Influenza viruses cause significant mortality and morbidity worldwide due to seasonal oubreaks as well as occassional, and sometimes devastating, pandemics. Estimates state that approximately 5% of the adult and 20% of the child population is infected yearly, leading to approximately a half-million deaths and three million severe infections in non-pandemic years. Aside from globally-circulating strains, zoonotic outbreaks caused by avian strains are a constant threat. In 1997, the first human cases of H5N1 infections occurred and since then strains of this subtype have killed approximately 700 people causing a severe disease with as high as 60% lethality rate. In March 2013, a strain of the H7N9 subtype started an epizootic in China causing a severe respiratory disease reminiscent of H5N1 infections and with a 20% case fatality rate. In this thesis, we have studied the host responses as well the viral replication kinetics of H5N1 and H7N9 strains and compared then to those of mild H1N1 seasonal and 2009 pandemic strains. During early infections of A549 cells, we have shown that the H5N1 virus induced a more profound and functional change to the host proteome. All viruses induced the NRF2-mediated oxidative stress responses and the H7N9 and H5N1 strains downregulated fibronectin, a host protein vital to infection for human strains. Using mathematical modeling and extensive growth kinetic analysis, we showed that the H5N1 and H7N9 strains had higher peak titers and faster growth kinetics. This was due to an higher infection rate for the H7N9 strain and an higher production rate for the H5N1 strain, compared to the human viruses. Conversely, the 2009 pandemic H1N1 strain had the poorest replication kinetics, longest eclipse phase and lowest infection rates. These results point towards the higher level of cellular disruption during infection with highly pathogenic strains of influenza, which may be indicative of the more profound changes required to support growth of viruses with faster kinetics to higher titers. Furthermore, the greater changes in the cellular proteome that we have characterized in vitro may be connected to the significantly greater virulence associated with infection by avian viruses in vivo, opening a novel and productive avenue of investigation to understand viral virulence mechanisms. / October 2015

Induction of an endogenous antibody response by therapeutic vaccination could provide an alternative to cost-intensive monoclonal antibody-based treatments for cancer. Since the target of a cancer vaccine will most likely be a self-antigen, self-tolerance of the immune system must be circumvented. Using fusion proteins consisting of the self-antigen to be targeted and a part derived from a foreign antigen, it is possible to break tolerance against the self-antigen. Furthermore, a potent adjuvant is required to support an immune response against a self-molecule. Currently no adjuvant suitable for this purpose is approved for use in humans. This thesis describes the development of a therapeutic vaccine targeting the vasculature of tumors. As tumor cells have developed strategies to escape immune surveillance, targeting of molecules associated with the tumor stroma is an interesting alternative. The alternatively spliced extra domain-A and B (ED-A and ED-B) of fibronectin and the glycan-binding protein galectin-1 are selectively expressed during events of tumor angiogenesis. We have designed recombinant proteins to target ED-B, ED-A and galectin-1, containing bacterial thioredoxin (TRX) as a non-self part, resulting in TRX-EDB, TRX-EDA and TRX-Gal-1. Vaccination against ED-B induced anti-ED-B antibodies and inhibited growth of subcutaneous fibrosarcoma. Immunization against ED-A decreased tumor burden and reduced the number of lung metastases in the MMTV-PyMT model for metastatic mammary carcinoma in a therapeutic setting. Analysis of the tumor tissue from ED-B and ED-A-immunized mice indicated an attack of the tumor vasculature by the immune system. Finally, we show that galectin-1 immunization reduced tumor burden and increased leukocyte numbers in the tumor tissue. Galectin-1 is pro-angiogenic and immunosuppressive, and therefore allows simultaneous targeting of fundamental characteristics of tumorigenesis. We furthermore show that the biodegradable squalene-based Montanide ISA 720 combined with CpG oligo 1826 (M720/CpG) is at least as potent as Freund’s adjuvant with respect to breaking self-tolerance, when comparing several immunological parameters. Freund’s is a potent but toxic adjuvant used in the majority of preclinical studies. The work presented in this thesis shows that therapeutic cancer vaccines targeting the tumor vasculature are a feasible and promising approach for cancer therapy.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [134]-154).

The composition and conformational state of biological molecules have a profound influence on cell behavior and large-scale processes including development and disease progression. Fibronectin fibers are a prevalent component of the extracellular matrix that are believed to adopt a wide array conformations with different functions. Two factors that are hypothesized to regulate fibronectin conformation, and hence fibronectin biological function, are allosteric regulators, such as heparan sulfates, and mechanical strain. However, the relative influence of allosteric regulators and mechanical forces on fibronectin conformation has not been determined. This conformational regulation is especially important in the context of the heparin 2 binding domain (modules III12 to III14), which is known to bind and present numerous growth factors, such as vascular endothelial growth factor, to cells. This thesis will highlight three contributions to this field. First, a new, and remarkably simple technique was developed that permits the detection of the non-equilibrium fibronectin conformations. This technique is founded on the identification of monoclonal antibodies that have altered affinities for fibronectin based on heparin treatment or mechanical strain dependence, or that bind fibronectin equally well in all conditions. Second, the impact of both heparin and mechanical strain on the binding of VEGF to the hep2 region of fibronectin was investigated. It was discovered that both strain and heparin co-regulate VEGF binding. Finally, studies of cell attachment and migration on single fibers of fibronectin with controlled strain states provided the first direct evidence that mechanical strain regulates cell attachment, spreading, and migration on a fibronectin matrix. This body of work demonstrating that the conformational changes in fibronectin lead to altered biological activity has broad impact in a number of fields due to the ubiquitous presence and requirement of fibronectin in cell and tissue function.

