Principal component and second generation wavelet analysis of Treasury yield curve evolution

Prices of U.S. Treasury securities vary over time and across maturities. When the market in Treasurys is sufficiently complete and frictionless, these prices may be modeled by a function time and maturity. A cross-section of this function for time held fixed is called the yield curve; the aggregate of these sections is the evolution of the yield curve. This dissertation studies aspects of this evolution. ^ There are two complementary approaches to the study of yield curve evolution here. The first is principal components analysis; the second is wavelet analysis. In both approaches both the time and maturity variables are discretized. In principal components analysis the vectors of yield curve shifts are viewed as observations of a multivariate normal distribution. The resulting covariance matrix is diagonalized; the resulting eigenvalues and eigenvectors (the principal components) are used to draw inferences about the yield curve evolution. ^ In wavelet analysis, the vectors of shifts are resolved into hierarchies of localized fundamental shifts (wavelets) that leave specified global properties invariant (average change and duration change). The hierarchies relate to the degree of localization with movements restricted to a single maturity at the base and general movements at the apex. Second generation wavelet techniques allow better adaptation of the model to economic observables. Statistically, the wavelet approach is inherently nonparametric while the wavelets themselves are better adapted to describing a complete market. ^ Principal components analysis provides information on the dimension of the yield curve process. While there is no clear demarkation between operative factors and noise, the top six principal components pick up 99% of total interest rate variation 95% of the time. An economically justified basis of this process is hard to find; for example a simple linear model will not suffice for the first principal component and the shape of this component is nonstationary. ^ Wavelet analysis works more directly with yield curve observations than principal components analysis. In fact the complete process from bond data to multiresolution is presented, including the dedicated Perl programs and the details of the portfolio metrics and specially adapted wavelet construction. The result is more robust statistics which provide balance to the more fragile principal components analysis. ^

Relevance of CD40/CD40L downstream pathways to Alzheimer's disease

The amyloid cascade hypothesis places amyloid-β at the origin of Alzheimer's disease (AD). Amyloid-β (Aβ) is the product of the sequential cleavage of the amyloid precursor protein (APP) by the enzymes β- and γ-secretases. An inflammatory component to AD has been suggested in association with CD40 (a member of the tumor necrosis factor receptor superfamily (TNFRS) and its cognate ligand CD40L. In this study, I hypothesized that the neutralization of pro-inflammatory cytokines produced downstream of CD40/CD40L interaction would reduce APP processing. I also hypothesized that blocking the binding of different adaptor proteins to CD40 by mutating its cytoplasmic tail would result in significant reduction of the APP metabolites: Aβ, sAPPβ, sAPPα, CTFβ and CTFα. ^ Treatment with CD40L of human embryonic kidney cells over-expressing both APP and CD40 (HEK/APPsw/CD40) significantly increased levels of the cytokine granulocyte macrophage colony stimulating factor (GM-CSF). Neutralizing antibodies against GM-CSF mitigated the CD40L-induced production of Aβ in these cells. Treatment of the HEK/APPsw/CD40 cells with recombinant GM-CSF significantly increased Aβ levels. GM-CSF receptor gene silencing with shRNA significantly reduced Aβ levels to below base line in non-stimulated HEK/APPsw/CD40 cells. Silencing of the GM-CSF receptor also decreased APP endocytosis (therefore reducing the availability of APP to be cleaved in the endosomes). ^ Using CD40 mutants, I show that CD40L can increase levels of Aβ(1-40), Aβ(1-42), sAPPβ, sAPPα and CTFβ independently of TRAF signaling. TRAFs had been shown to be necessary for most CD40/CD40L-dependent signaling. An increase in mature/immature APP ratio after CD40L treatment of CD40wt and CD40-mutant cells was observed, reflecting alterations in APP trafficking. CD4OL treatment of a neuroblastoma cell line over-expressing CTFβ suggested that CD40L affected γ-secretase activity. Inhibition of γ-secretase activity significantly reduced sAPPβ levels in the CD40L treated HEK/APPsw CD40wt and the CD40-mutant cells. The latter suggests CD40/CD40L interaction primarily acts on γ-secretase and affects β-secretase via a positive feedback mechanism. ^ Taken together, the results of this dissertation suggest that GM-CSF operates downstream of CD40/CD40L interaction and that GM-CSF modulates Aβ production by influencing APP trafficking. Moreover, the data presented suggest that CD40/CD40L interaction can modulate APP processing via a mechanism independent of TRAF signaling. ^

Tyrosine Phosphorylation-Mediated Signaling Pathways in Dictyostelium

While studies on metazoan cell proliferation, cell differentiation, and cytokine signaling laid the foundation of the current paradigms of tyrosine kinase signaling, similar studies using lower eukaryotes have provided invaluable insight for the understanding of mammalian pathways, such as Wnt and STAT pathways. Dictyostelium is one of the leading lower eukaryotic model systems where stress-induced cellular responses, Wnt-like pathways, and STAT-mediated pathways are well investigated. TheseDictyostelium pathways will be reviewed together with their mammalian counterparts to facilitate the comparative understanding of these variant and noncanonical pathways.

NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icretransgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63genes' expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation.

