A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

Interferon-β gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Asian genotypes of JC virus in Native Americans and in a Pacific Island population: Markers of viral evolution and human migration

The human polyomavirus JC (JCV) causes the central nervous system demyelinating disease progressive multifocal leukoencephalopathy. Previously, we showed that 40% of Caucasians in the United States excrete JCV in the urine as detected by PCR. We have now studied 68 Navaho from New Mexico, 25 Flathead from Montana, and 29 Chamorro from Guam. By using PCR amplification of a fragment of the VP1 gene, JCV DNA was detected in the urine of 45 (66%) Navaho, 14 (56%) Flathead, and 20 (69%) Chamorro. Genotyping of viral DNAs in these cohorts by cycle sequencing showed predominantly type 2 (Asian), rather than type 1 (European). Type 1 is the major type in the United States and Hungary. Type 2 can be further subdivided into 2A, 2B, and 2C. Type 2A is found in China and Japan. Type 2B is a subtype related to the East Asian type, and is now found in Europe and the United States. The large majority (56–89%) of strains excreted by Native Americans and Pacific Islanders were the type 2A subtype, consistent with the origin of these strains in Asia. These findings indicate that JCV infection of Native Americans predates contact with Europeans, and likely predates migration of Amerind ancestors across the Bering land bridge around 12,000–30,000 years ago. If JCV had already differentiated into stable modern genotypes and subtypes prior to first settlement, the origin of JCV in humans may date from 50,000 to 100,000 years ago or more. We conclude that JCV may have coevolved with the human species, and that it provides a convenient marker for human migrations in both prehistoric and modern times.

The Cell Surface Flocculin Flo11 Is Required for Pseudohyphae Formation and Invasion by Saccharomyces cerevisiae

Diploid yeast develop pseudohyphae in response to nitrogen starvation, while haploid yeast produce invasive filaments which penetrate the agar in rich medium. We have identified a gene, FLO11, that encodes a cell wall protein which is critically required for both invasion and pseudohyphae formation in response to nitrogen starvation. FLO11 encodes a cell surface flocculin with a structure similar to the class of yeast serine/threonine-rich GPI-anchored cell wall proteins. Cells of the Saccharomyces cerevisiae strain Σ1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids. In rich media, FLO11 is regulated by mating type; it is expressed in haploid cells but not in diploids. Upon transfer to nitrogen starvation media, however, FLO11 transcripts accumulate in diploid cells, but not in haploids. Overexpression of FLO11 in diploid cells, which are otherwise not invasive, enables them to invade agar. Thus, the mating type repression of FLO11 in diploids grown in rich media suffices to explain the inability of these cells to invade. The promoter of FLO11 contains a consensus binding sequence for Ste12p and Tec1p, proteins known to cooperatively activate transcription of Ty1 elements and the TEC1 gene during development of pseudohyphae. Yeast with a deletion of STE12 does not express FLO11 transcripts, indicating that STE12 is required for FLO11 expression. These ste12-deletion strains also do not invade agar. However, the ability to invade can be restored by overexpressing FLO11. Activation of FLO11 may thus be the primary means by which Ste12p and Tec1p cause invasive growth.

Observing the epoch of galaxy formation

Significant observational progress in addressing the question of the origin and early evolution of galaxies has been made in the past few years, allowing for direct comparison of the epoch when most of the stars in the universe were forming to prevailing theoretical models. There is currently broad consistency between theoretical expectations and the observations, but rapid improvement in the data will provide much more critical tests of theory in the coming years.

Galaxy formation

It is argued that within the standard Big Bang cosmological model the bulk of the mass of the luminous parts of the large galaxies likely had been assembled by redshift z ∼ 10. Galaxy assembly this early would be difficult to fit in the widely discussed adiabatic cold dark matter model for structure formation, but it could agree with an isocurvature version in which the cold dark matter is the remnant of a massive scalar field frozen (or squeezed) from quantum fluctuations during inflation. The squeezed field fluctuations would be Gaussian with zero mean, and the distribution of the field mass therefore would be the square of a random Gaussian process. This offers a possibly interesting new direction for the numerical exploration of models for cosmic structure formation.

Leukocyte lipid body formation and eicosanoid generation: cyclooxygenase-independent inhibition by aspirin.

