Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 69-82).

Thesis (Ph. D. in Cancer Biology)--Vanderbilt University, May 2009. / Title from title screen. Includes bibliographical references.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-11-27T18:25:19Z No. of bitstreams: 1 Djalma Gomes Ferrão Carvalhal Um ensaio de adesao... 2001.pdf: 31649822 bytes, checksum: cea5fde124b280c0b60a4b69bc0b6cb3 (MD5) / Made available in DSpace on 2012-11-27T18:25:19Z (GMT). No. of bitstreams: 1 Djalma Gomes Ferrão Carvalhal Um ensaio de adesao... 2001.pdf: 31649822 bytes, checksum: cea5fde124b280c0b60a4b69bc0b6cb3 (MD5) Previous issue date: 2001 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / O objetivo deste trabalho é desenvolver um ensaio de adesão, baseado no ensaio de Stamper- Woodruff, para o estudo das interações entre macrófagos e matriz conjuntiva. Foram induzidos bolsões inflamatórios em camundongos da linhagem BALB/c para obtenção de secções de pele inflamada, e células macrofágicas da linhagem J774G.8 foram utilizadas na padronização do ensaio. Secções de 7 i^m de espessura foram colocadas em lâminas de vidro, fixadas com acetona e bloqueadas com BSA. Foi estabelecida a concentração de 10® células/100 fil como a mais apropriada para a realização do ensaio, através de diluições seriadas. Células J774G.8 sem tratamento prévio ou tratadas com: ácido tetraacético diamino etileno, Mn++, lipopolissacarídeo, forbol miristato acetato, zimosan, os peptídios CS-1 e RGD, os anticorpos anti-CD49d ou contra a cadeia (32 de integrinas, foram adicionadas ás secções e incubadas por 30 minutos á temperatura ambiente. Como resultado, as células macrofágicas aderem preferencialmente ás áreas de inflamação. A adesão das células é dependente de cátions divalentes e pode ser modulada por substâncias que promovam a ativação celular. Além disso, a adesão mediada por integrinas pode ser inibida por peptídios RGD e CS-1 ou com anticorpos contra integrinas da família (31 e (32. A adesão das células macrofágicas é inibida mais intensivamente pela infecção com Leishmania mas não com a infecção por Mycobacterium ou por fagocitose de partículas de látex. Desta forma, o ensaio desenvolvido foi capaz de demonstrar a especificidade da adesão de células macrofágicas pela matriz conjuntiva bem como de sugerir a existência de mecanismos específicos de regulação das interações entre macrófagos e a matriz conjuntiva durante a infecção por Leishmania. / The aim of this work was to develop an adhesion assay, based on the Stamper-Woodruff’s assay to study the interactions between macrophages and the connective matrix. Inflammatory pouches were produced in BALB/c mice to obtain inflammed skin sections, and J774G.8 macrophage cell line was applied to standardize the assay. Sections of 7|im thick were placed onto glass slides, fixed with acetone and blocked with bovine serum albumin. The cell concentration of 10®/100 |xl was proved to be most appropriate for the assay, through serial dilutions. J774G.8 cells, with or without ethylene diamine tetraacetic acid, Mn'"'", Lipopolysaccharide, phorbol myristate acetate, zymosan, CS- 1, RGD peptide, antibodies anti-CD49d or against (32 chain integrin treatment, were added onto the sections and incubated for 30 minutes at room temperature. Macrophage cells adhered preferentially to inflammed areas. The adhesion of cells was dependent on divalent ions and could be modulated by substances that promote cell activation. Moreover, the adhesion mediated by integrin can be inhibited either by RGD or CS-1 peptides or by antibodies against integrins of the |31 or |32 sub-family. Adhesion of macrophage cells was intensively inhibited by infection with Leishmania and was not affected by infection with Mycobacterium or by endocytosis of latex beads. Thus, the developed assay was able to show the specific adhesion of macrophages to connective matrix, as well to illustrate a specific downregulation of macrophage adhesion to inflammed skin in Leishmania infection.

Chronic peritonitis contributes to technique failure in peritoneal dialysis (PD), an effective replacement therapy for chronic kidney failure. Staphylococcus aureus infection is one of the most common causes of peritonitis in PD. Interestingly, mice deficient in the cell surface heparan sulfate proteoglycan, syndecan-1, were reported to clear S. aureus corneal infection more effectively than wild-type mice. The objectives of this study were to examine the protein expression and role of syndecan-1 in leukocyte recruitment, chemokine presentation and S. aureus infection in the microcirculation underlying the parietal peritoneum in wild-type and syndecan-1-/- mice. Immunofluorescence intravital microscopy (IVM) of the parietal peritoneum microcirculation revealed that syndecan-1 was localized to the subendothelial region of venules and the mesothelial layer but does not regulate leukocyte recruitment and is not necessary for presentation of the chemokine MIP-2 in peritoneal venules. IVM was also used to study the effects of a conventional PD solution injected through a peritoneal catheter in a mouse PD model. After 6 weeks of dialysis, the peritoneal catheter implant increased leukocyte rolling and extravasation, fibrosis and angiogenesis in the parietal peritoneum independently from the dialysis solution treatment. Furthermore, the role of syndecan-1 was examined using a 4 week PD model. Four hours after infection with S. aureus through the dialysis catheter or intraperitoneal injection, the dialyzed syndecan-1-/- mice were more susceptible to S. aureus infection than undialyzed syndecan-1-/- controls and wild-type animals. IVM showed that in S. aureus infection, syndecan-1 was removed from the subendothelial surface of peritoneal venules but syndecan-1 deficiency did not affect leukocyte recruitment during S. aureus infection. This study indicates that syndecan-1 in the peritoneum and microcirculation is not a regulator of inflammatory responses but is crucial for providing a barrier to S. aureus infection, which may have important implications for susceptibility to S. aureus infections in PD. / Dissertation / Doctor of Philosophy (Medical Science)

