The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1. To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation. Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography. Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.

The budding yeast Saccharomyces cerevisiae exhibits exquisite control of cellular size in response to the nutritional composition of its environment. Size control is mediated at the G1/S phase transition, termed Start: passage through Start represents an irreversible commitment to cell division and is contingent on achieving a critical size. When nutrients are plentiful, yeast increase their critical size set-point resulting in larger cells; in contrast, in poor nutrients, yeast pass Start at a smaller size. The genetic basis for nutrient-dependent size control and the means by which yeast sense their size remain elusive. One measure of growth potential is ribosome biogenesis, the rate of which correlates with cell size. I characterized a G-patch domain containing protein, Pfa1, which has been shown to activate the helicase activity of the pre-rRNA processing factor Prp43. Intriguingly, Pfa1 is multiply phosphorylated in response to inhibition of the TOR kinase, the central player in growth regulation. This phosphorylation occurs in a region required for Pfa1 function in ribosome biogenesis, independent of its role as a helicase activator. Consistently, phosphorylation correlates with loss of physical interactions with ribosome biogenesis and altered interactions with the ribosome. Mutation of these phosphorylation sites eliminates TOR-dependent phospho-regulation, and confers sensitivity to TOR inhibition. I propose a model wherein Pfa1 is phosphorylated in response to nutrient stress, leading to relocalization of essential processing factors, and inhibition of both ribosome biogenesis and tRNA maturation. Further, I constructed and verified a non-covalent short oligonucleotide barcode microarray platform, and applied it to genome-scale parallel analyses of both the DNA damage response and cell size control in S. cerevisiae. Through these studies, I uncovered novel connections between size control and numerous cellular processes including: the large subunit of the ribosome; the mitochondrial pH gradient; and proteins involved in oxidant-induced cell cycle arrest.

The ubiquitin system plays a pivotal role in regulating protein degradation, endocytosis and numerous other cellular functions. E3 ubiquitin ligases, which mediate the final step in the ubiquitylation reaction cascade, are responsible for substrate recognition and ubiquitin attachment to them, underscoring the importance of identifying their substrates. Nedd4 family members are E3 ubiquitin ligases comprised of a C2-WW-HECT domain architecture. This thesis was aimed at first globally delineating the substrate specificity of the closely related Nedd4 family members in humans, hNedd4-1 (Nedd4) and hNedd4-2 (Nedd4L), and second, to follow up on one of the novel hits identified, the FGFR1, and study in detail how it is regulated by its E3 ligase hNedd4-1. To globally identify substrates for Nedd4 proteins, a high throughput proteomic screening technology using protein microarrays was employed. Despite the obvious homology in their domain architecture, the results presented here suggest that these Nedd4 family members may function non-redundantly, since they demonstrate a selective preference towards substrate ubiquitylation. Our focus on FGFR1, a substrate identified for hNedd4-1, has revealed an important functional role for this ubiquitin ligase in promoting FGFR1 endocytosis and downregulation of its signaling activity. The evidence presented indicates that this interaction has important consequences for developmental processes that are dependent on FGF signaling: human neural stem cell differentiation and zebrafish embryonic patterning and brain development. We demonstrate that the WW3 domain of Nedd4-1 recognizes a novel, non-canonical binding surface (peptide2) within the juxtamembrane region of FGFR1, and we are currently in the process of solving the 3 dimensional structure of the hNedd4-1 WW3: FGFR1 peptide2 complex using X-ray crystallography. Furthermore, in characterizing the interaction between hNedd4-1 and FGFR1, we have provided evidence for a novel mechanism for regulating the catalytic activity of hNedd4-1 by FGFR1 activation. This involves the formation of hNedd4-1 dimers upon removal of the autoinhibitory C2 domain from the HECT domain. Dimerized hNedd4-1, in turn, exhibits enhanced interactions with FGFR1 and enhanced receptor ubiquitylation. From these data, we proposed a negative feedback inhibitory model for FGFR1 downregulation, whereby activated receptor enhances the activation of its suppressor, hNedd4-1, to ensure timely termination of receptor signaling.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.

Campylobacter jejuni is a major cause of bacterial gastroenteritis in the developed world; however, many aspects of its biology remain poorly understood, including its colonization of the mucus layer lining the gastrointestinal tract. In this study, we utilized microarray transposon tracking to compile a list of 195 genes essential for the growth of C. jejuni in vitro under microaerophilic conditions. Then we characterized C. jejuni growing in an extracted intestinal mucus medium. We found that C. jejuni will grow efficiently in a medium comprised of either chick and piglet intestinal mucus, and that these media have a dramatic impact on its transcriptome. Within the genes identified as differentially expressed during growth in a mucus medium, we identified a single operon, (cj0481-cj0490), which we have subsequently characterized as being responsible for both the uptake and metabolism of L-fucose. This represents the first observation of carbohydrate metabolism by the otherwise asaccharolytic C. jejuni. We further found that the inability to utilize L-fucose puts C. jejuni at a competitive disadvantage when colonizing the piglet intestine, but not the chick cecum. Finally, we examined C. jejuni’s ability to utilize mucins as a carbon source while growing within the mucus layer. We found that despite mucins being a major source of L-fucose and amino acids within the intestine, C. jejuni has a minimal ability to degrade and utilize mucins on its own. However, close proximity to mucolytic bacteria within the microbiota of the intestine, allows for increased C. jejuni growth. Together, this paints the picture of an organism that is well adapted to survival within the mucus lining of the intestine and establishing itself as part of the intestinal microbiota.

Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are added to polyurethane foams in a variety of industrial and consumer products to prevent flame ignition. The gradual release of these flame retardants (FRs) from such products leads to contamination of various abiotic and biotic media, including wild birds. Recent studies demonstrated endocrine-disrupting effects of TCPP and TDCPP, including alteration of circulating thyroid hormone (TH) levels. The TH-pathway is essential for normal growth and development in birds. There are limited data on the toxicological effects of TCPP and TDCPP in avian species and, prior to this work, no study has examined their effects in avian embryos. This M.Sc. thesis investigates the developmental, molecular and biochemical effects of TCPP and TDCPP in chicken (Gallus gallus domesticus) embryos via egg injection studies. TCPP delayed pipping at doses ≥9.24 μg/g, both TCPP and TDCPP reduced embryo growth at the highest dose (51.6 μg TCPP/g and 45 μg TDCPP/g), and TDCPP decreased free plasma thyroxine and gallbladder size at 7.64 μg/g and 45 μg/g, respectively. Real-time reverse transcription polymerase chain reaction was used to measure changes in mRNA levels of hepatic genes that were responsive to these FRs in a previous in vitro study. TCPP dysregulated the expression of TH-responsive genes and xenobiotic metabolizing enzymes (cytochrome P450s; CYPs), whereas TDCPP only affected CYPs. Less than 1% of the administered TCPP or TDCPP was detected in egg contents following 19 days of incubation, indicating extensive metabolism of the parent compounds. DNA microarrays were used to perform a global transcriptional analysis on liver samples from embryos that exhibited adverse effects following TDCPP injection. 47 differentially expressed genes were identified at the 45 μg/g dose. Functional analysis revealed that immune function and lipid and steroid metabolism were major targets of TDCPP toxicity and indicated a state of cholestatic liver/biliary fibrosis. Since the TH-pathway is a key regulator of metabolic homeostasis, its disruption early in development is a potential cause of the observed adverse effects. This thesis demonstrates, for the first time, developmental and endocrine-disrupting effects of TCPP and TDCPP in an avian species and attempts to link phenotypic changes to molecular-level disruptions in hopes to improve the understanding of their modes of action.

Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.

Severe manifestations of malaria, including cerebral malaria (CM) and respiratory distress, result in approximately three million deaths annually worldwide. Currently, relatively little is known about severe disease pathogenesis. The development and outcome of severe malaria is determined by host-pathogen interactions, a complex interface of genetics and immune responses. Hypothetically, a spectrum of genetic susceptibility and resistance to severe disease exists within the host population, and malaria infection results in diverse host and parasite responses that impact disease outcome. The aim of this study was to identify differential host and parasite responses in a murine model of severe malaria, Plasmodium berghei ANKA (PbA), in CM-susceptible and CM-resistant mice; and to analyze host genetics in patients with severe disease due to Plasmodium falciparum. In vivo, expression microarray analysis showed that, in malaria target organs, differential responses were related to immune response – primarily interferon and complement pathways – and apoptosis. Histopathological examination of the brain confirmed an increased prevalence of apoptosis in CM-susceptible mice. Further examination of the role of complement in CM-susceptibility determined that early complement 5 (C5) activation conferred susceptibility to CM, and that C5 deficiency conferred resistance, which could be recapitulated by antibody blockade of activated C5 or its receptor in susceptible mice. Additionally, single nucleotide polymorphism (SNP) studies identified that complement receptor 1 SNPs were associated with disease severity in patients with P. falciparum malaria. PbA parasites displayed a unique transcriptional signature in each tissue examined (brain, liver, spleen and lung), showed differential gene expression between CM-resistant and susceptible hosts, and were most prominent in lung tissue. Closer examination of lung involvement in PbA infection revealed that PbA-infected C57BL/6 mice develop acute lung injury (ALI), defined by disruption of the alveolar-capillary membrane barrier. ALI susceptibility did not correlate with CM susceptibility, but was influenced by peripheral parasite burden and CD36-mediated parasite sequestration in the lung. PbA provides a clinically relevant experimental model for CM and ALI, through which important disease mechanisms can be identified and modulated. Ideally, the use of such models aids in the discovery of disease biomarkers and novel therapeutic strategies, which may be applied to human severe and cerebral malaria.

