Receptor-like kinases (RLKs) are encoded for by one of the largest gene families in Arabidopsis and represent the predominant form of cell surface receptors in plants. RLKs mediate signal transduction in diverse processes including steroid-mediated growth pathways, pathogen-triggered innate immune responses. Here I present characterization of mutant phenotypes, expression patterns, and genetic interactions for the BAK1 INTERACTING RECEPTOR (BIR) family of Leucine-rich Repeat-RLKs, three members of which have had no previous characterization. Furthermore, I show that cell death, aerial growth, and lateral root development defects in bir1-1 are suppressed by mutations of the LRR-RLK co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1); I identify a novel primary root growth phenotype in bir1-1 mutants, as well as a lateral root development phenotype for bir3 mutants; and primary root growth and aerial defects in bir3.bir4;bak1 triple mutants. Using an allelic series of bak1 mutations I show that bir phenotypes are dependent upon particular functions of BAK1, and propose that the BIR family exhibits a novel function, previously undescribed for LRR-RLKs, as regulators of co-receptor/ligand-binding receptor complex specificity.

During Arabidopsis embryogenesis, cell-cell signaling plays an essential role in establishing an organized body plan centered around two major axes of development: apical-basal and radial. Two topics of great interest are how the layered structure is initiated and maintained during and after embryogenesis and how communication between layers is achieved to allow for coordinated development. Recent research involving Receptor-Like Kinases (RLKs) in plants suggests that their roles in integrating various signals are important in many aspects of development, including embryonic and post-embryonic patterning. The research presented here describes the roles of two pairs of RLKs with independent roles in two different signaling environments. The first RLK pair, GSO1 and GSO2, function in root development at the transition to photoautotrophic nutrition to integrate sugar signals and regulate root growth. GSO1 and GSO2 regulate root epidermal cell identity by controlling the pattern of cell division of stem cells. The second pair of RLKs, RPK1 and TOAD2, function to control root development by regulation of meristem proliferation and a coordinated response to signaling molecules of the CLE family. The response of wild-type roots to treatment with CLE peptides (A-type) is meristem growth arrest, resulting in short roots. toad2 mutants are insensitive to the effect of CLE peptides in reducing meristem size and TOAD2 also regulates RPK1 upon CLE stimulation. Although responding to different signals, the two pairs of RLK share a common output of regulating cell proliferation in and around the root meristem, especially in the epidermis of the root.

Bacteriophages are viruses that infect bacteria. This thesis describes studies of gp74 from the bacteriophage HK97, which functions as an HNH endonuclease. HNH endonucleases are DNA digestion proteins characterized by two highly conserved His residues and an Asn residue. Like other HNH endonucleases, the activity of gp74 is dependent on binding of divalent metal ions to the HNH motif. Current work focused on confirming the identity of conserved HNH motif residues of gp74. We hypothesized the catalytic His residue is H43, the structural Asn residue is N73, and that H82 is involved in metal–binding. Additional residues in the ββα–fold, such as D42, may also bind the metal. Our bound metal analysis and the sequence of gp74 also suggest the presence of a Zn2+–finger motif. Mutations of D42 and H82 decrease the activity of gp74, without affecting the structure. These studies advance our understanding of the gp74 activity.

Bacteriophages are viruses that infect bacteria. This thesis describes studies of gp74 from the bacteriophage HK97, which functions as an HNH endonuclease. HNH endonucleases are DNA digestion proteins characterized by two highly conserved His residues and an Asn residue. Like other HNH endonucleases, the activity of gp74 is dependent on binding of divalent metal ions to the HNH motif. Current work focused on confirming the identity of conserved HNH motif residues of gp74. We hypothesized the catalytic His residue is H43, the structural Asn residue is N73, and that H82 is involved in metal–binding. Additional residues in the ββα–fold, such as D42, may also bind the metal. Our bound metal analysis and the sequence of gp74 also suggest the presence of a Zn2+–finger motif. Mutations of D42 and H82 decrease the activity of gp74, without affecting the structure. These studies advance our understanding of the gp74 activity.

Wallace Stevens (1879-1955) is probably the greatest and most prolific exponent of purist estheticism in all of American letters. Born in Reading, Pennsylvania, his first book of poetry, Harmonium (1923) won him the respect and admiration of his fellow writers, but brought him little popular acclaim and less remuneration. His career as a poet spanned four decades, the 1923 volume being followed by Ideas 2( Order (1935); The Man with the Blue Guitar (1937); Parts of â World (1942); Transport to Symmer (1947); The Auroras gt Autumn (1950); The Necessary Angel (a collection of essa7s, 1951); The Collected Poems of Wallace Stevens (1954); and Opus Posthumous (a miscellany of poems, plays, and prose works, edited by Samuel French Morse, 1957). Unlike most of his contemporaries, Stevens chose to divide his time between the world of poetry and that of business, keeping in the process very much to himself, and refusing to mix with literary or academie society. From 1916 to his death in 1955, he was associated with the Hartford Accident and Indemnity Company, of which he became vice-president in 1934. [...]

