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Typescript (photocopy). / Digitized by Kansas Correctional Industries

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<p>The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its activity as a kinase. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1-mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.</p> / Dissertation

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.