Aortic valve disease is among the third most common cardiovascular disease worldwide, and is also a strong predictor for other cardiac related deaths. Altered mechanical forces are believed to cause changes in aortic valve biosynthetic activity, eventually leading to valve disease, however little is known about the cellular and molecular events involved in these processes. To gain a fundamental understanding into aortic valve disease mechanobiology, an ex vivo experimental model was used to study the effects of normal and elevated cyclic stretch on aortic valve remodeling and degenerative disease. The hypothesis of this proposal was that elevated cyclic stretch will result in increased expression of markers related to degenerative valve disease. Three aspects of aortic valve disease were studied: (i) Altered extracellular matrix remodeling; (ii) Aortic Valve Calcification; and (iii) Serotonin-induced valvulopathy. Results showed that elevated stretch resulted in increased matrix remodeling and calcification via a bone morphogenic protein-dependent pathway. In addition, elevated stretch and serotonin resulted in increased collagen biosynthesis and tissue stiffness via a serotonin-2A receptor-mediated pathway. This work adds to current knowledge on aortic valve disease mechanisms, and could pave the way for the development of novel treatments for valve disease and for the design of tissue engineered valve constructs.

Algae are photosynthetic microorganisms that convert carbon dioxide and sunlight into biomass that can be used for biofuel production. Although they are usually cultivated in suspension, these microorganisms are capable of forming productive biofilms over substrata given the right conditions. This dissertation focuses on algal biofilms and their application in biofuel feedstock production. In particular it reports the construction and performance of an algae biofilm photobioreactor, the physico-chemical surface properties of different algal species and adhesion substrata, and cell-surface interactions based on experimental results and theoretical models. A novel algae biofilm photobioreactor was constructed and operated (i) to demonstrate the proof of concept, (ii) to analyze the performance of the system, and (iii) to determine the key advantages and short comings for further research. The results indicated that significant reductions in water and energy requirements were possible with the biofilm photobioreactor. Although the system achieved net energy ratio of about 6, the overall productivity was low as Botryococcus branunii is notoriously slow growing algae. Thus, further studies were focused on identification of algal species capable of biofilm growth with larger biomass and lipid productivities. Adhesion of cells to substrata precedes the formation of all biofilms. A comprehensive study has been conducted to determine the interactions of a planktonic and a benthic algal species with hydrophilic and hydrophobic substrata. The physico-chemical surface properties of the algal cells and substrata were determined and using these data, cell-substrata interactions were modeled with the thermodynamic, Derjaguin, Landau Verwey, Overbeek (DLVO) and Extended Derjaguin, Landau, Verwey, Overbeek (XDLVO) approaches and critical parameters for algal adhesion were identified. Finally, the adhesion rate and strength of algal species were quantified with parallel plate flow chamber experiments. The results indicated that both cell and substrata surface hydrophobicity played a critical role for the adhesion rate and strength of the cells and XDLVO approach was the most accurate model. Finally, based on these findings the physico-chemical surface properties of ten algal species and six substrata were quantified and a screening was done to determine algae species substratum couples favoring adhesion and biofilm formation. / text

published_or_final_version / Zoology / Master / Master of Philosophy

Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus. Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form. We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, the active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells. Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction.

During the growth phase of oogenesis, mammalian oocytes increase several hundred-fold in volume. Although it is known that ovarian granulosa cells send growth promoting signals, neither these external signals nor the transduction pathways that become activated in the oocyte are known. Therefore, the presence and the activity of candidate signaling pathways in growing murine oocytes were investigated. By immunoblotting, the MAP kinases, ERK1 and ERK2, as well as their activating kinase MEK, were detected in oocytes at all stages of growth. However, using a phospho-specific anti-ERK antibody, no immunoreactive species were detectable in isolated granulosa cells or oocytes at any stage of growth, except metaphase II. Phosphorylated ERK was also present, although in smaller quantities, in oocyte-granulosa cell complexes at the later stages of growth. Furthermore, when ovarian sections were stained with an anti-ERK antibody, the protein was found to be highly concentrated in the cytoplasm of oocytes at all stages of growth, with lower levels in the nucleus. Another member of the MAP kinase family, Jun kinase (JNK), was investigated. By immunoblotting, JNK was detected in growing oocytes. Experiments using an anti-JNK antibody on ovary sections revealed the protein to be uniformly distributed in non-growing and growing oocytes with no evidence of preferential nuclear localization. These results imply that an interaction between the oocyte and the granulosa cells may be required to generate phosphorylated ERK. They also imply that growth signals probably are not relayed through ERK, but do not exclude a role for Jun kinase in mediating oocyte growth.