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.

Blood contacting devices are frequently limited by complications such as surface-induced thrombosis. This thesis investigated the feasibility of using a family of recombinant elastin-like polypeptides (ELPs), namely ELP1, ELP2 and ELP4 that differ by molecular weight and sequence length, as potential thromboresistant coatings. The ELP coatings were prepared by physical adsorption onto the surface of Mylar, with surface modification confirmed by goniometry, X-ray photoelectron spectroscopy (XPS), and chemical force microscopy (CFM). Both surface wettability and hydrophilic adhesion force increased as the ELP sequence length decreased. The ELP adsorption process monitored by using quartz crystal microbalance with dissipation (QCM-D) showed that the ELPs adsorbed within a monolayer. Additionally, ELP surface coverage was found to increase with the polypeptide sequence length. The QCM-D studies also revealed that the longer polypeptides (ELP2 and ELP4) exhibited higher specific dissipation values indicating that they established adsorbed layers with greater structural flexibility and associated water content compared to ELP1. Exposure of the ELP coatings to flowing reconstituted blood demonstrated that both the ELP2 and ELP4 coatings reduced the quantity of adsorbed fibrinogen (Fg), with the ELP4 coating resulting in the lowest levels of adherent platelets. Energy dissipation versus frequency shift plots obtained from QCM-D studies indicated that adsorbed Fg on the ELP4 coating maintained a softer, more flexible film then on the other ELPs. The ELP4 coating also demonstrated an altered binding activity for GPIIb/IIIa where only the AGDV motif in the adsorbed Fg gamma-chain appeared to be exposed and bioactive. Conversely, on the other ELP coatings both the AGDV and RGD motifs (found within the Fg alpha-chain) were available for binding, suggesting that a different Fg conformational state exists on the ELP1 and ELP2 coatings. Moreover, both the ELP2 and ELP4 coatings displayed minimal bulk platelet reactivity following extended whole blood shear exposure (up to an hour) compared to Mylar. This was not observed with the ELP1 coating. Overall, the results suggest that the structural flexibility and associated water content of the ELP coatings appear to be important criteria influencing their thrombogenicity, with ELP4 displaying the most favourable blood-material response compared to ELP1 and ELP2.

Glucagon-like peptide-2 (GLP-2) augments intestinal adaptation in animal models of short bowel syndrome (SBS) and in adult patients with SBS. However, GLP-2 has not been used as a therapy for pediatric SBS. In this thesis, it is hypothesized that exogenous GLP-2 therapy will improve outcomes of intestinal adaptation in proximal intestinal resection (JI) and distal intestinal resection (JC) neonatal piglet models of SBS. Improvements in morphological parameters (increased small intestinal length) and histological parameters (increased jejunal villus length or jejunal crypt depth) of intestinal adaptation in JI and JC neonatal piglets treated with GLP-2 were observed. However, improved clinical outcomes (fewer days of diarrhea, fewer days on parenteral nutrition, more days on enteral nutrition alone) were only observed in GLP-2 treated JC animals. Since the JC anatomical subtype (no remnant ileum) represents the majority of clinical cases of neonatal SBS, these results support a potential role for GLP-2 therapy in pediatric SBS.

Oocyte maturation and ovulation are two major events that occur in fish prior to spawning. While earlier studies have shown that 17α, 20β-dihydroxy-4-pregnen-3-one (17,20β-P) and the insulin-like growth factor (IGF) system are regulators of oocyte maturation in the zebrafish (Danio rerio), it is not known whether these hormones play a role in regulating prostaglandin synthesis which is thought to mediate ovulation. I determined if 17,20β-P and human IGF-1 affect the expression of genes involved in prostaglandin biosynthesis including phospholipase A2 (cpla2) and cyclooxygenase-1/2 (ptgs1/ptgs2), or prostaglandin F2α (PGF2α) levels. 17,20β-P and IGF-1 stimulated oocyte maturation in mid-vitellogenic (MV) and full grown (FG) follicles. In FG follicles, 17,20β-P increased cpla2 expression, whereas IGF-1 increased cpla2 and ptgs2 expression. Both 17,20β-P and IGF-1 increased PGF2α production. The phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling pathways were shown to mediate IGF-1- and 17,20β-P-induced oocyte maturation and cpla2 and ptgs2 expression. Collectively, these results demonstrate that 17,20β-P and IGFs are important regulators of oocyte maturation and prostaglandin synthesis in zebrafish.