Mature αβ and γδ T cells arise from a common precursor population in the thymus. Much debate has focused on the mechanism of T cell lineage choice made by these multi-potential precursor cells. It is widely believed that the decision of these precursor cells to commit to the γδ or αβ T cell lineages is regulated primarily by a specific instructive signal relayed through the appropriate T cell receptor. Contrary to this model, we present evidence for a TCR-independent lineage commitment process. Comparison of global gene expression profiles from immature αβ and γδ lineage thymocytes identified Sox13, an HMG-box transcription factor, as a γδ T cell-specific gene. Unlike other HMG-box transcription factors such as TCF1, LEF1 and SOX4, that are critical for proper αβ T cell development, Sox13 expression is restricted to early precursor subsets and γδ lineage cells. Importantly, SOX13 appears to influence the developmental fate of T cell precursors prior to T cell receptor expression on the cell surface. Transgenic over-expression of Sox13 in early T cell precursors strongly inhibits αβ lineage development, in part, by inhibiting precursor cell proliferation and concomitantly, leading to increased cell death among αβ lineage subsets. Steady-state γδ T cell numbers, however, appear unaffected. Strikingly, the DP αβ lineage cells that do develop in Sox13 transgenic mice are imprinted with a γδ- or precursor-like molecular profile, suggesting that SOX13 plays an active role in the lineage fate decision process or maintenance. Sox13-deficient mice, on the other hand, have selectively reduced numbers of γδ thymocytes, indicating that SOX13 is essential for proper development of γδ T cells. We present additional data demonstrating that SOX13 is a canonical WNT signaling antagonist modulating TCF1 activity, raising a strong possibility that WNT signals, and their modulators, are at the nexus of γδ versus αβ T cell lineage commitment.

DNA damage response pathways are a complicated network of proteins that function to remove and/or reverse DNA damage. Following genetic insult, a signal cascade is generated, which alerts the cell to the presence of damaged DNA. Once recognized, the damage is either removed or the damaged region is excised, and the original genetic sequence is restored. However, when these pathways are defective the cell is unable to effectively mediate the DNA damage response and the damage persists unrepaired. Thus, the proteins that maintain the DNA damage response pathway are critical in preserving genomic stability. One essential DNA repair protein is the Breast Cancer Associated gene, BRCA1. BRCA1 is essential for mediating the DNA damage response, facilitating DNA damage repair, and activating key cell cycle checkpoints. Moreover, mutations in BRCA1 lead to a higher incidence of breast and ovarian cancer, highlighting the importance of BRCA1 as a tumor suppressor. In an effort to better understand how BRCA1 carried out these functions, researchers sought to identify additional BRCA1 interacting proteins. This led to the identification of several proteins including the BRCA1 Associated C-terminal Helicase, BACH1. Due to the direct interaction of BACH1 with a region of BRCA1 essential for DNA repair and tumor suppression, it was speculated that BACH1 may help support these BRCA1 function(s). In fact, initial genetic screenings confirmed that mutations in BACH1 correlated not only with hereditary breast cancer, but also with defects in DNA damage repair processes. The initial correlation between BACH1 and cancer predisposition was further confirmed when mutations in BACH1 were identified in the cancer syndrome Fanconi anemia (FA) (complementation group FA-J), thus giving BACH1 its new name FANCJ. These findings supported a previously established link between the FA and BRCA pathways and between FA and DNA repair. In particular, we demonstrated that similar to other FA/BRCA proteins, suppression of FANCJ lead to a substantial decrease in homologous recombination and enhanced both the cellular sensitivity to DNA interstrand cross-linking agents and chromosomal instability. What remained unknown was specifically how FANCJ functioned and whether these functions were dependent on its interaction with BRCA1 or other associated partners. In fact, we identified that FANCJ interacted directly with the MMR protein MLH1. Moreover, we found that the FANCJ/BRCA1 interaction was not required to correct the cellular defects in FA-J cells, but rather that the FANCJ/MLH1 interaction was required. Although both the FA/BRCA and MMR pathways undoubtedly mediate the DNA damage response, there was no evidence to suggest that these pathways were linked, until recently. Our findings not only indicate a physical link between these pathways by protein-protein interaction, but also demonstrated a functional link.

