Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references.

Thesis (M.Sc.). / Written for the Dept. of Anatomy and Cell Biology. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 128 pages. Includes vita. Includes bibliographical references.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 203-212; 260-261). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 187 pages. Includes vita. Includes bibliographical references.

Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 200-233). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Abstract The response of the innate immune system is triggered through Toll-like transmembrane receptors (TLR) that recognize a variety of microbial products. TLR4 is the principal mediator for Gram negative bacterial endotoxin (LPS), whereas TLR2 mediates the response to Gram positive bacteria, mycobacteria, and yeast. Stimulation of TLR activates complex cascades leading first to the production of inflammatory mediators, such as proinflammatory cytokines IL-1 α/β and TNF-α. Overproduction of inflammatory cytokines as well as failure in the activation of innate immunity are detrimental to the host. Excess inflammatory stimulation leads to a septic shock, which may cause multi-organ failure and even death. The lack of any innate response exposes the host to overwhelming bacterial infections. Appropriate regulation of the innate immune response could be a target for attempts to find therapeutics to septic shock. This experimental study focuses on functional activation of the signaling receptors TLR4 and TLR2 upon a LPS challenge. An acute inflammation model was used for both in vivo and in vitro experiments. LPS was used to stimulate a mouse macrophage cell line. It was administred intraperitoneally or intra-amniotically to non-pregnant or time-mated mice. The basal and induced mRNA expression levels and the protein production of TLRs as well as the mRNA expression of several inflammatory mediators were studied. The present study showed that the expression of TLR4 and TLR2 is strain and tissue-specific. At the mRNA level, the levels of TLR4 expression limited the extent of the acute cytokine response. The quality of the cytokine response was modulated by protein aggregates formed by TLR4 on the cell surface. The LPS challenge caused a marked increase in the expression of TLR2 mRNA but not the protein; the significance of this remains to be studied. The study further showed that the expression of TLRs is regulated during the perinatal period, and that the acute cytokine response to LPS in the lung develops during antenatal differentiation. The present study provides information about how the activation of TLR regulates the acute inflammatory response and further helps to elucidate new targets for the anti-inflammatory strategies in controlling inflammatory events. / Tiivistelmä "Toll-like"-reseptorit (TLR) ovat solukalvon proteiineja, jotka spesifisesti tunnistavat erilaisia bakteerirakenteita. Infektiossa tällainen bakteerirakenne sitoutuu reseptoriin ja seurauksena solussa käynnistyy synnynnäinen immuunivaste eli tulehdusvälittäjäaineiden tuotto. Liiallinen tulehdusvälittäjäaineiden tuotto voi johtaa septiseen shokkiin eli verenmyrkytykseen, elinvaurioihin ja jopa kuolemaan. Septisen shokin synty voisi olla estettävissä immuunivasteen voimakkuuden tarkoituksenmukaisella säätelyllä. Väitöskirjassa on tutkittu, miten TLR4 ja TLR2 aktivoituvat bakteeri-infektiossa, tarkoituksena selvittää, säätelevätkö reseptorit immuunivasteen käynnistystä ja voimakkuutta solussa. Tutkimuksessa todettiin, että TLR4:n ja TLR2:n geenien ilmentymistä säädellään eri tavoin eri hiirikannoilla ja eri kudoksissa. TLR4-tason nousu aiheutti voimakkaamman immuunivasteen, kun taas reseptorin matala esiintymistaso laski immuunivasteen voimakkuutta. Lisäksi TLR4:aan solukalvolla sitoutuvat muut proteiinit vaikuttivat immuunivasteen laatuun. Tutkimuksessa todettiin myös, että TLR:n määrä sikiön keuhkoissa rajoittaa keuhkojen immuunivasteen kehittymistä. Tutkimus antaa tietoa siitä, miten TL-reseptorien aktivaatio säätelee synnynnäistä immuunipuolustusta ja selventää mahdollisuutta kontrolloida immuunivasteen voimakkuutta vaikuttamalla TL-reseptoriin.

