The objective of this study was to identify possible roles of the class II antigen-associated, electrophoretically invariant chain (Ii) in class II antigen functions through analysis of the kinetics of synthesis, processing, and turnover of Ii in various cellular systems of Ii hyperexpression. Using [35S]methionine metabolic labeling of microsomal membrane proteins and analysis in one-dimensional SDS polyacrylamide gradient gels and two-dimensional electrophoretic gels, enhanced expression of Ii was demonstrated in leukemic cells of a subset of patients with hairy cell leukemia (HCL), in normal peripheral blood cells freshly transformed with EBV, and in Burkitt's lymphoma cell lines treated with n-butyrate. As part of an initial effort to identify leukemic cell subset-defining, membrane-associated molecules which might then be tested as targets for, or regulators of, the anti-leukemic cell immune response, subsets of HCL patients were identified on the basis of leukemic cell enhanced expression of 35,000 and 15,000 dalton proteins (p35 and p15). A similar enhanced expression of the p35 molecule was demonstrated in peripheral blood or cord blood lymphocytes freshly transformed with Epstein-Barr virus (EBV), as compared to long-term cultured EBV cell lines. Further structural characterization of the HCL p35 by one-dimensional SDS-PAGE and two-dimensional gel electrophoresis of HCL total microsomal membrane proteins, anti-class II antigen immunoprecipitates, and anti-Ii immunoprecipitates showed that the HCL p35 molecule was the human class II antigen-associated Ii chain. In order to determine the functional significance of altered Ii expression on class II antigen function, the relative kinetics of Ii synthesis, processing, and turnover was examined in an in vitro model system of regulated Ii synthesis. Treatment of the Burkitt's lymphoma cell line, Jijoye, and its class II antigen-negative, mutant, daughter cell line, P3HR-1, with 4 mM n-butyrate for 48 hr, enhanced the rate of synthesis of the Ii chain. One-dimensional SDS-PAGE and two-dimensional gel, electrophoretic analysis of anti-Ii and anti-class II antigen immunoprecipitates isolated from pulse-/pulse-chase-/or continuously labeled, control and butyrate-treated P3HR-1 and Jijoye cells demonstrated enhanced levels of synthesis of the dominant, most basic form of Ii in the butyrate-treated samples (to a greater degree in Jijoye cells). The butyrate enhancement of Ii synthesis occurred in the presence or absence of detectable class II alpha and beta chains, as did the relative turnover, although the terminal processing of Ii to an electrophoretically slower and more acidic form was dependent upon its association with class II antigens. The level of the dominant, most basic form of Ii expressed in the hairy leukemic cells equaled that in butyrate-treated Jijoye cells. However, hairy leukemic cell, class II antigen-associated Ii was not terminally processed. The results of these experiments were consistent with the following. In lymphoblastoid cells, two pathways for Ii turnover might exist. One is through association with class II antigen complexes and progressive carbohydrate-chain terminal processing, and a second is not associated with class II antigens and such processing. Class II antigens may direct the processing of Ii towards some function (rather than vice versa). Butyrate treatment rather uniquely enhances the synthesis of Ii in some lymphoid cells in the presence or absence of class II antigens. Hairy leukemic cells demonstrate an inability to terminally process Ii which might be relevant to the function of Ii and/or class II antigens on those cells.

Early post-implantation vertebrate embryos are shaped by complex cellular and molecular mechanisms. In mice, the visceral endoderm, an extraembryonic cell lineage that appears before gastrulation, provides several important functions such as nutrition and mechanical protection. My thesis research focused on the role of the visceral endoderm in embryo patterning, a newly discovered function for this tissue. My results showed that an interplay between two subpopulations of visceral endoderm the anterior and posterior visceral endoderm, located on the opposite sides of the developing conceptus, are critical for the establishment of the anteroposterior body axis of the embryo. I also found that senescence-associated β-galactosidase activity delineates the visceral endoderm marking apical vacuole, a lysosomal-like organelle. This however indicates the nutritional function of visceral endoderm cells rather than a senescent population. My studies highlight the fundamental role of extraembryonic tissues in patterning mammalian embryos as opposed to housekeeping roles. They also reveal important difference when conducting studies at the organismal level rather than in cells in culture.