Damage to the endothelium is an important contributor to the initiation and progression of atherosclerosis. GRP78 is an endoplasmic reticulum (ER)-resident molecular chaperone in normal healthy endothelium that functions to assist in the correct folding of newly synthesized proteins and to prevent the aggregation of folding intermediates. In addition, GRP78 is present as a transmembrane protein on the surface of lesion-resident endothelial cells. Surface GRP78 is known to act as a surface signaling receptor in cancer cells and is activated by anti-GRP78 autoantibodies (GRP78a-Abs) isolated from the serum of cancer patients. However, the role of cell surface GRP78 on endothelial cells and the influence of GRP78a-Abs in atherosclerosis is unknown. The objectives of this study were to investigate the effects of GRP78a-Abs on lesion development, examine whether engagement of cell surface GRP78 by GRP78a-Abs modulates endothelial cell function, and determine whether GRP78a-Abs were associated with cardiovascular disease (CVD) in humans. This research showed that ApoE-/- mice with advanced atherosclerotic lesions have elevated serum levels of GRP78a-Abs and ApoE-/- mice immunized against recombinant GRP78 demonstrated a significant increase in GRP78a-Abs titers as well as accelerated lesion growth. Furthermore, this work demonstrated that activation of surface GRP78 on endothelial cells by GRP78a-Abs significantly increases gene expression of adhesion molecules ICAM-1 and VCAM-1 as well as leukocyte adhesion through the NFκB pathway. Additionally, middle-aged to elderly adults at risk for CVD showed a tendency toward elevated circulating GRP78a-Ab levels. Our results suggest that signaling through cell surface GRP78 can activate intracellular pathways that contribute to endothelial cell activation and augment atherosclerotic lesion development. These findings demonstrate a novel role for GRP78a-Abs and surface GRP78 receptor activity in endothelial cell function and the early stages of lesion development, as well as establish an initial framework for future work involving circulating GRP78a-Abs and atherosclerotic disease in humans. Furthermore, this work indicates inhibiting the interaction of GRP78a-Abs with cell surface GRP78 could present a novel therapeutic strategy to modulate lesion growth, thereby reducing the risk for atherosclerosis and cardiovascular disease. / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

No / Our objectives were to obtain monoclonal anti-endothelial cell antibodies (AECA) from systemic lupus erythematosus (SLE) patients, to characterize their antigen specificity, and their capability to induce a pro-inflammatory and pro-adhesive endothelial phenotype, and to investigate the mechanism of endothelial cell (EC) activation in vitro. Monoclonal IgG AECA were generated by hybridoma formation with human SLE B cells. Antigen specificity was characterized by immunoblotting with enriched cell membrane fractions, by cytofluorimetry and by cell solid-phase ELISA. Endothelial activation was evaluated by measuring increases in U937 cell adhesiveness, adhesion molecule (E-selectin and ICAM-1) expression and IL-6 production. In addition, mechanisms of endothelial activation were investigated by assessment of NF-B by measuring the loss of its inhibitor I-B. mAb E-3 bound live EC and recognized a 42 kDa EC membrane protein, it enhanced U937 adhesiveness, E-selectin and ICAM-1 expression and IL-6 production, and caused the loss of I-B. We conclude this is the first in vitro demonstration that a human monoclonal AECA from a SLE patient reacts with a constitutive endothelial membrane antigen and induces a pro-inflammatory endothelial phenotype through NF-B activation.