A potential approach to in vivo gene therapy is to target retrovirus to specific receptors through a ligand-receptor interaction. Previous studies have placed a ligand at or close to the N-terminus of the ecotropic Moloney murine leukemia virus envelope and require co-expression of a wild type envelope on the pseudotyped virus for successful transduction of human cells. In this study, over forty chimeric envelopes were generated, which have single or multiple insertions of a 13 or 21 amino acid RGD containing sequence, flanked by cysteine residues, that target the cellular integrin receptors (Chapter III). Virus displaying only the chimeric envelopes was generated from packaging cell lines that express the gag and pol genes. Many of the mutant envelopes demonstrated the formation of syncytia when they were transfected into the XC indicator cell line, which is frequently used to determine envelope binding and fusion capabilities. Pseudotyped virus for several of the chimeric envelopes, transduced both NIH 3T3 mouse fibroblasts and human A375 melanoma cells. Ligands placed in the N-terminal region, within the VRA variable domain, and close to the N-terminus of the proline-rich region (PRR), demonstrated transduction into human melanoma cells. Ligands placed within the PRR and the C-terminus of the envelope did not demonstrate transduction into melanoma cells, although host cell transduction was demonstrated. Pseudotyped virus expressing an RGE containing target sequence, replacing the RGD sequence, had significantly lower transduction efficiency of melanoma cells. These data indicate that the MLV envelope tropism can be altered by insertion of short ligands at various locations throughout the envelope. These initial results were promising and helped to define regions within the envelope that could accommodate the insertion of small targeting ligands, that could redirect the tropism of pseudo typed virus to human cells. In the second part of this study, the focus shifted to targeting receptors that were expressed on specific cells, such as carcinoma cells. We inserted short ligands, flanked with cysteines, into the envelope to generate numerous targeting constructs that bind to receptors over-expressed on a variety of carcinoma cells. These pseudotyped retroviral vectors were generated by packaging cell lines that express only the viral Gag and Pol genes, with no wild-type envelope present. Select chimeric envelopes that express the 21 amino acid bombesin (BN)/gastrin releasing protein (GRP) binding sequence successfully transduced human melanoma cells, breast cancer cells, and cells that express the cloned GRP receptor gene. Nine additional chimeric envelopes were generated, that express a modified 56 amino acid heregulin sequence (HRG), that targets c-rbB-3 (Her-3) and c-erbB-4 (Her-4) receptors on breast carcinoma cells. Pseudotyped virus expressing only the BN/GRP mutant envelopes, transduced NIH 3T3 host cells, and two human carcinoma cell lines; A375 melanoma and MDA-MB-231 breast cells. The HRG chimeric envelopes demonstrated transduction of NIH 3T3 cells and human MDA-MB-453 breast carcinoma cells. Finally, a pseudotyped virus that expressed the chimeric BN/GRP envelopes and packaged the thymidine kinase gene, transduced melenoma and breast carcinoma cells and demonstrated ganciclovir cytotoxicity. Collectively, these data indicate that ligands of various sizes can be used to target pseudotyped virus to a variety of human cancer cells and transfer genes of interest. These findings may expand the feasibility and potential scope of gene therapy.

Enterohemorrhagic Escherichia coli 0157:H7 (EHEC) and Enteropathogenic E. coli (EPEC) are intestinal pathogens that induce characteristic lesions on mammalian cells called actin pedestals. Attachment to host cells by both EPEC and EHEC is an essential step towards colonization and is associated with the formation of highly organized actin cytoskeletal elements termed as attaching and effacing (AE) lesions beneath bound bacteria. The outer membrane protein intimin is required for the formation of these structures and binds its own translocated mammalian cell receptor called Translocated intimin receptor (Tir). These interactions induce a cascade of events that result in actin pedestal formation. In this thesis, we characterized pedestal formation and the requirements of pedestal formation by host adapted and in vitro cultivated EHEC. Our data indicate that growing EHEC in the mammalian host enhances bacterial cell attachment, expression and translocation of virulence effectors and actin signaling, and this enhancement is likely to entail more than one bacterial activity involved in host cell interactions. We also focused on the interaction between the two key bacterial players involved in pedestal formation, intimin and Tir. We randomly mutagenized the Tir-binding domain of intimin and isolated point mutants that disrupted Tir recognition. The ability of intimin mutants to bind to recombinant Tir correlated with their ability to trigger AE lesions on pre-infected mammalian cells. Half of the mutations fell within the previously identified 50 amino acid C-terminal region of intimin, and alanine scanning mutagenesis of this region identified four residues of EHEC intimin that are critical for Tir recognition. In a model of the EHEC intimin-Tir complex that is based on EPEC intimin and Tir, these four amino acids are predicted to be located at the intimin-Tir interface, indicating that these residues play a functional role in intimin recognition by Tir. To identify critical residues involved in intimin recognition and intimin mediated actin signaling, we generated point mutations in the extracellular domain of EHEC Tir. Based on our data, we conclude that Tir-intimin interaction is essential for triggering actin pedestals, and intimin function in the context of Tir signaling can be replaced by proteins that are entirely unrelated to intimin but that bind to Tir. These data are concordant with the model that intimin functions to cluster Tir in the membrane to induce actin assembly. Finally, as a step to study downstream actin signaling processes after Tir translocation, we mapped the domain of Tir involved in host cell signaling. We found that the clustering of a 12 amino acid stretch of C-terminus encompassing the Nck binding sequence of Tir generated actin nucleation indistinguishable from that mediated by the entire C-terminus, and abrogation of Nck binding by mutation of Y474 to Phenylalanine abolished actin assembly. Although these results do not rule out a role for other domains of Tir involved in actin pedestal formation, this suggests that the essential element of Tir consists of the Nck binding domain.