In 1990, Richard Jorgensen’s lab initiated a study to test if they could create a more vivid color petunia (Napoli et al. 1990). Their plan was to transform plants with the chalcone synthase transgene––the predicted rate limiting factor in the production of purple pigmentation. Much to their surprise, the transgenic plants, as well as their progeny, displayed a great reduction in pigmentation. This loss of endogenous function was termed “cosuppression” and it was thought that sequence-specific repression resulted from over-expression of the homologous transgene sequence. In 1998, Andrew Fire and Craig Mello described a phenomenon in which double stranded RNA (dsRNA) can trigger silencing of cognate sequences when injected into the nematode, Caenorhabditis elegans (Fire et al. 1998). This data explained observations seen years earlier by other worm researchers, and suggested that repression of pigmentation in plants was caused by a dsRNA-intermediate (Guo and Kemphues 1995; Napoli et al. 1990). The phenomenon––which soon after was coined RNA interference (RNAi)––was soon discovered to be a post-transcriptional surveillance system in plants and animals to remove foreign nucleic acids.

Pain signaling involves transmission of nociceptive stimuli in the spinal cord where a critical balance between excitatory and inhibitory inputs determines the response to noxious stimuli. The neuropeptide, substance P (SP), mediates transmission of pain in part by binding to the tachykinin receptor (NK-1R) in the dorsal horn (DH) of the spinal cord. One of SP’s downstream effects is to modulate N-type Ca2+(N-) channels. While phospholipid breakdown is a part of the inflammatory process that accompanies tissue damage, the role of this metabolic pathway has not been completely described with respect to N-channel modulation during pain signaling. Despite the incomplete understanding of this modulation, pharmacological antagonists of both NK-1R and N-channels have been used to treat pain. In Chapter II, using whole-cell patch clamp recording techniques, the SP signaling cascade that mediates inhibition of recombinant N-channel activity was characterized. By adopting a pharmacological approach, I show that this pathway resembles the slow pathway that was earlier described for modulation of N-current by the M1 muscarinic receptor (M1R). M1R couples to Gq to stimulate phospholipid breakdown. Together with previous observations, the data presented in this chapter provide evidence for involvement of the extracellular receptor kinase (ERK1/2), phospholipase A2 and release of phospholipid metabolites in the modulation of N-current by SP. Overall, this chapter shows that phospholipid metabolism involved in modulation of N-currents is not specific to M1Rs but that other Gq-coupled receptors may also modulate N-currents via the same signal transduction pathway. In Chapter III, enhancement of N-current by SP was studied as part of a collaborative project to understand current enhancement that occurs when a palmitoylated accessory CaVβ2a subunit is co-expressed with the pore-forming subunit CaV2.2 and the accessory subunit α2δ-1. When CaVβ3 is present, SP inhibits N-current as described in Chapter II. However, when palmitoylated CaVβ2a is co-expressed with CaV2.2 (and α2δ-1), current enhancement is observed at negative test potentials, demonstrating that both M1Rs and NK-1Rs exhibit the same profile of N-current modulation. This change in modulation by muscarinic agonists is not observed in the presence of a depalmitoylated CaVβ2a. However a chimeric CaVβ2aβ1b subunit that contains the palmitoylated N-terminus from CaVβ2a confers enhancement. Normally expression of the β1b subunit resulted in current inhibition. These findings indicated that the palmitoylated CaVβ2a participates in enhancement of current. Our data support a model where inhibition dominates over enhancement; when inhibition is blocked, enhancement may be observed. Lastly, we show that N-current inhibition by SP is minimized when exogenous palmitic acid is applied to cells co-expressing CaVβ3 subunits with N-channels. These results indicate that the presence of palmitic acid can prevent N-current inhibition when SP is applied most likely by interacting with CaV2.2. We propose a model where palmitic acid occupies the inhibitory site and serves to antagonize inhibition by a lipid metabolite, which is most likely arachidonic acid. The CaVβ2a protein seems to have a role in positioning the palmitoyl groups near CaV2.2. This chapter provides a new role for protein palmitoylation where the palmitoyl groups of CaVβ2a are both necessary and sufficient to block inhibition of another protein: CaV2.2. In Chapter IV, I probe the role of the relative orientation of CaVβ2a and the pore-forming subunit of the N-channel in N-current modulation. Evidence is presented that shows that not just the presence of a palmitoylated CaVβ2a is necessary, but the relative orientation of CaVβ2a to CaV2.2 is critical for blocking inhibition. Using N-channel mutants that cause a change in the orientation of CaVβ2a relative to CaV2.2, I show that the block of inhibition is disrupted; inhibition by the slow pathway is rescued. These findings further support my model that the palmitoyl groups of CaVβ2a normally reside in a specific location that overlaps with the slow pathway inhibitory site on CaV2.2. Lastly I present data showing that the enhancement of N-current, observed when palmitoylated CaVβ2a is present, occurs via the slow pathway. In Chapter V the effect of CaVβ’s orientation on N-channel modulation by the dopamine D2 receptor is tested. In this form of modulation, inhibition is rapid and voltage-dependent. The signaling pathway is membrane-delimited since Gβγ, released after receptor stimulation, directly interacts with the N-channel at a site that overlaps with a high affinity binding site for CaVβs. While N-currents are modulated by this pathway, the deletion mutants show aberrant membrane-delimited modulation. The findings in this chapter further underscore the importance of proper positioning of CaVβ to CaV2.2 for eliciting proper N-current modulation after GPCR stimulation. Overall, the data presented in this dissertation provides a mechanistic approach into examining modulation of N-current by different GPCRs via two different signaling pathways as well as the role CaVβ subunits serve in each modulatory pathway.

