The 5-hydroxytryptamine (5-HT) system modulates numerous physiological and behavioural processes, and dysfunction within this system underlies many behavioural disorders, such as major depression. The 5-HT1A receptor is the primary somatodendritic autoreceptor that controls the firing rate of 5-HT neurons, but is also coupled to numerous signalling pathways. An understanding of 5-HT1A receptor signalling may lead to the development of antidepressant drugs that selectively target therapeutic pathways in treating depression. The 5-HT1A receptor is coupled to inhibitory G-proteins via its intracellular loops 2 and 3. Point mutations within these loops selectively uncouple receptor signalling pathways. In this thesis, I addressed whether mutant receptors’ uncoupling from signalling pathways is associated with alteration in G-protein interaction and coupling. Using bioluminescence resonance energy transfer (BRET) to monitor receptor-G-protein interactions, we show that both wild-type and mutant receptors demonstrate a saturable interaction with Gαi protein in unstimulated conditions. Addition of 5-HT increased the BRET signal for the wild-type 5-HT1A receptor, and this increase was blocked by a 5-HT1A receptor antagonist and G-protein blocker (pertussis toxin). Mutant receptors that were deficient in Gαi signalling, but not those that still signalled to Gαi, failed to respond to receptor activation with increased receptor-Gαi interaction. Pull down studies verified the basal and agonist-induced interaction of 5-HT1A receptors with Gαi proteins. In conclusion, we have shown that the 5-HT1A receptor interacts with Gαi consistent with a pre-coupled model and that 5-HT-induced activation enhances this interaction and requires specific residues in the intracellular loops.

The gulf toadfish (Opsanus beta) is a member of a group of teleosts that have retained their ornithine urea cycle (OUC) allowing them to excrete nitrogenous waste in the form of urea (ureotely). Urea-­N for the entire day is excreted in 1-­2 quick pulsing events (1-­3 h). This study evaluated the hypothesis that urea-­N pulsing events in gulf toadfish can be triggered by social signals from conspecifics via a specific waterborne messenger. Using a crowding protocol, we found that pre-­conditioned seawater induced a secondary urea pulsing event in naïve conspecifics. Furthermore, it was revealed that other factors such as signal concentration and donor body mass relay information to recipients as well. Fractionation of pre-­conditioned seawater was carried out to narrow possible signal candidates and the aqueous portion was found to contain the active molecule. Ammonia was found to be an important factor controlling the response of toadfish to pre-conditioned seawater.

In vertebrate and invertebrate systems, the role of glia-neuron interactions during development and behavior is becoming apparent. Recent studies have been aimed at characterizing glial-expressed proteins that affect the modulation of activities traditionally thought to be regulated by the neuron itself. The soil nematode Caenorhabditis elegans has recently emerged as an important invertebrate model to study glial roles in nervous system function and development. My dissertation work focuses on the characterization of HLH-17, a C. elegans basic helix-loop-helix transcription factor that is strongly and constitutively expressed in the glial cells that associate with four of the cephalic (CEP) neurons in the head of the animal. The CEP neurons are four of eight dopaminergic neurons with well characterized roles in the modulation of a number of behavioral activities in the worm. Although HLH-17 is required for neither the specification nor the development of the CEPsh glia or the CEP neurons, it does have a defined role during dopamine responses. We show that HLH-17 functions upstream of the dopamine receptors DOP-1, DOP-3 and the dopamine transporter DAT-1 to affect DA-dependent behaviors. Also, our microarray analyses provide preliminary evidence that HLH-17 targets factors responsible for receiving and transducing signaling molecules that are involved in the modulation of synaptic events in the worm nervous system. Together these results point to a role for HLH-17 in glia-neuron interactions in C. elegans. My dissertation studies therefore provide further support for the role of glial-expressed proteins in the regulation of activities mediated by the nervous system.

Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS-mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors.

B-cell acute lymphoblastic leukemia (B-ALL) represents a collection of diseases that are categorized into subtypes based on the presence of recurrent cytogenetic abnormalities. These abnormalities often result in the expression of oncogenic drivers that denote a standard- or high-risk for relapse. Currently, survival rates boarder 40% for adult patients and relapses are often observed in patients lacking high-risk markers. Thus, there is an unmet need for biomarkers that can identify all high-risk leukemia, and development of novel therapies based on a better understanding of the molecular drivers of B-ALL. To address this need, I designed a multi-parameter phospho-flow cytometry platform and characterized basal and cytokine-potentiated signaling in adult B-ALL samples. I identified patterns of cytokine-responsiveness across B-ALL patients that correlated with the presence of high-risk oncogenic drivers. Furthermore, I demonstrated that small-molecule inhibitors could abrogate cytokine-induced signaling in high-risk patients suggesting these inhibitors may compliment current chemotherapeutic protocols.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