Lipid bodies, cytoplasmic inclusions that develop in cells associated with inflammation, are inducible structures that might participate in generating inflammatory eicosanoids. Cis-unsaturated fatty acids (arachidonic and oleic acids) rapidly induced lipid body formation in leukocytes, and this lipid body induction was inhibited by aspirin and nonsteroidal antiinflammatory drugs (NSAIDs). Several findings indicates that the inhibitory effect of aspirin and NSAIDs on lipid body formation was independent of cyclooxygenase (COX) inhibition. First, the non-COX inhibitor, sodium salicylate, was as potent as aspirin in inhibiting lipid body formation elicited by cis-fatty acids. Second, cis-fatty acid-induced lipid body formation was not impaired in macrophages from COX-1 or COX-2 genetically deficient mice. Finally, NSAIDs inhibited arachidonic acid-induced lipid body formation likewise in macrophages from wild-type and COX-1- and COX-2-deficient mice. An enhanced capacity to generate eicosanoids developed after 1 hr concordantly with cis-fatty acid-induced lipid body formation. Arachidonic and oleic acid-induced lipid body numbers correlated with the enhanced levels of leukotrienes B4 and C4 and prostaglandin E2 produced after submaximal calcium ionophore stimulation. Aspirin and NSAIDs inhibited both induced lipid body formation and the enhanced capacity for forming leukotrienes as well as prostaglandins. Our studies indicate that lipid body formation is an inducible early response in leukocytes that correlates with enhanced eicosanoid synthesis. Aspirin and NSAIDs, independent of COX inhibition, inhibit cis-fatty acid-induced lipid body formation in leukocytes and in concert inhibit the enhanced synthesis of leukotrienes and prostaglandins.

On the formation and evolution of the first Be star in a black hole binary MWC 656

We find that the formation of MWC 656 (the first Be binary containing a black hole) involves a common envelope phase and a supernova explosion. This result supports the idea that a rapidly rotating Be star can emerge out of a common envelope phase, which is very intriguing because this evolutionary stage is thought to be too fast to lead to significant accretion and spin up of the B star. We predict ∼10–100 of B-BH binaries to currently reside in the Galactic disc, among which around 1/3 contain a Be star, but there is only a small chance to observe a system with parameters resembling MWC 656. If MWC 656 is representative of intrinsic Galactic Be-BH binary population, it may indicate that standard evolutionary theory needs to be revised. This would pose another evolutionary problem in understanding black hole (BH) binaries, with BH X-ray novae formation issue being the prime example. Future evolution of MWC 656 with an ∼5 M⊙ BH and with an ∼13 M⊙ main-sequence companion on an ∼60 d orbit may lead to the formation of a coalescing BH–NS (neutron star) system. The estimated Advanced LIGO/Virgo detection rate of such systems is up to ∼0.2 yr−1. This empirical estimate is a lower limit as it is obtained with only one particular evolutionary scenario, the MWC 656 binary. This is only a third such estimate available (after Cyg X-1 and Cyg X-3), and it lends additional support to the existence of so far undetected BH–NS binaries.

Dairy Consumption and Incident Type 2 Diabetes Among American Indians: The Strong Heart Family Study

Thesis (Master's)--University of Washington, 2016-06

Inter-subjective and transnational racial effects: The role of the United States in the formation and evolution of the collective perception and racial relations in Cuba, 1898–1902

Since the arrival of the first African slaves to Cuba in 1524, the issue of race has had a long-lived presence in the Cuban national discourse. However, despite Cuba’s colonial history, it has often been maintained by some historians that race relations in Cuba were congenial with racism and racial discrimination never existing as deep or widespread in Cuba as in the United States (Cannon, 1983, p. 113). In fact, it has been argued that institutionalized racism was introduced into Cuban society with the first U.S. occupation, during 1898–1902 (Cannon, 1983, p. 113). This study of Cuba investigates the influence of the United States on the development of race relations and racial perceptions in post-independent Cuba, specifically from 1898-1902. These years comprise the time period immediately following the final fight for Cuban Independence, culminating with the Cuban-Spanish-American War and the first U.S. occupation of Cuba. By this time, the Cuban population comprised Africans as well as descendants of Africans, White Spanish people, indigenous Cubans, and offspring of the intermixing of the groups. This research studies whether the United States’ own race relations and racial perceptions influenced the initial conflicting race relations and racial perceptions in early and post-U.S. occupation Cuba. This study uses a collective interpretative framework that incorporates a national level of analysis with a race relations and racial perceptions focus. This framework reaches beyond the traditionally utilized perspectives when interpreting the impact of the United States during and following its intervention in Cuba. Attention is given to the role of the existing social, political climate within the United States as a driving influence of the United States’ involvement with Cuba. This study reveals that emphasis on the role of the United States as critical to the development of Cuba’s race relations and racial perceptions is credible given the extensive involvement of the U.S. in the building of the early Cuban Republic and U.S. structures serving as models for reconstruction. U.S. government formation in Cuba aligned with a governing system reflecting the existing governing codes of the U.S. during that time period.

