Browsing by Subject "Epithelial Cells"
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Item Effects of Radiation Exposure on Lung Carcinogenesis(2010-01-12T18:50:48Z) Delgado, Oliver; Shay, Jerry W.Lung cancer is one of the most prevalent forms of cancer in both men and women with over 1.3 million annual related deaths worldwide. Analysis of several human populations exposed to radiation reveals that the lung is remarkably susceptible to the carcinogenic effects of radiation exposure. The considerable lung surface area and slow rate of epithelial turnover may have causal roles in this vulnerability. This may be due to the increased probability that a progenitor cell of the lung, which is proposed to be the cancer-initiating cell, may acquire multiple carcinogenic alterations from radiation exposure. Currently, the lung is believed to have several facultative progenitor cells, situated throughout the lung epithelium, that are regionally restricted in their regenerative capacity. Normal human bronchial epithelial cells (HBECs), immortalized through the expression of Cdk4 and hTERT, provide a sustainable cell reagent for the evaluation of the radiation effects in vitro. These HBECs retain a novel multipotent capacity in vitro (capable of differentiating into both central and peripheral lung cell types) and thus may represent an unrestricted progenitor of the adult lung that resembles an embryonic progenitor. Studies to determine whether the differentiation state influences radiation exposure effects, such as DNA damage and repair, are ongoing. As cellular responses change upon the acquisition of oncogenic mutations, the effects of fractionated or acute radiation exposure on lung carcinogenesis in vivo were determined utilizing the transgenic LA1 K-ras mouse model of lung cancer compared to wildtype littermates. Radiation-induced carcinogenesis is a major concern not only for cancer patients being treated with therapeutic radiation but also for astronauts on long-term space missions. X-ray radiation did not affect the incidence or progression of lung carcinogenesis in this mouse model of lung cancer. High-energy 56Fe- particle irradiation (a type of radiation present in deep space), however, significantly increased the incidence of invasive carcinoma when administered as a fractionated dose but not as a single acute dose. These results demonstrate that pre-initiated lesions may be more susceptible to malignant transformation upon exposure to radiation. Thus, radiation may have an impact on both lung cancer initiation and progression. [Keywords: radiation; lung; cancer; stem cells; progression]Item Intracellular Trafficking of Influenza Hemagglutinin and Members of the Low Density Lipoprotein Receptor Family(2004-12-15) Tall, Renee Danielle; Lehrman, MarkPolarized epithelial cells usually line body cavities, providing a barrier between two dissimilar environments. Distinct apical and basolateral membrane surfaces are maintained by sorting proteins in the biosynthetic and endocytic pathways. Influenza hemagglutinin is sorted to the apical membrane in polarized epithelial cells by a mechanism mediated by an association with lipid microdomains known as lipid rafts. Lipid rafts and their associated proteins are operationally defined by their resistance to detergent solubilization at cold temperatures. By systematic mutagenesis of the transmembrane domain of hemagglutinin, I show that ten consecutive amino acids are required to confer resistance to detergent extraction. Although some of the hemagglutinin transmembrane mutants were sorted apically without incorporation into detergent-resistant membranes, I determined that these mutants were transiently associated with lipid rafts. A small fraction of hemagglutinin coprecipitates with MAL/VIP17, a protein required for apical transport. The hemagglutinin and MAL that co-precipitated were contained in a detergent-resistant vesicle in an orientation consistent with a transport intermediate, suggesting that MAL might sort hemagglutinin into apical vesicles in the Golgi. However, the time course of the association of hemagglutinin and MAL in the biosynthetic pathway indicate that the two proteins do not associate until the majority of the HA reached the cell surface. Both the timing and limited extent of coprecipitation suggest that MAL may not sort hemagglutinin into apical vesicles in the biosynthetic pathway. Megalin, a member of the low density lipoprotein family of receptors, is sorted apically in polarized epithelial cells via a sorting signal present in its cytosolic domain. I show that the cytosolic domain of megalin associates with sorting nexin 17, a protein that interacts with all core members of the low density lipoprotein receptor family. Although sorting nexin 17 may not sort megalin apically in the biosynthetic pathway, I show