Orientador: Maria José Soares Mendes Giannini / Banca: Alexandre Melo Bailão / Banca: Christiane Pienna Soares / Resumo: Paracoccidioides brasiliensis (Pb) é o agente etiológico da paracoccidioidomicose, micose sistêmica de grande importância no Brasil, país que possui a maior concentração de áreas endêmicas para essa doença no mundo. Uma das estratégias possivelmente utilizadas pelo patógeno seria a expressão de genes envolvendo adaptação às condições do hospedeiro, que pode também estar relacionadas à captação de micronutrientes. A matriz extracelular (MEC) desempenha um papel importante na regulação da adesão celular, diferenciação, migração e proliferação das células. Este estudo propõe uma análise de transcritos e de proteínas expressas em condições de depleção de cobre, na presença de quatro matrizes extracelulares - laminina, fibronectina e colágeno I e IV, mimetizando as condições de infecção por P. brasiliensis por meio das técnicas de RDA (Representational Difference Analysis) e eletroforese bidimensional. Para isso, o isolado Pb01 foi cultivado por 3 horas no meio quimicamente definido (MVM), depletado de cobre (Cu) e a seguir colocado em contato com os quatro diferentes componentes da MEC e a adesão foi avaliada por citometria de fluxo. Um aumento significativo (p ≤ 0,05) na adesão frente a todos os componentes da MEC foi observado quando o fungo foi cultivado sem Cu. Então, o RNA e os extratos protéicos foram obtidos de Pb sem Cu e Pb sem Cu em contato com os diferentes componentes da MEC. Ensaios de RDA foram realizados para demonstrar os genes envolvidos neste processo. Duas hibridizações foram realizadas nas proporções de 1:10 e 1:100 de tester e driver, respectivamente, com um excesso de driver para remover as seqüências comuns em ambas condições. Os produtos diferencialmente expressos foram amplificados, resultando em padrões distintos que foram seqüenciados... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioides brasiliensis (Pb) is the etiologic agent of paracoccidioidomycosis, a systemic mycosis of great importance in Brazil, which has the highest concentration of endemic areas for this disease in the world. One of the strategies used by the pathogen may be the expression of proteins related to the adaptation to the host conditions, which may also be related to the uptake of micronutrients. Extracellular matrix (ECM) plays an important role in the regulation of cell adhesion, differentiation, migration and proliferation of cells. This study proposes an analysis of transcripts and proteins expressed in condition of copper depletion in the presence of four components of extracellular matrix - laminin, fibronectin and collagen I and IV, mimicking the conditions of infection by P. brasiliensis, using techniques of RDA (Representational Difference Analysis) and two-dimensional electrophoresis. For this, we cultured the Pb 01 strain at 3 hours in a chemically defined media (MVM) with depletion copper (Cu). After, the fungus was placed at contact with the four differents ECM components and the adhesion was evaluated by flow cytometry. A significant increase of binding (p ≤ 0,05) to all ECM components was observed when the fungal was cultured without Cu. So RNA and protein extracts were obtained of Pb without Cu, and Pb without Cu in contact with the differents ECM components. RDA assay was developed to demonstrate the genes involved in this process. Two hybridizations were performed in the proportions of 1:10 and 1:100 of tester and driver respectively, with an excess of driver to remove the common sequences in both conditions. The differentially expressed products were amplified, resulting in distinct patterns that were sequenced revealing genes involved in differents process like virulence (25%), protein synthesis... (Complete abstract click electronic access below) / Mestre