Obesity, cardiovascular disease risk factors and weight loss in a population of adult Mexican Americans

The study of obesity has evolved into one of the most important public health issues in the United States (U.S.), particularly in Hispanic populations. Mexican Americans, the largest Hispanic ethnic subgroup in the U.S., have been significantly impacted by obesity and related cardiovascular diseases. Mexican Americans living in the Lower Rio Grande Valley (the Valley) in the Texas-Mexico border are one of the most disadvantaged and hard-to-reach minority groups. Demographic factors, socioeconomic status, acculturation, and physical activity behavior have been found to be important predictors of health, although research findings are mixed when establishing predictors of obesity in this population. Furthermore, while obesity has long been linked to cardiovascular disease (CVD) risk factors such as hypertension, type 2 diabetes, and dyslipidemia; information on the relationships between obesity and these CVD risk factors have been mostly from non-minority population groups. Overall, research has been mixed in establishing the association between obesity and related CVD risk factors in this population calling attention to the need for further research. Nevertheless, identifying predictors of success for weight loss in this population will be important if health disparities are to be addressed. The overall objective of the findings presented in this dissertation was to attain a more informed profile of obesity and CVD risk factors in this population. In particular, we examined predictors of obesity, measures of obesity and association with cardiovascular disease risk factors in a sample of 975 Mexican Americans participating in a health promotion program in the Valley region. Findings suggest acculturation factors to be one of the most important predictors of obesity in this population. Results also point to the need of identifying other possible risk factors for predicting CVD risk. Finally, initial body mass index is an important predictor of weight loss in this population group. Thus, indicating that this population is not only amenable to change, but that improvements in weight loss are feasible. This finding strengthens the relevance of prevention programs such as Beyond Sabor for Mexican populations at risk, in particular, food bank recipients.

The Effect of Carbohydrate Amount, Quality and Type on Arterial Pulse Pressure in Cuban-Americans with and Without Type 2 Diabetes

Background: Arterial pulse pressure, the difference between systolic and diastolic blood pressure, has been used as an indicator (surrogate measure) of arterial stiffness. High arterial pulse pressure (> 40) has been associated with increased cardiovascular disease and mortality. Several clinical trials have reported that the proportion of calories from carbohydrate has an effect on blood pressure. The primary objective of this study was to assess arterial pulse pressure and its association with carbohydrate quantity and quality (glycemic load) with diabetes status for a Cuban American population. Methods: A single point analysis included 367 participants. There was complete data for 365 (190 with and 175 without type 2 diabetes). The study was conducted in the investigator’s laboratory located in Miami, Florida. Demographic, dietary, anthropometric and laboratory data were collected. Arterial pulse pressure was calculated by the formula systolic minus the diastolic blood pressure. Glycemic load, fructose, sucrose, percent of average daily calories from carbohydrate, fat and protein, grams of fiber and micronutrient intakes were calculated from a validated food frequency questionnaire. Results: The mean arterial pulse pressure was significantly higher in participants with (52.9 ± 12.4) than without (48.6 ± 13.4) type 2 diabetes. The odds of persons with diabetes having high arterial pulse pressure (>40) was 1.85 (95% CI =1.09, 3.13); p=0.023. For persons with type 2 diabetes higher glycemic load was associated with lower arterial pulse pressure. Conclusions: Arterial pulse pressure and diet are modifiable risk factors of cardiovascular disease. Arterial pulse pressure may be associated with carbohydrate intake differently considering diabetes status. Results may be due to individuals with diabetes following dietary recommendations. The findings of this study suggest clinicians take into consideration how medical condition, ethnicity and diet are associated with arterial pulse pressure before developing a medical nutrition therapy plan in collaboration with the client.

