Activation of the NLRP3 inflammasome has been shown in response to numerous activators; here we show that the oxazolidinone antibiotic linezolid results in both the NLRP3-dependent in vitro release of the proinflammatory cytokine IL-1 Α; and in vivo neutrophilic influx following its intraperitoneal administration. Clinical use of linezolid is commonly limited by hematologic side effects; herein we also show NLRP3-deficiency protected animals against linezolid-induced effects on the bone marrow. Importantly, all previously described activators of the NLRP3 inflammasome have required the generation of reactive oxygen species (ROS). Linezolid is however unique amongst NLRP3 agonists in that its ability to activate the NLRP3 inflammasome in a ROS-independent manner. The pathways for ROS-dependent and ROS-independent NLRP3 activation converge upon mitochondrial dysfunction and specifically the mitochondrial lipid cardiolipin. We demonstrated that interference with cardiolipin synthesis specifically inhibits NLRP3 inflammasome activation. These findings firstly suggests that ROS generation is not the canonical activator of NLRP3 but rather an intermediary step leading to the mitochondrial perturbation that is tied to NLRP3 inflammasome activation and also implicate the involvement of mitochondrial lipid cardiolipin in this process; secondarily, linezolid-induced NLRP3 activation may account for thetoxicity associated with prolonged usage of this antibiotic.

The pharyngeal foregut endoderm (PFE) gives rise to several important organs including the thyroid, thymus and parathyroid glands. In mice and humans, defects in the development of PFE can lead to thymic aplasia and aberrations in thymic epithelial cell (TEC) function can lead to immunodeficiency or autoimmune disease. Successful differentiation of pluripotent stem cells (PSCs) to PFE could provide a renewable cell source that enables the study of human diseases that originate in the PFE. Here, I identify signaling pathways that influence the differentiation of PSCs to PFE. Firstly, using a novel mouse reporter PSC line we develop a protocol that generates a Pax9 expressing population that is enriched for PFE markers and upon transplantation can form organized epithelial structures. However, since this protocol was inefficient for human PSCs, we subsequently identified additional signaling pathways required for the efficient generation of human PFE and determined a key role for retinoic acid. Upon transplantation, the human PFE gives rise to TECs, a ventral PFE derivative. Finally, to facilitate future investigation into the gene regulatory networks in PFE, we develop a CRISPR-effector system to modulate endogenous gene expression in PSCs. We demonstrate that developmentally relevant genes can be repressed or induced, thereby influencing the cellular state. These data present strategies to generate cells of the PFE lineage from PSCs, facilitating the production of cells for patient-specific disease modeling or cell replacement therapies, and a method to interrogate gene and regulatory element function in PFE and its derivatives.

Inhibitor of DNA binding/differentiation protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. ID4 shares the homology of HLH domain with other ID proteins (ID1, ID2, and ID3) and lack the basic DNA binding region. Evidence suggested that unlike ID1, ID2 and ID3, ID4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promoting apoptosis. Consistent with these observations ID4 is epigenetically silenced in DU145 prostate cancer cell line. In this study we investigated whether ID4 is also epigenetically silenced in prostate cancer. We also examined association between ID4 promoter hyper-methylation and its expression in prostate cancer cell lines. ID4 protein expression was analyzed in human prostate adenocarcinoma samples by Immunohistochemistry (IHC). ID4 promoter methylation pattern on prostate cancer cell lines was examined by methylation specific PCR. In addition, we performed methylation specific PCR on the human prostate tissues and genomic DNA to correlate cell line studies with clinical studies. IHC demonstrated decreased ID4 protein expression in human prostate tissue samples, whereas higher nuclear ID4 expression was found in normal prostate tissues. ID4 methylation specific PCR (MSP) on prostate cancer cell lines, showed ID4 methylation in DU145, but not in LNCaP and C33 cells. C81 and PC3 cells showed partial methylation. Increased ID4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with ID4 promoter hyper-methylation showed distinct loss of ID4 expression. However, the underlying mechanism involved in epigenetic silencing of ID4 is currently unknown. We hypothesized that ID4 promoter methylation is initiated by an EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27Me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27Me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Increased enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines was compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2, but decreased ID4 expression in prostate cancer strongly supports this model.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells , while the steroid 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by l,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