Background: The initial systolic blood pressure (SBP) in patients with acute heart failure (AHF) can be used as a guide when choosing specific pharmacologic treatments by helping identify the underlying type of HF (e.g., HF with preserved ejection fraction). Clinical experience and research data from our medical center suggests that AHF with elevated SBP may be presenting less frequently than in the past. This may call into question the utility of initial SBP as a clinical guide. The goal of this Master’s Thesis is to test the hypothesis that the frequency of AHF patients with a SBP>160mmhg has declined over time. Methods: This observational study compares data from 4 cohorts of adult patients admitted with AHF in central MA. Data were obtained from a contemporary (2011-2013) study of patients with AHF as well as from the 1995, 2000, 2006 Worcester Heart Failure Study (WHFS) cohorts. The Framingham criteria the diagnostic criterion for AHF. The main outcome was the proportion of patients with AHF with a SBP > 160 mmHg who presented in each of the 4 study cohorts and was examined by multivariate logistic regression. Results: 2,366 patients comprised the study population. The average age was 77 years, 55% were female, 94% white, and 75% had prior HF. In 1995 33.6% of AHF patients had a SBP >160 mmHg compared to 19.5% in 2011-2013 (p160 mmHg in 2006 (0.64, (0.42-0.96)) and 2011-13 (0.46, (0.28-0.74)). Conclusion: The proportion of patients with AHF and an initial SBP >160 mmHg has significantly declined over time. This may warrant a reexamination of the utility of SBP to inform diagnosis and treatment in patients with AHF.

During regeneration, cells must coordinate proliferation and differentiation to rebuild tissues that are lost. Understanding how source cells execute the regeneration process has been a longstanding goal in regenerative biology with implications in wound healing and cell replacement therapies. Melanocytes are pigment-producing cells in the skin of vertebrates that can be lost during hair graying, injury and disease-related depigmentation. Melanoma is an aggressive skin cancer that develops from melanocytes, and it is hypothesized that melanoma cells have properties that are similar to melanocyte stem cells. To gain insight into melanocyte regeneration we set out to identify the source of regeneration melanocytes in adult zebrafish and the path through which progenitor cells reconstitute the pigment pattern. Using targeted cell ablation and single cell lineage-tracing analyses we identified that a majority of regeneration melanocytes arise through direct differentiation of mitfa-expressing progenitor cells. Concurrently, other mitfa-expressing cells divide symmetrically to generate additional mitfa-positive progenitors, thus maintaining regeneration capability. Using reporter assays and drug studies, we found that Wnt signaling gets turned on in progenitor cells during regeneration and Wnt inhibition after melanocyte ablation blocks regeneration. Based on our finding that Wnt signaling is active in differentiated melanocytes but not in the progenitor cells, we explored the role of Wnt signaling in tumor initiation. We found that approximately half of the melanomas are Wnt silent, and overexpression of dkk1b, a negative regulator of canonical Wnt signaling, accelerates melanoma onset. This work defines an unappreciated contribution by direct differentiation in melanocyte regeneration and suggests a broader role for this process in the maintenance of epithelial sheets. This study also identifies a shared pathway between melanocyte progenitors and melanoma cells, which could be applicable to other cancers.

The integrin α6β4 (referred to as β4) is expressed in epithelial cells where it functions as a laminin receptor. Integrin β4 is important for the organization and maintenance of epithelial architecture in normal cells. Particularly, β4 is shown to be essential for mammary gland development during embryogenesis. Integrin β4 also plays important roles in tumor formation, invasion and metastasis in breast cancer. However, the mechanism of how integrin β4 mediates breast tumor formation has not been settled. A few studies suggest that integrin β4 is involved in cancer stem cells (CSCs), but the mechanism is not clear. To address this problem, I examined the expression of β4 in breast tumors and its potential role involved in regulating CSCs. My data shows that β4 is expressed heterogeneously in breast cancer, and it is not directly expressed in CSCs but associated with a basal epithelial population. This work suggests that β4 can regulate CSCs in a non-cell-autonomous manner through the interactions between β4+ non-CSC population and β4- CSC population. My data also shows that β4 expression is associated with CD24+CD44+ population in breast tumor. To further study the role of β4 in breast cancer progression, I generated a β4 reporter mouse by inserting a p2A-mCherry cassette before ITGB4 stop codon. This reporter mouse can be crossed with breast tumor models to track β4+ population during tumor progression.