The star formation and extinction coevolution of UV-selected galaxies over 0.05 < z < 1.2

We use a new stacking technique to obtain mean mid-IR and far-IR to far-UV flux ratios over the rest-frame near-UV, near-IR color-magnitude diagram. We employ COMBO-17 redshifts and COMBO-17 optical, GALEX far- and near-UV, and Spitzer IRAC and MIPS mid-IR photometry. This technique permits us to probe the infrared excess (IRX), the ratio of far-IR to far-UV luminosity, and the specific star formation rate (SSFR) and their coevolution over 2 orders of magnitude of stellar mass and over redshift 0.1 < z < 1.2. We find that the SSFR and the characteristic mass (Script M_0) above which the SSFR drops increase with redshift (downsizing). At any given epoch, the IRX is an increasing function of mass up to Script M_0. Above this mass the IRX falls, suggesting gas exhaustion. In a given mass bin below Script M_0, the IRX increases with time in a fashion consistent with enrichment. We interpret these trends using a simple model with a Schmidt-Kennicutt law and extinction that tracks gas density and enrichment. We find that the average IRX and SSFR follow a galaxy age parameter ξ, which is determined mainly by the galaxy mass and time since formation. We conclude that blue-sequence galaxies have properties which show simple, systematic trends with mass and time such as the steady buildup of heavy elements in the interstellar media of evolving galaxies and the exhaustion of gas in galaxies that are evolving off the blue sequence. The IRX represents a tool for selecting galaxies at various stages of evolution.

Physics of Hexagonal Limit-Periodic Phases: Thermodynamics, Formation and Vibrational Modes

Limit-periodic (LP) structures exhibit a type of nonperiodic order yet to be found in a natural material. A recent result in tiling theory, however, has shown that LP order can spontaneously emerge in a two-dimensional (2D) lattice model with nearest-and next-nearest-neighbor interactions. In this dissertation, we explore the question of what types of interactions can lead to a LP state and address the issue of whether the formation of a LP structure in experiments is possible. We study emergence of LP order in three-dimensional (3D) tiling models and bring the subject into the physical realm by investigating systems with realistic Hamiltonians and low energy LP states. Finally, we present studies of the vibrational modes of a simple LP ball and spring model whose results indicate that LP materials would exhibit novel physical properties.

A 2D lattice model defined on a triangular lattice with nearest- and next-nearest-neighbor interactions based on the Taylor-Socolar (TS) monotile is known to have a LP ground state. The system reaches that state during a slow quench through an infinite sequence of phase transitions. Surprisingly, even when the strength of the next-nearest-neighbor interactions is zero, in which case there is a large degenerate class of both crystalline and LP ground states, a slow quench yields the LP state. The first study in this dissertation introduces 3D models closely related to the 2D models that exhibit LP phases. The particular 3D models were designed such that next-nearest-neighbor interactions of the TS type are implemented using only nearest-neighbor interactions. For one of the 3D models, we show that the phase transitions are first order, with equilibrium structures that can be more complex than in the 2D case.

In the second study, we investigate systems with physical Hamiltonians based on one of the 2D tiling models with the goal of stimulating attempts to create a LP structure in experiments. We explore physically realizable particle designs while being mindful of particular features that may make the assembly of a LP structure in an experimental system difficult. Through Monte Carlo (MC) simulations, we have found that one particle design in particular is a promising template for a physical particle; a 2D system of identical disks with embedded dipoles is observed to undergo the series of phase transitions which leads to the LP state.

LP structures are well ordered but nonperiodic, and hence have nontrivial vibrational modes. In the third section of this dissertation, we study a ball and spring model with a LP pattern of spring stiffnesses and identify a set of extended modes with arbitrarily low participation ratios, a situation that appears to be unique to LP systems. The balls that oscillate with large amplitude in these modes live on periodic nets with arbitrarily large lattice constants. By studying periodic approximants to the LP structure, we present numerical evidence for the existence of such modes, and we give a heuristic explanation of their structure.

