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Background : Plasmodium vivax merozoites specifically invade reticulocytes. Until recently, two reticulocyte-binding proteins (Pvrbp1 and Pvrbp2) expressed at the apical pole of the P. vivax merozoite were considered to be involved in reticulocyte recognition. The genome sequence recently obtained for the Salvador I (Sal-I) strain of P. vivax revealed additional genes in this family, and in particular Pvrbp2a, Pvrbp2b (Pvrbp2 has been renamed as Pvrbp2c) and two pseudogenes Pvrbp2d and Pvrbp3. It had been previously found that Pvrbp2c is substantially more polymorphic than Pvrbp1. The primary goal of this study was to ascertain the level of polymorphism of these new genes. Methodology/Principal Findings : The sequence of the Pvrbp2a, Pvrbp2b, Pvrbp2d and Pvrbp3 genes were obtained by amplification/cloning using DNA purified from four isolates collected from patients that acquired the infection in the four cardinal regions of Thailand (west, north, south and east). An additional seven isolates from western Thailand were analyzed for gene copy number variation. There were significant polymorphisms exhibited by these genes (compared to the reference Sal-I strain) with the ratio of mutations leading to a non-synonymous or synonymous amino acid change close to 3:1 for Pvrbp2a and Pvrbp2b. Although the degree of polymorphism exhibited by these two genes was higher than that of Pvrbp1, it did not reach the exceptional diversity noted for Pvrbp2c. It was interesting to note that variations in the copy number of Pvrbp2a and Pvrbp2b occurred in some isolates. Conclusions/Significance : The evolution of different members of the Pvrbp2 family and their relatively high degree of polymorphism suggests that the proteins encoded by these genes are important for parasite survival and are under immune selection. Our data also shows that there are highly conserved regions in rbp2a and rbp2b, which might provide suitable targets for future vaccine development against the blood stage of P. vivax.

Corticotropin-releasing hormone (CRH) is an important mediator of the mammalian stress response. Functioning both as a neurotransmitter and endocrine hormone, it signals through two receptors, CRH-R1 and CRH-R2. CRH is also bound with high-affinity by CRH-Binding Protein (CRH-BP), a secreted glycoprotein. As soluble binding proteins can play an important role in modulating the availability and activity of ligands at the receptors, this thesis focuses on the characterization of multiple binding proteins in the CRH system, including both truncated splice forms of CRH-R2 and the classical CRH-BP. First, we identified splice variants of CRH-R2 that were predicted to serve as soluble-decoy receptors because they encode the extracellular, ligand-binding domain of CRH-R2 but terminate prior to the transmembrane domains. These splice variants, called soluble CRH-R2 (sCRH-R2) α and β, encode similar proteins but have unique N-termini. We demonstrated that the α isoform of sCRH-R2 was efficiently translated in vivo, despite being a predicted substrate for nonsense-mediated RNA decay; however, the resulting protein was not trafficked for secretion due to an ineffective signal peptide, and was consequently degraded by the proteasome. In contrast, the β isoform of sCRH-R2, with its unique signal peptide, was properly trafficked for secretion and escaped degradation. Therefore, unlike sCRH-R2α, sCRH-R2β is positioned to function as a soluble CRH-binding protein. For analysis of CRH-BP, current models suggest that CRH-BP and CRH-receptors compete for available ligand, yet the rate of ligand association and dissociation, which are paramount to this competition, were not established. We determined these kinetic parameters for CRH with CRH-BP and CRH-R2, showing that CRH binds faster and releases more slowly for CRH-BP, which suggests CRH-BP is an efficient ligand trap. Furthermore, we demonstrated that CRH-BP inhibits CRH-R2 activation in cell culture. Strikingly, the amplitude and duration of the inhibition was dependent on the time of ligand interaction with CRH-BP prior to encountering the receptors, highlighting the importance of kinetic and temporal considerations in defining the function of CRH-BP. Together, these studies further characterize the role of both the classic and alternative binding proteins in the CRH system and advance our understanding of their function in stress biology.