The studies presented in this thesis addressed mainly two aspects of Piwi-interacting RNA (piRNA) biology in the Drosophilagermline. We investigated the role of the piRNA pathway in embryonic axis specification. piRNAs mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila piRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization and asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellatesilencing. Furthermore, piRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. We conclude that piRNA based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline. We have also identified a new member of the piRNA pathway. We show that mutations in rhino, which encodes a rapidly evolving Heterochromatin Protein 1 (HP1) chromo box protein, lead to germline specific DNA break accumulation, trigger Chk2 kinase dependent defects in axis specification, and disrupt germline localization of Piwi proteins. Mutations in rhino and the piRNA pathway gene armitage disrupt silencing of all major transposon families, but do not alter expression of euchromatic or heterochromatic protein coding genes. Deep sequencing studies show that rhino mutations significantly reduce or eliminate anti-sense piRNAs derived from the majority of transposable elements in the Drosophila genome, and lead to a dramatic reduction in piRNAs derived from major piRNA production clusters on chromosomes 2R and 4. Rhino protein localizes to distinct nuclear foci, and associates with the chromosome 2R and 4 clusters by chromatin immunoprecipitation. The Rhino HP1 homologue is therefore required for piRNA biogenesis, transposon silencing, and maintenance of germline genome integrity.

CIS/SOCS (cytokine-inducible SH2 protein/suppressor of cytokine signaling) are a family of proteins that are thought to act as negative regulators of signaling by erythropoetin, interleukin-6 and other cytokines whose receptors are related to the growth hormone receptor (GHR), and like growth hormone (GH), signal through the JAK/STAT pathway. We examined the possibility that CIS/SOCS proteins may also be involved in GH signaling, in particular, in termination of the transient insulin-like effects of GH. mRNAs for CIS, SOCS3, and to a lesser extent SOCS1 were detectable by Northern blot analysis of rat adipocyte total RNA, and the expression of CIS and SOCS3 was markedly increased 30 min after incubation with 500 ng/ml hGH. Both CIS and SOCS3 were detected in adipocyte extracts by immunoprecipitation and immunoblotting with their corresponding antisera. GH stimulated the tyrosine phosphorylation of a 120 kDa protein (p120) that was co-precipitated from adipocyte extracts along with αCIS and detected in Western blots with phospho-tyrosine antibodies. However, no tyrosine phosphorylated proteins in these cell extracts were immunoprecipitated with antibodies to CIS3/SOCS3. p120 was later identified as the GHR based on the observations that two GHR antibodies recognized p120 in scale-up experiments and that p120 and the GHR share several characteristics, including their molecular weights, tyrosine phosphorylation upon GH stimulation, interaction with CIS, similar extent of glycosylation as judged by electrophoretic mobility shift after Endo F digestion, comparable mobility shifts upon thrombin digestion, and N-terminal histidine-tagging. The findings, however, do not rule out the possibility that there might be other tyrosine phosphorylated 120 kDa protein(s) that interact with CIS and contribute to the p120 signal, as well as the GHR. Further studies of the association of CIS with the GHR revealed that CIS might selectively interact with multiply tyrosine phosphorylated forms of the GHR, and these tyrosines are likely located near the carboxyl end of the GHR. Overexpression of CIS partially inhibited GH-induced STAT5 phosphorylation in CHO cells. Studies in freshly isolated and GH-deprived (sensitive) adipocytes revealed that the abundance of CIS does not correlate with the termination of the insulin-like effects of GH or the emergence of refractoriness. Neither the association of CIS with the GHR nor the tyrosine phosphorylation status of the GHR, JAK2 and STAT5 appear responsible for refractoriness in adipocytes. These data imply that some negative regulators other than CIS might contribute to the termination of GH-induced insulin-like effects in adipocytes.

