The goal of this thesis was to explore the effects of dose resolution, respiratory variation and dose calculation method on dose accuracy. To achieve this, two models of lung were created. The first model, called TISSUE, approximated the connective alveolar tissues of the lung. The second model, called BRANCH, approximated the lungs bronchial, arterial and venous branching networks. Both models were varied to represent the full inhalation, full exhalation and midbreath phases of the respiration cycle.<p> To explore the effects of dose resolution and respiratory variation on dose accuracy, each model was converted into a CT dataset and imported into a Monte Carlo simulation. The resulting dose distributions were compared and contrasted against dose distributions from Monte Carlo simulations which included the explicit model geometries. It was concluded that, regardless of respiratory phase, the exclusion of the connective tissue structures in the CT representation did not significantly effect the accuracy of dose calculations. However, the exclusion of the BRANCH structures resulted in dose underestimations as high as 14\% local to the branching structures. As lung density decreased, the overall dose accuracy marginally decreased.<p> To explore the effects of dose calculation method on dose accuracy, CT representations of the lung models were imported into the Pinnacle$^3$ treatment planning system. Dose distributions were calculated using the collapsed cone convolution method and compared to those derived using the Monte Carlo method. For both lung models, it was concluded that the accuracy of the collapsed cone algorithm decreased with decreasing density. At full inhalation lung density, the collapsed cone algorithm underestimated dose by as much as 15\%. Also, the accuracy of the CCC method decreased with decreasing field size.<p> Further work is needed to determine the source of the discrepancy.

Glucocorticoid (GC) signalling via the GC receptor (GR) regulates many aspects of lung development. To determine the need for epithelial GC-GR signalling, triple transgenic (TT) mice with doxycycline (dox) – inducible suppression of GR exclusively in the distal lung epithelium (DLE) were created. Following exposure to dox, E18.5 TT fetuses showed a reduction in GR mRNA levels and elimination of GR protein expression exclusively in the DLE. Newborn TT pups had decreased viability and TT fetal lungs had increased tissue to airspace ratios, decreased levels of proximal epithelial protein CC10, of all surfactant proteins (ATII cell proteins), of ion conductance channels β and γENaC, of water channel AQP5, and of ATI cell protein T1α. Thus DLE GC-GR signalling is important for neonatal viability and increased mortality of TT pups could be due to impaired epithelial differentiation, leading to decreased surfactant protein expression, delayed fluid clearance and/or increased lung cellularity.

A growing demand for information on the human health and environmental effects of materials produced using nanotechnology has led to a new area of investigation known as nanotoxicology. Research in this field has widespread implications in facilitating the medical applications of nanomaterials but also in addressing occupational and environmental toxicity concerns. Improving our understanding of these issues also has broad appeal in the stewardship of nanotechnology and its acceptance by the public. This work represents some of the early research in burgeoning field of nanotoxicology. Using a variety of in vivo and in vitro models, as well as cellular and molecular techniques I first studied a possible role for the novel cytokine endothelial monocyte activating polypeptide-II (EMAP-II) in acute lung inflammation in rats (Chapter 2). This work demonstrated a significant increase in total EMAP-II concentration in lipopolysaccharide inflamed lungs as early as 1h post-treatment (P<0.05). Increased numbers of monocytes and granulocytes were also observed in lungs treated with mature EMAP-II relative to control rats (P<0.05), and the recruitment of cells did not occur via upregulation of either Interleukin-1β or Macrophage inflammatory protein-2. I further studied whether mature EMAP-II can be induced in pulmonary nanotoxicity studies by exposure to rosette nanotubes (RNT) (Chapters 3-5). In the first in vivo experiments in mice on the RNT(1)-G0 (Chapter 3) I showed an acute inflammatory response at the 50 µg dose by 24h, but this response was resolving by 7d post-exposure as evidenced by a decreased number of cells in the bronchoalveolar lavage fluid (P<0.05) and from histological examination. The results of this study indicated that water soluble and metal-free rosette nanotubes can demonstrate a favorable acute pulmonary toxicity profile in mice. Subsequently, I studied the responses of the pulmonary epithelium using the human Calu-3 cell line (Chapter 4). This experiment indicated that RNT(2)-K1 neither reduces cell viability at 1 or 5 µg/ml doses nor does it induce a dose-dependent inflammatory cytokine response in pulmonary epithelial cells in vitro. My final experiment (Chapter 5) studied the human U937 pulmonary macrophage cell line since the macrophage is one of the key defense mechanisms to encounter RNT in the lung environment. The data indicate that this cell line lacks a robust inflammatory response upon exposure to RNT and that when RNT length is changed by altering the conditions of nanotube self-assembly, cytokine release into the supernatant is not affected profoundly. Although, EMAP-II is upregulated in a lipopolysaccharide model of lung inflammation, it does not serve as a good marker of RNT exposure. The data indicate that RNT have a favourable toxicity profile and these experiments provide a framework upon which rosette nanotubes can be investigated for a range of biomedical applications. Furthermore, in light of media and scientific reports of nanomaterials showing signs of toxicity, this work demonstrates that a biologically inspired nanostructure such as the RNT can be introduced to physiological environments without acute toxicity.