For nearly five decades, the simple eukaryote Saccharomyces cerevisiae has been used as a model for understanding the eukaryotic cell cycle. One vein of this research has focused on understanding how chromosome structure is regulated in relation to the cell cycle. This work characterizes a new mechanism that modulates the chromatin organizing condensin complex, in hopes of furthering the understanding of chromosome structure regulation in eukaryotes. During mitosis, chromosomes are condensed to facilitate their segregation through a process mediated by the condensin complex. Upon interphase onset, condensation is reversed, allowing for efficient transcription and replication of chromosomes. This work demonstrates that Ycg1, the Cap-G subunit of budding yeast condensin, is cell-cycle regulated with levels peaking in mitosis and decreasing as cells enter G1 phase. The cyclical expression of Ycg1 is unique amongst condensin subunits, and is established by a combination of cell cycle-regulated transcription and constitutive proteasomal degradation. Interestingly, when cyclical expression of Ycg1 is disrupted, condensin formation and chromosome association increases, and cells exhibit a delay in cell-cycle entry. These results demonstrate that Ycg1 levels limit condensin function, and suggest that regulating the expression of an individual condensin subunit helps to coordinate chromosome conformation with the cell cycle. These data, along with recent corroborating results in Drosophila melanogaster suggest that condensin regulation through limiting the expression of a single condensin subunit may be broadly conserved amongst eukaryotes.

Chemotaxis, the ability of a cell to migrate towards a directional stimulus, is a basic property of virtually all cells at some stage in their development. Chemotaxis is preceded by the development of a polarized cellular morphology. The region of the cell closest to the attractant forms a broad lamellipod. The contents of the cell flow forward into this lamellipod and the rear of the cell becomes constricted into a narrow uropod. These local differences in cell structure and function presumably reflect local differences in cell chemistry, but the chemical processes involved are poorly understood. Ca+2 is known to play a ubiquitous role as an essential second messenger in many cellular processes, but its role in chemotaxis is unclear. While many chemotactic stimuli cause Ca+2 to rise intracellularly, the relationship between this rise in Ca+2 and local changes in cell behavior has been difficult to understand. In my dissertation work, I directly tested the role of cellular Ca+2 changes in polarization and chemotaxis by simultaneously imaging intracellular Ca+2 and cell morphology. This work was carried out on single eosinophils isolated from the newt, Taricha granulosa, because of their large size (~100 um, when polarized) and rapid responsiveness (~20 um/min) to chemotactic stimuli present in newt serum. An imaging system was developed to simultaneously image cell behavior, and intracellular Ca+2 following microinjection of the Ca+2 sensitive fluorescent probe, Fura-2. Cell behavior was quantified from time lapse video images captured by a SIT video camera, stored on a video optical disk recorder, and later digitized for analysis. Quantitation was accomplished by interactively tracing the cell's outline and determining the position of the geometric centroid. Variation in the radius of the outline from the centroid was used to calculate a "polarization index", which could be monitored over time. Cell speed was calculated from the movement of the centroid over time. Agents which are known to interfere with Ca+2 signalling significantly inhibited both the polarization and the movement of cells in response to 10% newt serum. These treatments included: chelation of extracellular Ca+2 with EGTA, the organic Ca+2 channel antagonist, verapamil, the inorganic Ca+2 channel blocker, cobalt, the Ca+2 ionophore, ionomycin, and caffeine, an agent known to release Ca+2 from internal stores. In contrast, the K+ ionophore, valinomycin, and treatment of cells with dibutryl cAMP had no effect on cell behavior. The development of a polarized, motile morphology following stimulation of newt eosinophils with 10% serum was accompanied by a rise in intracellular Ca+2. In addition, Ca+2 in a polarized, moving cell was non-uniformly distributed and periodic elevations in intracellular Ca+2 were seen during changes in cell behavior. In turning cells, Ca+2 was significantly higher than in cells moving in a straight line and there was a clearly detectable gradient of Ca+2 within the cell. The region closest to the new direction of movement had the lowest Ca+2 and the rear of the cell was significantly higher. This gradient persisted following a turn, even though Ca+2 was much lower overall in cells moving in a straight line. A gradient of Ca+2 along the long axis of the cell might be important for the differential regulation of different regions of the moving cell. Loading cells with the cell-permeant, esterified form of Fura-2 revealed a region of high Ca+2 associated with the microtubule organizing center (MTOC). This region was surrounded by a membrane system labeled by the lipid soluble, membrane potential sensitive dye, DiOC6(3). This region of Ca+2 was depleted by caffeine treatment. These observations, coupled with the effects of caffeine on cell behavior, suggest that a Ca+2 storage site associated with the MTOC may play a role in regulating cell polarization and chemotaxis. The effects of releasing "caged calcium" on cell behavior and [Ca2+]i were examined as a means of directly testing the ability of changes in [Ca2+]i to regulate cell behavior. Although photolysis of the compound inhibited cell polarization and movement, technical problems made it difficult to attribute these effects entirely to the release of Ca2+. The results presented here, particularly the gradients of [Ca2+]i which were observed, suggest that local regulation of the cytoplasmic components involved in cell movement by local differences in [Ca2+]i could, in part, explain the regional specialization seen during this process. This form of regulation will be discussed in detail, as will potential mechanisms to test for its function during cell polarization and chemotaxis.