Bibliography: leaves 271-325. / xxxiii, 325, [249] leaves, [23] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The date presented in this thesis is directed toward the molecular characterisation of cell surface molecules (CSMs) that mediate interactions between human haemopoietic progenitor cells (HPC) and cells of the bone marrow (BM) stroma. The research focuses on the role of selectins in the regulation of haemopoiesis, the identification and molecular characterisation of novel structures expressed at the surface of primitive human HPC and cultured BM stromal cells, the molecular characterisation of the antigen identified by the mAb HCC-1 which delineates a subset of the CD34+ cell population, and the molecular cloning of a novel mucin-like transmembrane glycoprotein termed MGC -24v. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997?

Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001. / "December 2001." Includes bibliographical references (leaves 178-225).

Thesis (Ph. D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 97-146).

O potencial oncogênico do papilomavírus humano (HPV) baseia-se na capacidade das oncoproteínas virais E6 e E7 alterarem o ciclo celular, levando à imortalização e malignidade das células. O importante papel das oncoproteínas na progressão tumoral e na interação com inúmeros alvos celulares tem relevância em estudos para o desenvolvimento de vacinas e terapias contra os cânceres associados ao HPV. Este estudo investigou a localização intracelular das oncoproteínas E6 e E7 de HPV16/18 e seus possíveis alvos celulares. Demonstrou a presença de E6 nuclear, citoplasmática e intramitocondrial, tanto em células naturalmente transformadas por HPV, como em células transfectadas com o oncogene E6 viral. E7 foi detectada no núcleo e citoplasma, porém nunca ocorreu E7 intramitocondrial. Confirmou a hipótese da presença intramitocondrial da oncoproteína viral E6 de HPV16/18 de alto risco. Dado inédito cuja relevância está relacionada com a aplicação clínica, no desenvolvimento de imunobiológicos e fármacos capazes de neutralizar a ação deste importante alvo terapêutico. / The oncogenic potential of HPV is based on the capacity of viral oncoproteins E6 and E7 to change cellular cycle leading to immortality and malignancy. The important role of oncoproteins in tumor progression and its interaction with numerous cellular targets have relevance in studies to the development of vaccines and therapies against HPV associated cancers. This study investigated intracellular localization of E6 and E7 HPV16/18 oncoproteins and its possible cellular targets. It showed the presence of E6 in the cellular nucleus, cytoplasm, and intramitochondrial in naturally HPV transformed cell, as well as in cells transfected with E6 viral oncogene. E7 was detected inside nucleus and cytoplasm, but E7 intramitochondrial did not occur. This study confirmed the hypothesis of the intramitochondrial presence of E6 viral oncoprotein from high risk HPV. This is an original data whose relevance is directly related to clinical application in the development of immunobiologicals and drugs, which are able to neutralize the action of this important therapeutic target.