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.

The co-dependence of nicotine and alcohol addiction occurs at high rates, complicates treatment, and is often associated with significant morbidity and mortality. Treatment options of alcohol and tobacco co-dependence are limited. Currently, there are drugs available for nicotine dependence or alcohol dependence. However, there are no therapeutic drugs available on the market for the co-dependence of nicotine and alcohol. Therefore, and important opportunity of new therapeutic options and drug development has presented itself. NT69L, a non-selective neurotensin (NT) agonist, provides a potential novel therapy for nicotine addiction in alcoholics by interacting with the common neurotransmitter circuits supporting the rewarding process for both nicotine and alcohol. Considering the behavioral effects of NT69L in attenuating nicotine self-administration in rats and alcohol consumption in mice, the present study was designed to assess the effects of NT69L as a new drug. NT69L was used in the treatment of nicotine addiction in an animal model of alcoholics and in attempts to attenuate withdrawal signs associated with nicotine and alcohol dependence. Wistar rats pre-exposed to alcohol vapor or air were allowed to self-infuse nicotine (0.03mg/kg/infusion) or saline. When the rats reached a stable level of responding, the effect of pretreatment with NT69L (1mg/kg i.p.) on the reinforcing effect of nicotine was determined. Animals self-infused nicotine at a significantly (p < .05) higher rate compared to saline in both air and alcohol vapor exposed groups. Acute pretreatment with a single injection of NT69L significantly (p < .05) reduced nicotine self-infusion in both the alcohol vapor and the air exposed groups for 5 days post-injection. Additionally, NT69L attenuated the alcohol- and nicotine-induced withdrawal signs associated with the discontinuation of alcohol and nicotine administration. Neurotensin agonist, NT69L, may represent a potential novel therapy to treat the co-addiction of alcohol and nicotine.

p53, known as the “guardian of the genome”, is the most well-characterized tumor suppressor gene. The central role of p53 is to prevent genome instability. p53 is the central node in an incredibly elaborate genome defense network for receiving various input stress signals and controlling diverse cellular responses. The final output of this network is determined not only by the p53 protein itself, but also by other p53 cooperating proteins. p300 and CBP (CREB-Binding Protein) act as multifunctional regulators of p53 via acetylase and ubiquitin ligase activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. In the nucleus, CBP and p300 exhibited differential regulation of p53 gene target expression, C-terminal acetylation, and biologic response after DNA damage. p300 activated, and CBP repressed, PUMA expression, correlating with activating acetylation of p53 C-terminal lysines by p300, and a repressive acetylation of p53 lysine-320 induced by CBP. Consistent with their gene expression effects, CBP deficiency augmented, and p300 deficiency blocked, apoptosis after doxorubicin treatment. Subcellular compartmentalization of p300/CBP’s ubiquitination and transcription activities reconciles seemingly opposed functions—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while nuclear p300/CBP direct p53 acetylation, target gene activation, and biological outcome after genotoxic stress. p53 is a prominent tumor suppressor gene and it is mutated in more than 50% of human tumors. Reactivation of endogenous p53 is one therapeutic avenue to stop cancer cell growth. In this thesis, we have identified S5as a critical regulator of p53 degradation and activity. S5a is a non-ATPase subunit in the 19S regulatory particle of the 26S proteasome. Our preliminary data indicates that S5a is required for p53 instability and is a negative regulator of p53 tranactivation. As a negative regulator of p53, S5a may therefore also represent a new target for cancer drug development against tumors that specifically maintain wild type p53.