The encompassing aim of this project was to gain a better understanding of the role of the cellular immune response to dengue virus (DV) infection. Dengue virus occurs as four distinct serotypes, called D1-D4. Symptomatic DV infection occurs as two forms of illness. The more severe form of DV infection, dengue hemorrhagic fever (DHF), is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. At its most severe, DHF can result in circulatory shock and death. Epidemiological studies indicate that DHF is more likely to occur following a secondary infection with a serotype of DV other than that which caused the primary infection, and there is evidence of increased T cell activation in more severe disease. These data and others indicate that DHF may be of an immunopathological nature. The memory CD4+ T cell response of a D4-immune donor was analyzed. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious DV antigens showed the highest proliferation to D4V antigen, with lesser, crossreactive proliferation to D2V antigen. CD4+ cytotoxic T lymphocyte (CTL) clones were established by stimulation with D4 antigen using a limiting dilution method. Seven out of 15 clones recognized the D4V capsid protein. The clones showed heterogeneity in their usage of T cell receptor Vα and Vβ genes. Six of these CTL clones were crossreactive between 02 and 04, and one clone was specific for D4. Using synthetic peptides, the D4V-specific clone was found to recognize an epitope between amino acids (aa) 47-55 of the capsid protein, while the crossreactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2 and D4. These results showed that the DV capsid protein can be a target of the cellular immune response following DV infection. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84-92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84-92. These results indicate that this donor exhibits memory CD4+ T cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level, but in bulk culture responses as well. Experiments were performed demonstrating that the CD4+ CTL clones were capable of mediating bystander lysis of non-antigen presenting target cells. Following activation on plate-bound anti-CD3 antibody or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Bystander lysis of neighboring, non-infected cells by activated CD4+ CTL clones might contribute to the pathology of DHF. The mechanisms of lysis employed by the T cell clones against both cognate and bystander target cells were assessed using chemical inhibitors of either the perforin- or Fas/FasL-mediated pathways. Three CD4+ CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/FasL interactions. In contrast, one clone used a Fas/FasL mechanism to lyse both cognate and bystander targets. These experiments indicated that the perforin- and FasL-mediated mechanisms of target cell lysis are not mutually exclusive, in that a single clone can kill target cells using either mechanism. Additionally, the ability of CD4+ CTL clones to lyse target cells by the perforin pathway indicates that, like CD8+ CTL, these clones might play a role in viral clearance and recovery from infection through lysis of virus-infected cells. Cytokine production by the capsid-specific CTL clones was also examined. Six of six clones studied produced high quantities of IFN-γ in response to either D2V antigen or the epitope peptide. IFN-γ was also produced by PBMC in a bulk culture from this donor stimulated with D4V antigen. All of the clones produced both TNF-α and TNF-β following stimulation. Four of six clones produced low amounts of IL-2, and only three of six clones produced detectable amounts of IL-4. Production of cytokines by activated CD4+ T cell clones in vivo could contribute to both viral clearance and immunopathology. To better understand the role that cytokine production might play in vivo in response to DV infection, cytokine mRNA levels were examined by PCR in DV-infected Thai children. mRNA for the cytokines IFN-γ, TNF-β, TNF-α, IL-1β, and IL-6 were detectable in the PBMC of DV-infected children. Semi-quantitative PCR analysis indicated that TNF-α mRNA levels were elevated in Thai children with DHF compared to children with classical dengue fever, the less severe form of illness (p=.013). All other cytokines showed no statistically significant difference between children with DHF and those with DF, although IFN-γ showed a trend toward elevation in more severe disease (p=.l). Increased production of TNF-α and/or IFN-γ in vivo could potentially contribute to the immunopathology of severe dengue illness. Taken as a whole, the data presented in this thesis provide a better understanding of the role of the cellular immune response to dengue virus infection and its potential contribution to the immunopathology of dengue hemorrhagic fever.