In this thesis, I address two fundamental questions related to our understanding of how DNA damage is processed and repaired during replication. Using Two-dimensional (2-D) agarose gel analysis, I first examine whether DNA damage on plasmids introduced by transformation is processed in a manner similar to that observed on endogenously replicating plasmids and the chromosome. The original intent for using this approach was to develop a technique that could examine how different DNA adducts would be repaired in various sequence contexts. However, I found that distinct differences exist between the processing of DNA damage on transforming plasmids and the chromosome. The 2-D agarose gel analysis shows that RecA-mediated processing does not contribute to the survival of transforming plasmids and that this effect is likely due to inefficient replication of the plasmids after they are initially introduced into cells. These observations, while important, place limitations on the usefulness of transforming plasmids to characterize cellular repair processes. In a second question, I characterize the composition of the replisome following arrest by UV-induced DNA damage. Using 2-D agarose gel analysis the structural changes that occur in DNA during processing and repair have been well characterized, however, little is known about the fate of the replisome itself during these events. I used thermosensitive replication mutants to compare the DNA structural intermediates induced after disruption of specific components of the replisome to those observed after UV damage. The results show that dissociation of subunits required for polymerase stabilization are sufficient to induce the same processing events observed after UV damage. By contrast, disruption of the helicase-primase complex induces abnormal structures and a loss of replication integrity, suggesting that these components remain intact and bound to the template following replication arrest. I propose that polymerase dissociation provides a mechanism that allows repair proteins to gain access to the lesion while retention of the helicase serves to maintain the integrity and licensing of the fork so that replication can resume from the appropriate site once the lesion has been processed.

Thousands of synthetic chemicals have been released into the environment, causing widespread exposures of wildlife and humans alike. Some of these chemicals are known to disrupt aspects of hormone action thus inducing abnormalities in endocrine tissues and organs. Bisphenol S (BPS) and tetrabromobisphenol A (TBBPA), two largely unstudied chemicals commonly used in consumer products, are suspected to have endocrine disrupting properties based on their similar chemical structures to known endocrine disrupting chemicals (EDCs). To determine whether developmental exposure to BPS or TBBPA induced abnormalities in the ovary, mice were administered oil, BPS or TBBPA during pregnancy and lactation. The ovaries of female offspring were examined for gross morphological, immunohistological and gene expression differences at postnatal day (PND) 22 and week 16. To determine whether these exposures alter responses to hormones, two females from each litter were administered either oil or ethinyl estradiol (EE2) from PND 19-21. Our study identified significant differences in ovarian follicular formation and gene expression after developmental TBBPA and BPS exposures. Most effects were observed at PND22 (pre-puberty), and were apparent after the EE2 challenge, suggesting that changes induced by BPS and TBBPA are age- and hormone- dependent. Our results are consistent with the hypothesis that TBBPA and BPS are EDCs; that these compounds can disrupt development of the ovary; and that the observed effects of TBBPA and BPS exposure are similar to risk factors for ovarian diseases including cancer. These findings may indicate that widespread exposures to BPS and TBBPA could have implications for public health.

Aspects of the behavioral ecology of bird song learning are examined in three parts. First, an approach from image analysis is extended to allow rapid, quantitative description of animal sounds. In this approach, sounds are summarized as sets of time-frequency-amplitude landmarks. Second, the role of dawn song in tree swallow (Tachycineta bicolor) breeding biology is examined. Song syllable sharing among tree swallows was found to be high among birds nesting at the same site, but sharing was lower between birds nesting at different sites. When birds nested at different sites, the distance between those sites was not related to the amount of difference between the birds' syllable repertoire compositions. All tree swallow song repertoires did not remain constant during the breeding season; some individuals added new syllable types, others modified existing types. Singing performance was correlated with reproductive success in tree swallows: males that sang more precise repetitions of their syllable types attracted more extra-pair mates. Furthermore, pairwise comparisons between the social and genetic fathers of extra-pair young found that the genetic fathers averaged higher syllable consistency than the cuckolded males. Third, a comparative study of the phylogenetic distribution of vocal mimicry examined the evolutionary history of song learning in oscine passerines. Vocal mimicry, defined as the habitual incorporation of heterospecific sounds into song displays, was found in twenty-eight separate clades of oscines. These clades were found in every major oscine superfamily, but made up a higher proportion of daughter groups within the most ancient superfamilies of oscines. The most plesiomorphic lineages of oscines were found to contain many highly-skilled mimics. These observations support the hypothesis that the course of song learning in oscines has run repeatedly from permissive learning rules that permit mimicry to restrictive learning rules that limit mimicry.