A sinalização no sistema nervoso pode ocorrer pelo acoplamento direto entre células, via canais de junção comunicantes (JCs). Estes canais permitem a passagem de moléculas de até 1 kDa, e são formados por subunidades proteicas denominadas conexinas (Cxs). A comunicação celular via JCs desempenha um importante papel durante o desenvolvimento e a sinalização visual. Além disso, o acoplamento celular provido pelas JCs tem sido relacionado a processos de sobrevivência/morte celular. Do mesmo modo, ciclinas e cinases dependentes de ciclinas (CDKs), além de seu papel clássico na regulação do ciclo e diferenciação celular, estão envolvidas em processos neurodegenerativos. Estudos recentes têm observado a reentrada no ciclo celular de células neuronais pós-mitóticas em apoptose. Neste contexto, analisamos a expressão gênica e proteica das Cxs e ciclinas em resposta às lesões no sistema visual, especificamente na retina. Estas análises foram realizadas após a indução de trauma mecânico, modelo experimental que permite a visualização do foco, penumbra e áreas adjacentes à lesão. Utilizando técnicas combinadas, como a reação em cadeia da polimerase em tempo real (PCR Real-Time), Western Blot e imuno-histoquímica, avaliamos a expressão espaço-temporal destes genes e as proteínas por eles codificadas, em diferentes tempos pós-lesão. Os resultados da PCR Real-Time revelaram uma ausência de modulação da expressão gênica de Cx36 para os diferentes tempos pós-lesão analisados. A Cx43 mostrou aumento dos transcritos, após três e sete dias pós-lesão. A ciclina D1 e B1 apresentaram modulação significativa após um, três e sete dias pós-lesão. As análises de imuno-histoquímica revelaram uma redistribuição da Cx36 em resposta à lesão em diferentes tempos pós-lesão. A Cx43, por sua vez, apresentou um aumento aparente de sua expressão no foco e zona de penumbra da lesão, nos período de um, três e sete dias pós-lesão. Em retinas, após um e três dias de lesão, a ciclina D1 encontrou-se presente em células próximas ao foco da lesão. Observamos a presença de ciclina B1 no foco da lesão após um dia de lesão. Por meio de análises de Western Blot não foi possível detectar alterações das diferentes proteínas estudadas nas retinas, em períodos variados de exposição à lesão. Os dados deste estudo sugerem que i) possivelmente, as células afetadas pela lesão se encontram acopladas; ii) expressam proteínas reguladoras do ciclo celular. Levando em consideração o conjunto de resultados, sugerimos que é possível induzir ou prevenir a reentrada do ciclo celular em células pós-mitóticas da retina, controlando o acoplamento mediado pelas proteínas formadoras das JC. / Communication in the nervous system can occur directly between the cells through structures known as gap junction (GJ) channels. These channels allow the passage of small molecules up to 1 kDa and are composed of protein subunits named connexins (Cxs). Cell communication through GJ plays an important role during the development and visual signaling. Furthermore, cell coupling provided by the GJs has been related to processes of survival/cell death. Similarly, in addition to the classic role of cyclins and cyclins dependent kinases (CDKs) in the cell cycle regulation and differentiation, they are also involved in neurodegenerative processes. Recent studies have demonstrated the reentry of apoptotic post mitotic neurons in the cell cycle. In this context, we analyzed the gene and protein expression of Cxs and cyclins after lesions in the visual system, specifically in the retina. For this purpose, a mechanic trauma was induced in the retina, which represents a model that allows us to visualize the lesion focus, penumbra and adjacent areas. Using combined techniques, such as the real time polymerase chain reaction (real-time PCR), Western blot and immunohistochemistry, we evaluated the spatio-temporal expression of these genes and their encoded proteins at different times post-lesion. The real-time PCR revealed no modulation of the Cx36 gene expression for all the times post-lesion analyzed. Our results showed increase in the Cx43 transcripts one, three and seven days post-lesion. The immunohistochemistry analysis indicated redistribution of Cx36 in response to the lesion in different times. On the other hand, Cx43 presented evident increased expression in the focus and penumbra areas of the lesion one, three and seven days post-lesion. In our experiments we could observe that cyclin D1 is expressed in cells located close to the lesion focus one and three days post-lesion, while cyclin B1 is expressed in these cells only one day post-lesion. The Western blot analysis did not show changes on the protein levels evaluated in this study in any of the post-lesion times analyzed. Data obtained from this study suggest i) the cells affected by the lesion are possibly coupled by GJ; ii) these cells express protein regulators of cell cycle. Altogether, the results indicate that it is possible to induce or prevent the reentry of post mitotic cells of the retina in the cell cycle, by controlling cell coupling provided by GJ.