Identification of genes downstream of endothelin-3 signaling: characterization of WSB1 and SPC12

Signaling of endothelin-3 (Edn3) through its receptor, endothelin receptor B (EdnrB), has been shown to be indispensable for the development of certain neural crest derivatives. Since no research has been directed to investigate what the downstream targets of this signaling pathway are, the purpose of this study was to identify and characterize genes that are transcriptionally regulated by Edn3 signaling. Data from Differential Display RT-PCR of Edn-3 induced cDNA vs. non-induced cDNA obtained from primary neural crest cultures was analyzed. Thirty bands that were differentially expressed were sequenced and submitted for a homology search (BLAST). Among the genes identified were WSB 1 (a member of the SOCS family of negative regulators) and SPC 12 (the smallest subunit, 12kDa, of mammalian signal peptidase). Using whole-mount in-situ hybridization, the expression patterns of EdnrB, WSB 1 and SPC 12 were characterized. WSB 1 and SPC 12 expression patterns were found to overlap with that of EdnrB, suggesting that Edn3 might regulate the transcription of these genes in specific neural crest derived lineages.

A model for the modular evolution of rna addressing open questions on the origin of life

A main unsolved problem in the RNA world scenario for the origin of life is how a template-dependent RNA polymerase ribozyme emerged from short RNA oligomers generated by random polymerization of ribonucleotides (Joyce and Orgel 2006). Current estimates establish a minimum size about 165 nt long for such a ribozyme (Johnston et al. 2001), a length three to four times that of the longest RNA oligomers obtained by random polymerization on clay mineral surfaces (Huang and Ferris 2003, 2006). To overcome this gap, we have developed a stepwise model of ligation-based, modular evolution of RNA (Briones et al. 2009) whose main conceptual steps are summarized in Figure 1. This scenario has two main advantages with respect to previous hypotheses put forward for the origin of the RNA world: i) short RNA....

Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction.

When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of betaARK1 (betaARKct), that the desensitization and down-regulation of betaARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical betaAR abnormalities seen in human heart failure, delivery of the betaARKct transgene at the time of myocardial infarction prevents the rise in betaARK1 activity and expression and thereby maintains betaAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer

BACKGROUND: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance.

METHODS: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided.

RESULTS: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner.

CONCLUSIONS: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.

Characterization of CDK5RAP2 as a novel regulator in the Hippo signaling pathway

CEP161 is a novel component of the Dictyostelium discoideum centrosome and is the ortholog of mammalian CDK5RAP2. Mutations in CDK5RAP2 are associated with autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder characterized by reduced head circumference, a reduction in the size of the cerebral cortex and a mild to moderate mental retardation. Here we show that the amino acids 1-763 of the 1381 amino acids of CEP161 protein are sufficient for centrosomal targeting and centrosome association. AX2 cells over-expressing truncated and full length CEP161 proteins have defects in growth and development. Furthermore, we identified the kinase SvkA (severinkinase A) as its interaction partner which is the D. discoideum Hippo related kinase designated here as Hrk-svk. Hrk-svk is the direct homolog of human MST1. Both proteins co-localize at the centrosome. We further demonstrate that this interaction is also conserved in mammals. We were able to show that CDK5RAP2 interacts with MST1 and TAZ and it also down-regulates the transcript levels of TAZ in HEK293T cells. Taken together, our data on Dictyostelium CEP161 and human CDK5RAP2 supports the hypothesis that CDK5RAP2 as a novel regulator of Hippo signaling pathway. We propose that CDK5RAP2 mutations may lead to a decrease in the number of neurons and the subsequent reduction of brain size by regulating the hippo signaling pathway.

The complexity of Drosophila innate immunity

Metazoans rely on efficient mechanisms to oppose infections caused by pathogens. The immediate and first-line defense mechanism(s) in metazoans, referred to as the innate immune system, is initiated upon recognition of microbial intruders by germline encoded receptors and is executed by a set of rapid effector mechanisms. Adaptive immunity is restricted to vertebrate species and it is controlled and assisted by the innate immune system. Interestingly, most of the basic signaling cascades that regulate the primeval innate defense mechanism(s) have been well conserved during evolution, for instance between humans and the fruit fly, Drosophila melanogaster. Being devoid of adaptive signaling and effector systems, Drosophila has become an established model system for studying pristine innate immune cascades and reactions. In general, an immune response is evoked when microorganisms pass the fruit fly’s physical barriers (e.g. cuticle, epithelial lining of gut and trachea), and it is mainly executed in the hemolymph, the equivalent of the mammalian blood. Innate immunity in the fruit fly consists of a phenoloxidase (PO) response, a cellular response (hemocytes), an antiviral response, and the NF-κB dependent production of antimicrobial peptides referred to as the humoral response. The JAK/STAT and Jun kinase signaling cascades are also implicated in the defence against pathogens.

Macrophage adaptation leads to parallel evolution of genetically diverseEscherichia colismall-colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.

Activation of Sonic hedgehog signaling in ventricular cardiomyocytes exerts cardioprotection against ischemia reperfusion injuries

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Sonic hedgehog signaling regulates mode of cell division of early cerebral cortex progenitors and increases

The morphogen Sonic Hedgehog (SHH) plays a critical role in the development of different tissues. In the central nervous system, SHH is well known to contribute to the patterning of the spinal cord and separation of the brain hemispheres. In addition, it has recently been shown that SHH signaling also contributes to the patterning of the telencephalon and establishment of adult neurogenic niches. In this work, we investigated whether SHH signaling influences the behavior of neural progenitors isolated from the dorsal telencephalon, which generate excitatory neurons and macroglial cells in vitro. We observed that SHH increases proliferation of cortical progenitors and generation of astrocytes, whereas blocking SHH signaling with cyclopamine has opposite effects. In both cases, generation of neurons did not seem to be affected. However, cell survival was broadly affected by blockade of SHH signaling. SHH effects were related to three different cell phenomena: mode of cell division, cell cycle length and cell growth. Together, our data in vitro demonstrate that SHH signaling controls cell behaviors that are important for proliferation of cerebral cortex progenitors, as well as differentiation and survival of neurons and astroglial cells.