Molecular Insights of CD4+ T Cell Differentiation, Effector Formation and Helper Function

CD4+ T cells play a crucial in the adaptive immune system. They function as the central hub to orchestrate the rest of immunity: CD4+ T cells are essential governing machinery in antibacterial and antiviral responses by facilitating B cell affinity maturation and coordinating the innate and adaptive immune systems to boost the overall immune outcome; on the contrary, hyperactivation of the inflammatory lineages of CD4+ T cells, as well as the impairments of suppressive CD4+ regulatory T cells, are the etiology of various autoimmunity and inflammatory diseases. The broad role of CD4+ T cells in both physiological and pathological contexts prompted me to explore the modulation of CD4+ T cells on the molecular level.

microRNAs (miRNAs) are small RNA molecules capable of regulating gene expression post-transcriptionally. miRNAs have been shown to exert substantial regulatory effects on CD4+ T cell activation, differentiation and helper function. Specifically, my lab has previously established the function of the miR-17-92 cluster in Th1 differentiation and anti-tumor responses. Here, I further analyzed the role of this miRNA cluster in Th17 differentiation, specifically, in the context of autoimmune diseases. Using both gain- and loss-of-function approaches, I demonstrated that miRNAs in miR-17-92, specifically, miR-17 and miR-19b in this cluster, is a crucial promoter of Th17 differentiation. Consequently, loss of miR-17-92 expression in T cells mitigated the progression of experimental autoimmune encephalomyelitis and T cell-induced colitis. In combination with my previous data, the molecular dissection of this cluster establishes that miR-19b and miR-17 play a comprehensive role in promoting multiple aspects of inflammatory T cell responses, which underscore them as potential targets for oligonucleotide-based therapy in treating autoimmune diseases.

To systematically study miRNA regulation in effector CD4+ T cells, I devised a large-scale miRNAome profiling to track in vivo miRNA changes in antigen-specific CD4+ T cells activated by Listeria challenge. From this screening, I identified that miR-23a expression tightly correlates with CD4+ effector expansion. Ectopic expression and genetic deletion strategies validated that miR-23a was required for antigen-stimulated effector CD4+ T cell survival in vitro and in vivo. I further determined that miR-23a targets Ppif, a gatekeeper of mitochondrial reactive oxygen species (ROS) release that protects CD4+ T cells from necrosis. Necrosis is a type of cell death that provokes inflammation, and it is prominently triggered by ROS release and its consequent oxidative stress. My finding that miR-23a curbs ROS-mediated necrosis highlights the essential role of this miRNA in maintaining immune homeostasis.

A key feature of miRNAs is their ability to modulate different biological aspects in different cell populations. Previously, my lab found that miR-23a potently suppresses CD8+ T cell cytotoxicity by restricting BLIMP1 expression. Since BLIMP1 has been found to inhibit T follicular helper (Tfh) differentiation by antagonizing the master transcription factor BCL6, I investigated whether miR-23a is also involved in Tfh differentiation. However, I found that miR-23a does not target BLIMP1 in CD4+ T cells and loss of miR-23a even fostered Tfh differentiation. This data indicate that miR-23a may target other pathways in CD4+ T cells regarding the Tfh differentiation pathway.

Although the lineage identity and regulatory networks for Tfh cells have been defined, the differentiation path of Tfh cells remains elusive. Two models have been proposed to explain the differentiation process of Tfh cells: in the parallel differentiation model, the Tfh lineage is segregated from other effector lineages at the early stage of antigen activation; alternatively, the sequential differentiation model suggests that naïve CD4+ T cells first differentiate into various effector lineages, then further program into Tfh cells. To address this question, I developed a novel in vitro co-culture system that employed antigen-specific CD4+ T cells, naïve B cells presenting cognate T cell antigen and BAFF-producing feeder cells to mimic germinal center. Using this system, I were able to robustly generate GC-like B cells. Notably, well-differentiated Th1 or Th2 effector cells also quickly acquired Tfh phenotype and function during in vitro co-culture, which suggested a sequential differentiation path for Tfh cells. To examine this path in vivo, under conditions of classical Th1- or Th2-type immunizations, I employed a TCRβ repertoire sequencing technique to track the clonotype origin of Tfh cells. Under both Th1- and Th2- immunization conditions, I observed profound repertoire overlaps between the Teff and Tfh populations, which strongly supports the proposed sequential differentiation model. Therefore, my studies establish a new platform to conveniently study Tfh-GC B cell interactions and provide insights into Tfh differentiation processes.