The temperature-sensitive α-factor receptor (Ste2-3p) and arginine permease (Can1tsp) were found to provide the model substrates for quality control of plasma membrane proteins in Saccharomyces cerevisiae. When the ste2-3 mutant cells were grown at 34°C, Ste2-3p failed to accumulate at the plasma membrane and was delivered to the vacuole for degradation without traversing the plasma membrane. Upon reaching the vacuole, cytoplasmic domains of both Ste2p and Ste2-3p appeared within the vacuolar lumen. Four stp mutants were identified to suppress temperature-sensitive defects in both Ste2-3p and Can1tsp. The stp22 and STP26 mutations also caused missorting of vacuolar protein carboxypeptidase Y, and a subset of vacuolar protein sorting mutants (vps) suppressed ste2-3 mutation. In the stp22 mutant, both Ste2p and Ste2-3p accumulated in the prevacuolar compartment (PVC) and on the plasma membrane. Three independent mutations that bypassed the phenotype of stp22Δ mutant were identified and mapped to the SNF8 locus, and they were found to affect a single amino acid residue (G209D). The mutant protein, Snf8bpp, but not Snf8p, was able to compensate for the lack of functional Stp22p and to restore PVC-to-vacuole trafficking. The order of function for some VPS genes involved in PVC-to-vacuole traffic (class E) was determined by using this special snf8bp allele. In addition, a PtdIns 4-kinase encoded by the PIK1 gene was found to be involved in Ste2-3p trafficking, possibly affecting the PVC function.

The mechanism for centrosome assembly in somatic cells has previously been proposed to be microtubule independent. Studies presented in this dissertation demonstrate that in somatic cells pericentrin and γ tubulin, two paradigm centrosome proteins, assemble onto the centrosome in a microtubule, and dynein/dynactin dependent manner. High resolution, three-dimensional, time-lapse digital imaging of pericentrin-GFP labeled centrosomes has revealed tiny particles that move vectorally towards the centrosome at rates exceeding 1μm/second. These pericentrin-GFP particles contain γ tubulin and are not readily visible by standard two-dimensional digital imaging microscopy. Further studies have shown that dynein colocalizes with tiny particles of endogenous pericentrin outside of the centrosome which may reflect assembly intermediates in transit towards the centrosome. Furthermore, when dynein function is disrupted in G1 cells by nocodazole treatment, dynamitin overexpression, or dynein IC antibody (70.1) injection, assembly of pericentrin and γ tubulin onto the centrosome throughout the cell cycle is greatly reduced. Moreover, microtubule co-sedimentation studies have demonstrated that pericentrin associates with microtubules in vitro and is dependent on functional dynein/dynactin. Together these data strongly suggest that pericentrin and γ tubulin are novel cargoes of the dynein/dynactin motor complex which transports these proteins -and likely other components of the 3MDa nucleating complex (Dictenberg et al., 1998)- to the centrosome via rnicrotubules.