Establishment of cell polarity requires the involvement of several posttranscriptional regulatory mechanisms, including mRNA localization and translational control. A family of highly conserved RNA binding proteins in vertebrates, VICKZ (V̲g1RBP/V̲era, I̲MP-1, 2, 3, C̲RD-BP, K̲OC, Z̲BP-1) proteins, has been shown to act in these two processes. Previous studies of the posttranscriptional mechanisms mediated by VICKZ family members have been largely limited by the lack of genetic approaches in certain vertebrate systems. Identification of Imp, the Drosophila member of the VICKZ family, opened the possibility to use genetic approaches to investigate the roles of a VICKZ family member in mRNA localization and translational control. In this dissertation, we show that Imp is associated with Squid and Hrp48, two heterogeneous proteins (hnRNP) that complex with one another to regulate localized expression of gurken (grk). In addition, Imp binds grk mRNA with high affinity in vitro and is concentrated at the site of grk localization in midstage oocytes. Mutation of the Imp gene does not substantially alter grk expression, but does partially suppress the grk mis-expression phenotype of fs(1)k10 mutants. In contrast, overexpression of Imp in germ line cells results in mislocalization of grk mRNA and protein. The opposing effects of reduced and elevated Imp activities on grk expression suggest that Imp acts in regulation of grk expression, but in a redundant way. To further explore the mechanisms by which localized expression of grk is regulated by Imp, a deficiency screen was conducted to search for dominant modifiers of the dorsalized phenotype resulting from Imp overexpression. Twelve genomic regions were identified to contain dominant modifiers of the Imp overexpression phenotype. Further characterization of mutants of genes within these genomic regions led to identification of five modifiers, including cyclin E (cycE), E2f transcriptional factor 1 (E2f1), lingerer (lig), snail (sna) and mushroom body expressed (mub). E2f1 encodes a transcriptional factor that is involved in regulating the G1 to S phase transition during mitosis. Mutation of E2f1 results in altered grk mRNA and protein distribution within oocyte, revealing a role for this gene in regulation of grk expression.

Mammals have developed a complex system to adapt to stressful situations. The endocrine component of the stress system is mediated via the hypothalamic-pituitary-adrenal (HPA) axis which controls the stress response and homeostatic mechanisms. Corticotropin-releasing hormone (CRH) is the primary hypothalamic regulator of the HPA axis and plays a role in coordinating neuronal, hormonal, immunological, and behavioral responses to stress. The activity of CRH is thought to be modulated by a CRH-binding protein (CRH-BP). CRH-BP is a 37 kilodalton secreted protein that exhibits high binding affinity for CRH and is expressed in a number of cells that express CRH or are CRH targets, such as anterior pituitary corticotropes. Regulation of CRH-BP gene expression was studied using both in vivo and in vitro systems. In response to stress, expression of CRH-BP mRNA in male rat pituitary increases 2--3 times over control levels, and adrenalectomy decreases CRH-BP mRNA levels to 8% of control levels. These data suggest that glucocorticoids positively regulate CRH-BP mRNA expression in the pituitary and that CRH-BP may play a modulatory role in the mammalian stress response. The regulation of pituitary CRH-BP gene expression by sex steroid hormones was also examined. Female mice showed significantly increased levels of pituitary CRH-BP mRNA compared to males, and female mice in proestrus have three-fold higher levels of pituitary CRH-BP mRNA than mice in diestrus. Ovariectomized mice exhibit significantly decreased levels of CRH-BP mRNA compared to control female mice, while ovariectomized mice with estrogen replacement have levels of CRH-BP mRNA levels equivalent to control mice. These data suggest that estrogen regulates pituitary CRH-BP mRNA levels in mice. The molecular mechanisms involved in regulation of CRH-BP gene expression were also examined using rat primary astrocyte cultures. Forskolin, TPA, and CRH increase CRH-BP mRNA levels between 6 and 30 times control levels, and glucocorticoids can inhibit these increases. These changes in steady-state CRH-BP mRNA levels are due in part to altered transcription of the gene. Together, these results demonstrate that Protein Kinase A, Protein Kinase C, adrenal and sex steroid mediated pathways are involved in transcriptional regulation of the CRH-BP gene expression.