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Athymic nude mouse models have been extensively used to study biological behaviour of normal and diseased human tissues. In such models, immune-deficient mice act as hosts for cysts constructed from human material. A unique biocyst model that entails transplantation of human vaginal cysts into athymic nude mice has been implemented to study diseases of oral mucosa. To date, only one immunohistochemical study of this biocyst model has been reported. Nevertheless, conclusions made in that study were only based on the observed expression patterns of human and murine markers. Statistical assessment of immunohistochemical data had been omitted by the investigator. Therefore, the objective of this study was to further delineate the immunohistochemical profile of normal human vaginal tissue and human vaginal tissue that had been xenografted into nude mice.Experimental cysts constructed from human vaginal mucosa were xenografted into athymic nude mice and harvested 9-weeks post transplantation. Immunohistochemical analysis of normal human vaginal tissue and human vaginal tissue that had been xenografted into nude mice was performed using a panel of human and murine markers. Expression patterns of human and murine markers were assessed. Human markers included cytokeratin 1,cytokeratin 5, cytokeratin 13, cytokeratin 14, collagen type IV, laminin, elastin, fibronectin,Langerhans cells and VEGFR-3. Murine markers included collagen type IV, laminin,fibronectin, Langerhans cells and VEGFR-2. Staining intensities were quantified and statistically analysed using one-way ANOVA with subsequent Friedman’s test for multiple comparisons. Since the sample size was small, the power of the test statistic was enhanced by including Dunn’s post-test for further multiple comparisons. A strong positive expression of all cytokeratins was detected in both normal and xenografted vaginal tissues. Human markers that exhibited weak to moderate positive expression were collagen IV, laminin, fibronectin and VEGFR-3. Human elastin and human Langerhans cells exhibited strong and varying expression patterns respectively. Weak expression patterns for all murine markers were reported, with an exception of VEGFR-2 which was negatively expressed in all xenografted vaginal tissues. Significant differences (P<0.05) in the mean staining intensities between normal and xenografted vaginal tissues were reported for cytokeratin 1, fibronectin and Langerhans cells. There were no statistical differences (P>0.05) in the mean staining intensities for other markers.In conclusion, immunohistochemical studies proved that human vaginal tissue could not only survive in nude mice, but could also become active and develop structures necessary for survival, in this case, a newly formed stromal layer. The epithelium and stromal layer exhibited a human ecosystem.

Liver fat-storing cells (FSC) play an important role in collagen deposition. During the induction of liver cirrhosis, FSC lose their fat droplets, acquire an actin-rich cytoskeleton and transform into myofibroblasts. Myofibroblasts have been associated with increased collagen production in cirrhotic livers. Cultured FSC resemble myofibroblasts. However, it is not known whether regulation of collagen gene expression is similar in FSC obtained from normal or cirrhotic livers. In this communication, we describe the characterization of two fat-storing cell lines, one from normal (NFSC) and one from CCl4-cirrhotic liver (CFSC), obtained after spontaneous immortalization in culture. We studied the effect of serum and various growth factors on cell proliferation. We determined the production of collagen and fibronectin and we analyzed the presence of mRNA transcripts of collagens type I, III, and IV, fibronectin laminin, transforming growth factor-β and interleukin-6. We found that CFSC have a greater serum-dependency than NFSC. NFSC grow with a mixture of insulin and epidermal growth factor, whereas CFSC proliferate only with platelet-derived growth factor. Although we did not find significant differences in the expression of mRNAs for collagen type I, fibronectin and transforming growth factor-β, collagen and fibronectin synthesis was increased 2- and 1.5-fold respectively. NFSC contained 1.6- and 2.0-fold more type III collagen and laminin mRNAs, respectively, than CFSC. Neither cell line expressed type IV collagen mRNA. NFSC but not CFSC produced interleukin-6. These results suggest that, except for the lack of transcripts of collagen type IV, both cell lines resemble primary cultures of FSC. However, significant differences in cell proliferation and interleukin-6 production between the two cell lines were found. We suggest that these cell lines could be useful tools to study possible differences in regulation of matrix production by FSC.

No description available.

Expression studies have implicated Fibronectin Leucine Rich Transmembrane protein 2 (FLRT2) in cranial neural crest cell migration and pre-chondrogenic cell condensation during craniofacial skeletogenesis. This aim of this study was to characterize the expression of FLRT2 and its relationship to the extracellular matrix (ECM) in ATDC5 chondroprogenitor cells. Immunofluorescence studies localized FLRT2 to the cell membrane as well as exracellularly, where it colocalized with fibronectin. FLRT2 was identified in the ATDC5-derived ECM after cell extraction. Further to its colocalization with fibronectin, FLRT2 associated with fibronectin-coated beads in cell cultures. Co-immunoprecipitation confirmed that FLRT2 and fibronectin interact, either directly or indirectly. Blocking fibronectin fibril formation in ATDC5 cell cultures demonstrated a concomitant decrease in extracellular FLRT2 accumulation. It appears that FLRT2 may exist in both a membrane-bound and a shed form. Either or both of these forms may participate in cell-ECM interactions in cooperation with fibronectin or other ECM proteins.

Expression studies have implicated Fibronectin Leucine Rich Transmembrane protein 2 (FLRT2) in cranial neural crest cell migration and pre-chondrogenic cell condensation during craniofacial skeletogenesis. This aim of this study was to characterize the expression of FLRT2 and its relationship to the extracellular matrix (ECM) in ATDC5 chondroprogenitor cells. Immunofluorescence studies localized FLRT2 to the cell membrane as well as exracellularly, where it colocalized with fibronectin. FLRT2 was identified in the ATDC5-derived ECM after cell extraction. Further to its colocalization with fibronectin, FLRT2 associated with fibronectin-coated beads in cell cultures. Co-immunoprecipitation confirmed that FLRT2 and fibronectin interact, either directly or indirectly. Blocking fibronectin fibril formation in ATDC5 cell cultures demonstrated a concomitant decrease in extracellular FLRT2 accumulation. It appears that FLRT2 may exist in both a membrane-bound and a shed form. Either or both of these forms may participate in cell-ECM interactions in cooperation with fibronectin or other ECM proteins.