The synapse, the minimal element required for interneuronal communication in the nervous sytems, is a structure with a great deal of plasticity, capable of undergoing changes that alter transmission strength, and even forming new connections. This property has great implications for a number of processes, including circuit formation and learning and memory. However, the proteins behind this synaptic plasticity are still not fully understood. To uncover and characterize the proteins that regulate the plastic nature of the synapse, I turned to the Drosophilalarval neuromuscular junction (NMJ), a powerful and accessible model system. I began by examining synaptic cell adhesion, as Cell Adhesion Molecules (CAMs) have long been implicated in synaptic outgrowth as well as learning and memory. CAMs have traditionally been thought of as molecules that mediate cell adhesion between the pre- and postsynaptic membrane. However, through the course of the studies presented here I demonstrate a CAM function that goes beyond simple cell adhesion, acting as a receptor that transduces adhesive signals to the intracellular space. In particular, I have demonstrated a role for the Drosophila CAM, Fasciclin II(FasII), in a signaling complex involving the Amyloid Precursor Protein-Like (APPL) and the Drosophila homolog of X11/MINT/Lin-10 (dX11). Further results show that deletion of either APPL or dX11 inhibits the FasII mediated outgrowth. These studies show that during NMJ expansion the transinteraction between FasII molecules in the pre- and postsynaptic membrane results in the recruitment of APPL and dX11 to the presynaptic cell surface, and the initiation of a signaling cascade that leads to bouton outgrowth. The next question addressed here was regarding the cytoskeletal changes that must occur during synapse remodeling. In particular I centered on the evolutionarily conserved cell polarity complex aPKC-Par3-Par6, which is know to regulate axon growth, the cell cytoskeleton during polarized cell division, and learning and memory. To understand the role of the cytoskeleton during NMJ expansion, I examined the organization of microtubules and actin during this process. Further, I identified atypical protein kinase C (aPKC) as a regulator of microtubule dynamics. I found that aPKC is required for regulating the degree of stabilization of synaptic microtubules. This stabilization requires the Microtubule Associated Protein-1B (MAP1B) homolog Futsch, which I demonstrated was required for aPKC to associate with and stabilize the microtubule cytoskeleton. The process of synaptic expansion not only requires modifications to the presynapse, but to the postsynapse as well. Previous work demonstrates that levels of the scaffolding proteins DrosophilaMembrane Associated Guanlyate Kinase (MAGUK) protein Discs-large (DLG), as well as the vertebrate homolog Postsynaptic Density-95 (PSD-95), which are concentrated at synapses, determine the size of postsynaptic membranes. To identify the underlying mechanisms of the regulation of postsynaptic size, we performed a yeast two hybrid screen, searching for DLG interacting proteins. We found a novel interaction between DLG, and a t-SNARE, GUK-interacting Syntaxin (Gtaxin; GTX), and went on to demonstrate that this interaction is required for proper postsynaptic membrane addition. Strong hypomorphic mutations in either dlg or gtx show a dramatic reduction in postsynaptic expansion. Overexpression of DLG produces an increase of synaptic GTX, as well as an increase in postsynaptic size, and an increased formation of GTX positive SNARE complexes. Taken together, these observations suggest that the MAGUK DLG regulates postsynaptic membrane addition by modulating the formation of a SNARE complex of the t-SNARE Gtaxin, and by targeting GTX to sites of postsynaptic membrane addition. In summary, the studies performed in this thesis probe a trans-synaptic adhesion based signaling complex required for presynaptic expansion, a specific pathway for dynamic microtubule stabilization required for pre- and postsynaptic expansion, and how a scaffolding protein regulates postsynaptic membrane expansion. These processes are all interconnected to maintain the efficacy of the synapse. The studies conducted revealed important information about how these processes are accomplished, and constitute an important step to elucidate the mechanisms by which synapse plasticity occurs at the level of single synaptic terminals.

Within the last few decades, epidemiological evidence has linked exposure to air pollution, both its particles and its organic components, with cardiovascular disease (CVD) progression. CVD is a life long disease with the disruption of the endothelium being the inaugural event in this inflammatory process. The vascular endothelium is extremely susceptible to environmental insults given its tremendous surface area and that it is in constant contact with blood and components circulating within the blood, including xenobiotics. The endothelium is important as a barrier from blood constituents however, dysfunction of this barrier leads to the influx of lymphocytes and granulocytes that lead to the fatty build‐up characteristic of atherosclerosis. The studies presented in this dissertation tested the hypothesis that two unique environmental contaminants, alumina nanoparticles and benzo[a]pyrene (B[a]P), lead to increased endothelial cell dysfunction, characterized by increased adhesion molecule expression. Alumina nanoparticles induced vascular cell adhesion molecule‐1 (VCAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and E‐selectin (ELAM‐1), as well as increased monocyte adhesion to activated endothelium. Polystyrene nanoparticles did not elicit this response. B[a]P induced ICAM‐1 expression, but only after toxification by aryl hydrocarbon receptor (AhR) controlled enzymes. Silencing of either AhR or the membrane microdomains called caveolae attenuated the B[a]P‐induced ICAM‐1 response. It was also shown that the induction of ICAM‐1 occurred by signaling through MEK, p‐38 MAPK, and activator protein‐1 (AP‐1). These data provide a novel mechanism by which air pollutants like B[a]P may cause increased atherosclerosis and describe a new toxicant, alumina nanoparticles, as a possible threat for the development of inflammatory diseases, such as atherosclerosis. Little is known about dietary interventions capable of alleviating xenobiotic‐induced toxicity. Nutrition is an obtainable and inexpensive means of possible preventative therapy. With this in mind, it was also hypothesized that plant polyphenols, such as flavonoids, can down‐regulate B[a]P‐induced ICAM‐1. Selective flavonoids, containing both a 4’ B‐ring hydroxyl substitution and a 2‐3 C‐ring double bond, protected against B[a]P‐induced ICAM‐1 activation, however this protection did not correlate with the flavonoid’s antioxidant capacity.