Spermatogonial stem cells (SSC) self-renew and differentiate into spermatozoa throughout the life of the male. SSC transplantation is a potential method for the propagation of genetically important males. These cells have been isolated in different mammalian species using specific cell surface markers, but not in felines. The goal of this study was to explore a relevant strategy for conservation of endangered felids by characterizing domestic cat (Felis catus) SSCs and assessing their ability for self-renewal after transplantation. Firstly, SSC and pluripotent surface markers, identified in non-feline species, were tested for expression in mixed germ cells from adults by immunocytochemistry and flow cytometry, with immunohistochemical confirmation of expression in prepubertal and adult testis tissue. Secondly, subpopulations were purified through fluorescence-activated cell sorting using spermatogonia-specific markers and molecularly characterized to ascertain levels of pluripotent transcription factors expressed in cat embryos. Thirdly, subpopulations of mixed germ cells and purified spermatogonial cells were transplanted to prepubertal cats to determine: 1) if SSCs capable of colonization were present, and 2) the value of using adolescent domestic cats without depletion of endogenous germ cells as recipients. Fourthly, various culture conditions were evaluated to identify proteins and factors required to maintain proliferation of cat SSCs. Lastly, adult lion testis tissue was characterized with the same surface markers, and mixed germ cells were transplanted to cat testes to evaluate the cat as a suitable host for lion SSC colonization and differentiation. Pluripotent surface markers were more reliable than the common SSC surface markers for isolating cat SSCs. Varying expression levels of pluripotent transcription factors between the different purified cell populations identified spermatogonial subpopulations. Cell purification was not necessary to colonize recipient testes, and transplantation validated the use of prepubertal males as recipients without depletion of endogenous cells. Unlike spermatogonia within mixed germ cells, purified spermatogonia were not maintained under various culture conditions; therefore, SSC culture conditions must be optimized. Similarities in the expression patterns of surface markers in lion and cat spermatogonia were revealed, and colonization of lion SSCs in cat testes provided further evidence of the domestic cat’s relevance as a model for exotic felid SSC transplantation.

The hypothesis to be tested states that the pathology of Alzheimer's disease (AD) involves elevated levels of oxidative stress, resulting in elevated levels of cellular oxidative defense mechanisms. If the premise is true, than AD pathologically afflicted cells should have a higher demand for glutathione (GSH) as an innate oxidative defense mechanism hence; greater GSH concentrations, increased GSH resynthesis capabilities, and increased levels of cystathionine gamma-lyase (CNase). Alzheimer diseased and age matched control lymphoblast cells, obtained from OHSU's Oregon Brain Aging Study, were cultured, and GSH biochemistry was subsequently evaluated. GSH was depleted by exposing cells to the GSH depleting agent diethylmaleate (DEM) and the resulting GSH concentrations were measured. GSH resynthesis was measured after depleting GSH with DEM, to a level of approximately half base GSH concentration, then removing the depleting agent, resuspending the cells in fresh medium (DEM-free), and subsequently measuring GSH levels. GSH concentrations were measured by HPLC, and all data was normalized to cellular protein concentration. Cellular CNase specific activity levels were measured by adding cytasthionine, the CNase substrate, and then measuring the amount of cysteine produced by means of the DTNB assay. The AD cell lines showed no increase in base levels of GSH as compared to control cell lines. The AD cell lines showed a statistically significant increase in GSH resynthesis capabilities and cystathionine gamma-lyase specific activity levels. These findings add further weight to the AD oxidative stress hypothesis, which is based on the premise that the causative agent of AD pathogenesis is an increase in the level of cellular free radicals produced.

Angiogenesis is a complex process coordinating cell migration, proliferation, and lumen formation. Changes to the microenvironment regulate angiogenesis through mechanotransduction and cytokine signals. In pulmonary hypertension, something in the process becomes abnormal, resulting in changes to the microenvironment and the formation of a glomerulus of dysfunctional capillaries, called a plexiform lesion. Endothelial cells, expressing CD117 (CD117+ EC clones) increase in the plexiform lesions of pulmonary hypertension, independent of pro-angiogenic VEGF signaling. We hypothesize that the mechanical environment and the macromolecular composition of the extracellular matrix, both, contribute to the aberrant angiogenesis. When we changed the mechanical environment, we changed the angiogenic potential and cellular phenotype of CD117+ Endothelial cell clones. Turbulent flow, pathologic substrate stiffness, and pathologic stretch increased Endothelial-to-mesenchymal markers, such as acta2, cnn1, snail, and slug in CD117+ EC clones while CD117- ECs showed minimal change. We perturbed the mechanical environment of CD117+ EC clones and identified changes in Bone Morphogenic Protein-2, an often overlooked pro-angiogenic cytokine. We coupled changes in the mechanical environment to Rho GTPase intracellular signaling, to predict how changes to the mechanotransduction would affect angiogenesis through a computational model. In our model of angiogenesis, we found vessel synchronicity to depend on both which cell undergoes mitosis, and also at which phase of GTPase cycling the cell undergoes mitosis. We believe changes to the GTPase cycling may be the mechanism linking mechanotransduction to the abnormal vessels found in pulmonary hypertension. We are the first group to look at the role of the ECM composition, independent of stiffness. Our results show diseased ECM composition alone leads to phenotypic changes indicative of PH progression. In conclusion, these results provide a possible cytokine implicated in the mechanotransduction of PH, established a computational model of angiogenesis which provides a mechanotransduction mechanism of disease progression, and established that the ECM composition alone is capable of phenotypic changes leading to disease progression.