Previous research discovered the existence of sedimentation-resistant mutants of E. coli. Genomic studies revealed that these mutants resisted sedimentation due to independent modifications to genes that influenced the Rcs signal transduction pathway, causing increased secretion of an exopolysaccharide capsule comprised primarily of colanic acid. The Rcs system is responsible for detecting envelope stressors; consequently, ampicillin and osmotic stress were used to perturb the cellular envelope and study the response of the mutants compared to wild-type cells. It was found that the overproduction of colanic acid in the mutants confers some resistance to envelope stress; however, the mutants still behaved similarly to wild-type cells. The doubling times of the strains grown in sodium chloride solutions were calculated. A wavelength scan from 400 nm to 800 nm was performed on strains grown in different salt concentrations to determine if there were significant differences in light scattering between the wild-type and mutant cells. Further analysis was performed that, along with the doubling time data, suggested that wild-type cells may have turned on genes for capsule production in response to being grown in high salt concentrations. Additional research could be conducted to test this hypothesis, perhaps through the quantification of colanic acid through a methyl pentose assay for wild-type cultures grown with high salt concentrations. The idea that wild-type cells could digest colanic acid as a carbon source when lacking resources was also investigated with different preparations of colanic acid. One preparation of colanic acid showed promising results, which could indicate that bacteria are able to digest their capsule in a novel method to produce energy when starved. Again, additional investigation should be conducted to confirm these results. Other future experiments could study the metabolome of these mutants to determine if they have increased quantities of alarmones related to biofilm formation.

B cell development in chicken takes place in a specific primary lymphoid organ, the bursa of Fabricius. The bursa is considered to provide a microenvironment that promotes B lymphocyte survival and maturation. The most important maturation step in the bursa is the immunoglobulin (Ig) gene conversion, a process that is responsible for immunoglobulin repertoire in avian species. The Ig-gene conversion is strictly regulated, and only progenitors that are able to initiate the process will develop into fully functional B lymphocytes. In this study the late embryonic B lymphocyte stages are investigated, the bursal stem cell stage and the onset of Ig-gene conversion stage. Previous studies identified functional and phenotypic differences between the two stages, showing high rate of proliferation at both, but a significant increase in apoptotic activity at the onset of gene conversion stage. The molecular basis behind the initiation of Ig-gene conversion is not well understood. Here two approaches are presented to provide information on the B lymphocyte developmental process. In chapter II proteomic analysis of the two cell stages was performed. The proteins were sorted into functional groups and signal transductions pathways were identified that are associated with proliferation, differentiation, cell adhesion and apoptosis. The project identified differences in protein profiles that might explain the changes in B lymphocyte physiology and bursal microenvironment at the initiation of Ig-gene conversion. In chapter III the antigen recognized by a bursal secretory dendritic cell specific monoclonal antibody, GIIF3 was identified and cloned. The antigen was shown to be expressed by bursal secretory dendritic cells only during the late embryonic period. The antigen was identified as smooth muscle gamma actin. Futher work will investigate what role the gene plays in dendritic cell funtion.

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Platelets play a vital role in processes of hemostasis and thrombosis under physiological and pathological conditions. Following vascular damage, platelets will accumulate and stably adhere to exposed subendothelial matrixes. The binding of platelet surface receptor Glycoprotein VI (GPVI) to exposed collagen initiates a signaling cascade, which culminates in platelet activation. Stimulation of GPVI pathways results in the generation of thromboxane and causes the platelets to secrete their granule contents. This generated thromboxane as well as constituents released from dense granules such as ADP, and serotonin, play an essential role in potentiating the platelet response through activation of other surface receptor mediated pathways. Importantly, downstream of all these separate pathways, kinases become activated and play a crucial role in phosphorylating their substrates to elicit critical cellular responses. Previously published studies have established the importance for myosin kinase in its role for phosphorylating the myosin light chain (MLC) downstream of ADP receptors. These studies have shown MLC phosphorylation occurs rapidly and is essential for shape change following the stimulation of ADP receptors. Technological advances in antibody development have resulted in the generation of commercially available phospho-specific antibodies for MLC phosphorylated on either threonine (Thr) 18 or serine (Ser) 19. These antibodies allowed us to revisit these prior studies and address whether phosphorylation on MLC (Ser) 19 would elicit one response while phosphorylation on (Thr) 18 may result in another functional response. Our result show, that MLC is phosphorylated rapidly on (Ser) 19 and plays an important role in shape change downstream of Gq pathways, while MLC (Thr) 18 phosphorylation occurs at a slower rate downstream of G12/13 pathways and contributes to platelet dense granule secretion. Protein kinase C's (PKC) are serine/threonine kinase, which become activated following the stimulation of many of the platelet surface receptors. PKCs are classified into three groups, classical (α, βI, βII, γ), novel (δ, ε, η, θ), and atypical (ζ, ι, λ, μ) based on their cofactor requirements for activation. The classical PKCs, which require diacylglycerol and calcium for their activation were investigated using the specific inhibitor Go6976. Much to our surprise, we demonstrated that downstream of GPVI pathways, Go6976 caused non-selective inhibition of Spleen tyrosine kinase (Syk) activity. This inhibition of Syk activity resulted in a concentration-dependent reduction in phosphorylation of downstream molecules Lat and PLCγ2 as well as platelet aggregation and secretion. Stimulation of surface receptors GPVI, CLEC-2, GPIb, and FcRIIa, all lead to the activation of tyrosine kinase pathways. The role for Syk in these pathways is essential and in the absence of its activity these pathways are completely shut down. We inadvertently discovered dextran sulfate (DxS) actives platelets. Our results show that DxS activates a Src-dependent pathway which does not utilize surface receptors GPVI, CLEC-2, GPIb, or FcRIIa. Platelets pretreated with Syk inhibitors OXSI-2 or Go6976 failed to cause αIIbβ3 activation in response to convulxin, however, platelets activated with DxS under the same conditions retained the ability to activate αIIbβ3. In response to DxS, platelet aggregation, intracellular calcium mobilization, and αIIbβ3 activation were significantly inhibited in platelets pre-treated with PI-3K inhibitors. Taken together these results for the first time establish a novel tyrosine kinase pathway in platelets that cause fibrinogen receptor activation in a PI-3K dependent manner without a role for Syk. In conclusion, we have evaluated the role of myosin light chain kinase, Syk, and PI-3 kinase downstream of platelet receptor-mediated pathways. We have examined the phosphorylation status of several of their effector molecules and have correlated these events with their functional responses in platelets. Here we have highlighted several roles for platelet kinases and their relative importance in regulating platelet functional outcomes. / Physiology