Severe manifestations of malaria, including cerebral malaria (CM) and respiratory distress, result in approximately three million deaths annually worldwide. Currently, relatively little is known about severe disease pathogenesis. The development and outcome of severe malaria is determined by host-pathogen interactions, a complex interface of genetics and immune responses. Hypothetically, a spectrum of genetic susceptibility and resistance to severe disease exists within the host population, and malaria infection results in diverse host and parasite responses that impact disease outcome. The aim of this study was to identify differential host and parasite responses in a murine model of severe malaria, Plasmodium berghei ANKA (PbA), in CM-susceptible and CM-resistant mice; and to analyze host genetics in patients with severe disease due to Plasmodium falciparum. In vivo, expression microarray analysis showed that, in malaria target organs, differential responses were related to immune response – primarily interferon and complement pathways – and apoptosis. Histopathological examination of the brain confirmed an increased prevalence of apoptosis in CM-susceptible mice. Further examination of the role of complement in CM-susceptibility determined that early complement 5 (C5) activation conferred susceptibility to CM, and that C5 deficiency conferred resistance, which could be recapitulated by antibody blockade of activated C5 or its receptor in susceptible mice. Additionally, single nucleotide polymorphism (SNP) studies identified that complement receptor 1 SNPs were associated with disease severity in patients with P. falciparum malaria. PbA parasites displayed a unique transcriptional signature in each tissue examined (brain, liver, spleen and lung), showed differential gene expression between CM-resistant and susceptible hosts, and were most prominent in lung tissue. Closer examination of lung involvement in PbA infection revealed that PbA-infected C57BL/6 mice develop acute lung injury (ALI), defined by disruption of the alveolar-capillary membrane barrier. ALI susceptibility did not correlate with CM susceptibility, but was influenced by peripheral parasite burden and CD36-mediated parasite sequestration in the lung. PbA provides a clinically relevant experimental model for CM and ALI, through which important disease mechanisms can be identified and modulated. Ideally, the use of such models aids in the discovery of disease biomarkers and novel therapeutic strategies, which may be applied to human severe and cerebral malaria.

Atherosclerosis occurs preferentially at branches and curves in arteries exposed to disturbed flow while sparing straight portions of arteries exposed to undisturbed flow. In vivo and in vitro studies have implicated NADPH oxidases in atherosclerosis and hypertension. Shear stress can induce reactive oxygen species production in endothelial cells from a variety of sources, including NADPH oxidases. Here, we examined the hypothesis that unidirectional laminar shear (LS) and oscillatory shear (OS) would differentially regulate gene expression profiles in NADPH oxidase-dependent and -independent manners, and that these genes would provide novel molecular targets in understanding endothelial cell biology and vascular disease. The p47phox subunit of the NADPH oxidase can be an important regulator of certain Nox isoforms, including Nox1 and Nox2 which may be responsible for shear-induced superoxide production. In order to isolate p47phox-dependent shear responses, we took advantage of the p47phox-/- transgenic mouse model which lacks a functional p47phox subunit. We developed a method to isolate murine aortic endothelial cells using an enzymatic digestion technique. These cells expressed characteristic endothelial markers, including VE-cadherin, PECAM1, and eNOS, and aligned in the direction of flow. We successfully isolated primary murine aortic endothelial cells from both wild-type C57BL/6 mice (MAE-WT) and p47phox-/- mice (MAE-p47). Furthermore, we established an immortalized cell line from each of these cell types, iMAE-WT and iMAE-p47. We carried out microarray studies using Affymetrix Mouse Genome 430 2.0 Arrays (39,000+ transcripts) on MAE-WT and MAE-p47 that were exposed to atheroprotective LS or atherogenic OS for 24 hours. In comparison to LS, OS significantly changed the expression of 187 and 298 genes in MAE-WT and MAE-p47, respectively. Of those, 23 genes showed similar gene expression patterns in both cell types while 462 genes showed different gene expression patterns in the two cell types, demonstrating a considerable role for p47phox-based NADPH oxidases in shear-dependent gene expression. Changes in expression of several genes, including Kruppel-like factor 2 (Klf2), endothelial nitric oxide synthase (eNOS), angiopoietin 2 (Ang2), junctional adhesion molecule 2 (Jam2), bone morphogenic receptor type II (Bmpr2), and bone morphogenic protein 4 (Bmp4) were confirmed by quantitative PCR and/or immunoblotting using both primary cells and immortalized cells. Of these genes, our data suggest that Jam2, Bmpr2, and Bmp4 may be shear-sensitive in a p47phox-dependent manner. Taken together, our studies have identified a set of shear- and p47phox-sensitive genes, including unexpected and novel targets, which may play critical roles in vascular cell biology and pathobiology.