A common bipotent thymocyte precursor gives rise to both lineages of T cells, αβ and γδ. This thesis addresses how the interplay between intrinsic T cell receptor (TCR) signals and cell extrinsic signals provided by Notch and TCR ligands help to assign and support a final lineage fate decision. Emerging data supports a model in which differential TCR signaling capacity plays an instructional role in specifying lineage fate, particularly through induction of the ERK - early growth response gene (Egr) - inhibitor of DNA binding 3 (Id3) pathway. In particular, Id3 expression serves to regulate adoption of the γδ fate. Moreover, Id3 is both necessary and sufficient to enable γδ-lineage cells to differentiate independently of Notch signaling and become competent interferon (IFN)-γ-producing effectors. These findings identify Id3 as a central player that controls both adoption of the γδ fate and their maturation in the thymus. While loss of Notch signaling in γδTCR-expressing CD4-CD8- (DN)3 cells does not affect development, Notch signals are critical for pre-TCR-bearing cells to transition to the CD4+CD8+ (DP) stage of αβ T cell development. Notch signals affect the activation of the PI3K/Akt pathway, which is required for pTα/TCRβ (pre-TCR)-induced survival, differentiation and proliferation of developing αβ-lineage thymocytes. Here, I identify the key molecular players responsible for the interaction between the Notch and PI3K pathways at this critical developmental stage. Notch induction of Hes1 expression is necessary to repress the expression of the PI3K/Akt pathway inhibitor, PTEN, which in turn facilitates pre-TCR-induced differentiation. c-Myc, another critical target of Notch, is required for proliferation during β-selection. Lastly, I find that the majority of DN3 cells expressing both pre-TCR and γδTCR follow the signal strength model for lineage development, and commit and mature along the γδ-lineage. However, manipulation of signal strength, through γδTCR ligand availability or Id3 expression, can skew this development outcome. Taken together, the results from this thesis provide a detailed examination of the molecular mechanisms that are instrumental in determining lineage fate, survival, and proliferation of differentiating thymocytes. Central to these outcomes is the interplay between the Notch, TCR, PI3K, and MAPK signaling pathways.

Tumorigenesis is driven by the sequential accumulation of genetic lesions within a cell, each which confer the cell with traits that enable its abnormal growth. The result is a mass of dysregulated cells, or tumor, which, upon further mutation, may spread, or metastasize, to other organs of the body. The dissemination of tumor cells makes treatment difficult, and thus confers cancer with its associated lethality. Over the past 30 years, the RAS genes have been critical in teaching us the mechanisms underlying the molecular progression of cancer. RAS is mutated in 33% of all cancers and is often an early event in its stepwise progression. As a result, the RAS genes are widely accepted as ‘drivers’ or ‘initiators’ of human tumorigenesis. Unfortunately, efforts directed at targeting RAS in the clinic have as of yet been unsuccessful. This has triggered a need to identify genes that are required for RAS tumorigenesis that are therapeutically tractable. My research has focused on deciphering the potential role of the Rho GEF Arhgef2 in RAS-mediated tumorigenesis. I have found that Arhgef2 is a bona fide transcriptional target of RAS and is upregulated in human tumors harboring RAS mutations. Importantly, depletion of Arhgef2 in RAS-mutated cells inhibits their survival, proliferation, and tumor growth in murine models. In search of the mechanism underlying the requirement of Arhgef2 in RAS tumorigenesis, I have uncovered a novel function for Arhgef2 as a positive regulator of a central RAS pathway, the mitogen-activated protein kinase (MAPK) pathway. Thus, Arhgef2 is part of a positive feedback loop in which RAS-dependent increases in Arhgef2 expression results in the amplification of RAS signaling. Moreover, Arhgef2 confers tumor cells with properties favoring their malignant conversion, thereby implicating Arhgef2 in the formation of metastases. Together, these studies suggest that Arhgef2 plays an important role at multiple stages of tumorigenic progression and may therefore be a promising therapeutic target in RAS-mutated tumors.

B-cell acute lymphoblastic leukemia (B-ALL) represents a collection of diseases that are categorized into subtypes based on the presence of recurrent cytogenetic abnormalities. These abnormalities often result in the expression of oncogenic drivers that denote a standard- or high-risk for relapse. Currently, survival rates boarder 40% for adult patients and relapses are often observed in patients lacking high-risk markers. Thus, there is an unmet need for biomarkers that can identify all high-risk leukemia, and development of novel therapies based on a better understanding of the molecular drivers of B-ALL. To address this need, I designed a multi-parameter phospho-flow cytometry platform and characterized basal and cytokine-potentiated signaling in adult B-ALL samples. I identified patterns of cytokine-responsiveness across B-ALL patients that correlated with the presence of high-risk oncogenic drivers. Furthermore, I demonstrated that small-molecule inhibitors could abrogate cytokine-induced signaling in high-risk patients suggesting these inhibitors may compliment current chemotherapeutic protocols.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.