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dc.contributor.advisorWright, William W.en_US
dc.contributor.authorAlvarado, David Michaelen_US
dc.date.accessioned2015-02-11T04:05:26Z
dc.date.available2015-02-11T04:05:26Z
dc.date.created2014-12en_US
dc.date.issued2014-04-17en_US
dc.date.submittedDecember 2014en_US
dc.identifier.urihttp://jhir.library.jhu.edu/handle/1774.2/37201
dc.description.abstractThe crucial role of structural support fulfilled by keratin intermediate filaments (IFs) in surface epithelia likely requires that they be organized into crosslinked networks. For IFs comprised of keratins 5 and 14, found in basal epidermal keratinocytes, formation of crosslinked bundles is, in part, self-driven through cis-acting determinants. Here, we targeted the expression of a bundling-competent KRT5/KRT8 chimeric cDNA (KRT8bc), or bundling-deficient wildtype KRT8 as a control, to the epidermal basal layer of Krt5 null mice to assess the functional importance of keratin IF self-organization in vivo. We report that targeted expression of K8bc rescued Krt5 null mice with ~47% frequency, while K8 failed to rescue. This outcome correlated with lower than expected levels of K8bc and especially K8 mRNA and protein in the epidermis of E18.5 replacement embryos. Electron microscopy of E18.5 embryonic skin revealed that the defects observed in filament bundling, cytoarchitecture, and mitochondria are partially restored by KRT8bc expression. As young adults, viable KRT8bc replacement mice develop alopecia and chronic skin lesions, indicating that the skin epithelia are not completely normal. These findings are consistent with a contribution of self-mediated organization of keratin IFs to structural support and cytoarchitecture in basal layer keratinocytes of the epidermis, and underscore the importance of context-dependent regulation for keratins in vivo. Structural support is not the only function fulfilled by K5 in skin. A recent genome-wide association study has identified novel single nucleotide polymorphisms (SNPs) altering the K5 coding sequence and conferring an increased susceptibility to basal cell carcinoma (BCC). We conducted an initial assessment of the properties of the K5 variants G138E and D197E in cultured HeLa cells. We found that these two keratin variants readily integrate into endogenous filament networks of HeLa cells, and do not exhibit any egregious assembly properties. We also show that these mutants do not alter the expression of early effectors in Sonic hedgehog (Shh) or Wnt signaling pathways. Relative to wildtype, expression of these KRT5 variants alter the RNA expression of two inflammatory cytokines, CCL2 and CXCL5. These findings point to a possible mechanism by which KRT5 SNPs may increase BCC susceptibility.en_US
dc.format.mimetypeapplication/pdfen_US
dc.publisherJohns Hopkins University
dc.subjectintermediate filamentsen_US
dc.subjectkeratinen_US
dc.subjectepidermisen_US
dc.subjectskinen_US
dc.subjectepidermolysis bullosa simplexen_US
dc.subjectgenodermatosisen_US
dc.subjectblisteringen_US
dc.subjecttransgenic miceen_US
dc.subjectmitochondriaen_US
dc.subjectBCCen_US
dc.titleON THE CONTRIBUTION OF SELF-MEDIATED ORGANIZATION OF KERATIN INTERMEDIATE FILAMENTS TO THEIR BIOLOGICAL FUNCTION IN VIVOen_US
dc.typeThesisen_US
thesis.degree.disciplineCell Biologyen_US
thesis.degree.grantorJohns Hopkins Universityen_US
thesis.degree.grantorSchool of Medicineen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePh.D.en_US
dc.type.materialtexten_US
thesis.degree.departmentCellular and Molecular Medicineen_US
dc.contributor.committeeMemberCoulombe, Pierre A.en_US
dc.contributor.committeeMemberCraig, Susan W.en_US
dc.contributor.committeeMemberReed, Randall R.en_US


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