The endoplasmic reticulum is the primary organelle in the cell which has the responsibility of properly folding proteins belonging to the secretory pathway. Secretory proteins are essential for a variety of functions within the body like metabolism, growth and survival. Hence, proper folding of the proteins in the ER is absolutely essential to maintain cellular and body function. The environment of the ER is substantially different from that of the cytoplasm and is primed essentially to provide the optimum conditions to fold newly synthesized polypeptides following translation by the ribosomes in the cytoplasm and on the surface of the ER. In order for secretory proteins to fold properly, ER homeostasis must be maintained. ER homeostasis is defined by the dynamic balance between the ER protein load and the ER capacity to process this load. The optimum environment of the ER, or ER homeostasis, can be perturbed by pathological processes such as hypoxia, glucose deprivation, viral infections, environmental toxins, inflammatory cytokines, and mutant protein expression, as well as by physiological processes such as aging. Disruption of ER homeostasis causes accumulation of unfolded and misfolded proteins in the ER. This condition is referred to as ER stress. Cells cope with ER stress by activating the unfolded protein response (UPR). The UPR is initiated by three ER transmembrane proteins: Inositol requiring 1 (IRE1), PKR-like ER kinase, and activating transcription factor 6 (ATF6). These three master regulators sense and interpret protein folding conditions in the ER and translate this information across the ER membrane to activate downstream effectors, spliced XBP1, phosphorylated eIF2α and ATF4, and cleaved active ATF6 respectively. These effectors have two distinct outputs, homeostatic and apoptotic. Homeostatic outputs are adaptive responses that function to attenuate ER stress and restore ER homeostasis. These responses include the attenuation of protein translation to reduce ER workload and prevent further accumulation of unfolded proteins, upregulation of molecular chaperones and protein processing enzymes to enhance the ER folding activity, and the increase in ER-associated degradation (ERAD) components to promote clearance of unfolded proteins. When ER stress reaches a point where the cells cannot tolerate the load of unfolded proteins any more, apoptosis sets in. One of the major secretory proteins in mammals, vascular endothelial growth factor VEGF, is essential for either normal or pathological angiogenesis (blood vessel development). VEGFA is the primary member of this family which is expressed in all endothelial cells and is responsible for sprouting and invasion of blood vessels into the interstitium and thus helps in supplying nutrients and oxygen to growing cells. Recent studies have indicated that cells suffering from insufficient blood supply experience ER stress. The ER needs energy and oxygen for the folding process, thus nutrient deprivation (low ATP production) and hypoxia caused by insufficient blood supply leads to inefficient protein folding and ER stress in cells, especially in cancer cells that grow and spread rapidly. This condition also occurs in the development of the mammalian placenta. The placenta is an essential tissue characterized by a lot of blood vessels. It is responsible for the exchange of nutrients and growth factors between maternal and fetal blood vessels and hence is essential for survival of the embryo. Nutrient deprivation and hypoxia stimulate the production of VEGFA and other angiogenic factors, leading to protection against ischaemic injury in both cancer cells as well as the developing placenta. In this dissertation, we report that the three master regulators of the UPR, IRE1α, PERK and ATF6α, mediate transcriptional regulation of VEGFA under ER stress in cancer cells. Inactivation of any of the three master regulators leads to attenuation of VEGFA expression under ER stress. We show that IRE1α is able to regulate VEGFA through its downstream transcription factor XBP1 which activates the VEGFA promoter. IRE1α mediated VEGFA regulation is also essential for normal development of labyrinthine trophoblast cells in the placenta. ATF6α also regulates VEGFA via its promoter. PERK is able to activate VEGFA by preferential activation of its downstream effector, ATF4, which binds intron 1 of the VEGFA gene. Thus our work reveals a twopronged differential regulatory action of the UPR sensors on VEGFA gene expression. This work suggests that a fully active UPR is essential for VEGFA upregulation under ER stress. All three regulators are required in cancer cells for normal VEGFA expression. This tight regulation of VEGFA by the UPR presents a wonderful opportunity for therapeutic intervention into angiogenic growth of tumors.