that overexpression of sorting nexin 17 increases the rate of low density lipoprotein receptor recycling to the plasma membrane. The increase in recycling does not affect the expression of low density lipoprotein receptor at the cell surface, suggesting that sorting nexin 17 decreases the cycling time of the receptor.Item The Ligand and Function of the RegIII Family of Bactericidal C-Type Lectins(2006-08-11) Cash, Heather Lynn; Hooper, LoraBeginning at birth, the intestines of humans and other mammals are colonized with a diverse society of resident bacteria that play a crucial role in host nutrient metabolism. To maintain this commensal relationship, resident microbes must be prevented from crossing the intestinal epithelium into host tissues where they can cause inflammation and sepsis. The innate immune system plays a crucial role in preventing bacterial incursions across gut epithelial surfaces. Mucosal epithelial cells produce a variety of secreted antimicrobial proteins that help to prevent bacterial attachment and encroachment at epithelial surfaces. Among these, Paneth cells are specialized small intestinal epithelial cells that have been shown to produce and secrete antimicrobial proteins and peptides. To gain new insights into the adaptation of mucosal surfaces to microbial challenges, the Hooper lab has used DNA microarrays to screen for Paneth cell genes whose expression is modulated by intestinal microbes. This screen revealed that expression of two C-type lectins, RegIIIbeta and RegIIIgamma , is strongly induced following intestinal colonization with resident microbes. Two features suggested that members of the RegIII family may have microbicidal functions. First, they are C-type lectin family members. Other C-type lectins, including the mannose binding lectin, have well-characterized innate immune functions and play critical roles in microbial killing by recruiting complement. Second, I have shown that the murine RegIII lectins localize to intestinal crypt cells, including Paneth cell secretory granules, and that they bind to luminal bacteria harvested from intestinal conditions. Based on these observations, we hypothesized that this family of proteins may perform an innate immune function, specifically antimicrobial defense. The studies reported in this thesis characterize a family of C-type lectins. Specifically, we determined that these proteins interact with peptidoglycan by binding with high affinity to its glycan structure, representing a unique blend of peptidoglycan recognition and lectin function. Additionally, we have demonstrated that this binding results in the specific disruption of the Gram positive bacterial cell wall, where peptidoglycan is exposed, which is the first example of a family of directly bactericidal C-type lectins. We also present evidence for the regulation of these bactericidal proteins by colonization with an intestinal microflora. Therefore, the research presented in this thesis elucidates the function of three members of the RegIII family, in both mice and humans.Item With No Lysine 1 (WNK1): A Potential Regulator of The Lysosomal Degradation Pathway(2007-05-22) Lenertz, Lisa Yvonne; Cobb, Melanie H.With no Lysine (K) 1 (WNK1) is an atypical serine/threonine protein kinase that has its catalytic lysine positioned in a unique location. This kinase, along with another member of the WNK family, WNK4, has been genetically linked to pseudohypoaldosteronism type II (PHAII), which is characterized by both hypertension and hyperkalemia. Several groups have used reconstitution assays in Xenopus oocytes and mammalian cell lines to show WNKs regulate the surface expression and/or activity of various ion transporters and channels, including the epithelial sodium channel (ENaC) and the sodium chloride co-transporter (NCCT). Although the mechanisms for regulating these cell surface proteins are not well defined, it appears that WNKs may modulate the intracellular trafficking of these channels and transporters. To help define the mechanisms WNK1 utilizes to influence blood pressure and to characterize this kinase biochemically, I performed a WNK1 kinase activation screen and a WNK1 yeast-two-hybrid screen. I have shown that WNK1 kinase activity increases in response to osmotic stress, which may imply its kinase activity is important for regulating ion homeostasis in response to a change in cell volume. I have also shown that a proline-rich region of WNK1 interacts with vacuolar protein sorting 4a (VPS4a), an ATPase that helps sort cargo from the plasma membrane to lysosomes. Cells expressing a VPS4 ATP-hydrolysis mutant trap cargo from the cell surface in an aberrant endosomal structure, slowing protein degradation via the lysosomal pathway. I hypothesize that WNK1 delivers cargo to VPS4a to facilitate the degradation of plasma membrane proteins.