokinin response by facilitating type-A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity. In addition, ARR1, a type-B ARR family member, also accumulated in the axr1 mutant background, suggesting that AXR1 regulates primary cytokinin signaling at multiple levels.

Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text