Glucocorticoid (GC) signalling via the GC receptor (GR) regulates many aspects of lung development. To determine the need for epithelial GC-GR signalling, triple transgenic (TT) mice with doxycycline (dox) – inducible suppression of GR exclusively in the distal lung epithelium (DLE) were created. Following exposure to dox, E18.5 TT fetuses showed a reduction in GR mRNA levels and elimination of GR protein expression exclusively in the DLE. Newborn TT pups had decreased viability and TT fetal lungs had increased tissue to airspace ratios, decreased levels of proximal epithelial protein CC10, of all surfactant proteins (ATII cell proteins), of ion conductance channels β and γENaC, of water channel AQP5, and of ATI cell protein T1α. Thus DLE GC-GR signalling is important for neonatal viability and increased mortality of TT pups could be due to impaired epithelial differentiation, leading to decreased surfactant protein expression, delayed fluid clearance and/or increased lung cellularity.

A growing demand for information on the human health and environmental effects of materials produced using nanotechnology has led to a new area of investigation known as nanotoxicology. Research in this field has widespread implications in facilitating the medical applications of nanomaterials but also in addressing occupational and environmental toxicity concerns. Improving our understanding of these issues also has broad appeal in the stewardship of nanotechnology and its acceptance by the public. This work represents some of the early research in burgeoning field of nanotoxicology. Using a variety of in vivo and in vitro models, as well as cellular and molecular techniques I first studied a possible role for the novel cytokine endothelial monocyte activating polypeptide-II (EMAP-II) in acute lung inflammation in rats (Chapter 2). This work demonstrated a significant increase in total EMAP-II concentration in lipopolysaccharide inflamed lungs as early as 1h post-treatment (P<0.05). Increased numbers of monocytes and granulocytes were also observed in lungs treated with mature EMAP-II relative to control rats (P<0.05), and the recruitment of cells did not occur via upregulation of either Interleukin-1β or Macrophage inflammatory protein-2. I further studied whether mature EMAP-II can be induced in pulmonary nanotoxicity studies by exposure to rosette nanotubes (RNT) (Chapters 3-5). In the first in vivo experiments in mice on the RNT(1)-G0 (Chapter 3) I showed an acute inflammatory response at the 50 µg dose by 24h, but this response was resolving by 7d post-exposure as evidenced by a decreased number of cells in the bronchoalveolar lavage fluid (P<0.05) and from histological examination. The results of this study indicated that water soluble and metal-free rosette nanotubes can demonstrate a favorable acute pulmonary toxicity profile in mice. Subsequently, I studied the responses of the pulmonary epithelium using the human Calu-3 cell line (Chapter 4). This experiment indicated that RNT(2)-K1 neither reduces cell viability at 1 or 5 µg/ml doses nor does it induce a dose-dependent inflammatory cytokine response in pulmonary epithelial cells in vitro. My final experiment (Chapter 5) studied the human U937 pulmonary macrophage cell line since the macrophage is one of the key defense mechanisms to encounter RNT in the lung environment. The data indicate that this cell line lacks a robust inflammatory response upon exposure to RNT and that when RNT length is changed by altering the conditions of nanotube self-assembly, cytokine release into the supernatant is not affected profoundly. Although, EMAP-II is upregulated in a lipopolysaccharide model of lung inflammation, it does not serve as a good marker of RNT exposure. The data indicate that RNT have a favourable toxicity profile and these experiments provide a framework upon which rosette nanotubes can be investigated for a range of biomedical applications. Furthermore, in light of media and scientific reports of nanomaterials showing signs of toxicity, this work demonstrates that a biologically inspired nanostructure such as the RNT can be introduced to physiological environments without acute toxicity.

The goal of this thesis was to explore the effects of dose resolution, respiratory variation and dose calculation method on dose accuracy. To achieve this, two models of lung were created. The first model, called TISSUE, approximated the connective alveolar tissues of the lung. The second model, called BRANCH, approximated the lungs bronchial, arterial and venous branching networks. Both models were varied to represent the full inhalation, full exhalation and midbreath phases of the respiration cycle.<p> To explore the effects of dose resolution and respiratory variation on dose accuracy, each model was converted into a CT dataset and imported into a Monte Carlo simulation. The resulting dose distributions were compared and contrasted against dose distributions from Monte Carlo simulations which included the explicit model geometries. It was concluded that, regardless of respiratory phase, the exclusion of the connective tissue structures in the CT representation did not significantly effect the accuracy of dose calculations. However, the exclusion of the BRANCH structures resulted in dose underestimations as high as 14\% local to the branching structures. As lung density decreased, the overall dose accuracy marginally decreased.<p> To explore the effects of dose calculation method on dose accuracy, CT representations of the lung models were imported into the Pinnacle$^3$ treatment planning system. Dose distributions were calculated using the collapsed cone convolution method and compared to those derived using the Monte Carlo method. For both lung models, it was concluded that the accuracy of the collapsed cone algorithm decreased with decreasing density. At full inhalation lung density, the collapsed cone algorithm underestimated dose by as much as 15\%. Also, the accuracy of the CCC method decreased with decreasing field size.<p> Further work is needed to determine the source of the discrepancy.

Die vorliegende Arbeit beschäftigte sich mit der Frage, inwieweit zytologische Untersuchungsmaterialien von Patienten mit nichtkleinzelligen Lungenkarzinomen geeignet sind, zur molekularbiologischen Analyse möglicher Mutationen des k-ras-Genes verwendet zu werden. Dafür wurden vier spezifische Polymerasekettenreaktionen mit anschließenden Restriktionsfragmentlängenpolymorphismus-Analysen kreiert und optimiert. In der Folge wurden die k-ras-Mutationen in den verschiedenen Codons nach einer Agarose-Gelelektrophorese unter der UV-Kamera detektiert. Es wurden vergleichende Untersuchungen mit Materialien tumorfreien Gewebes durchgeführt. Ebenso wurden, um die Eignung des zytologischen Materiales zur k-ras-Mutationsdiagnostik nachzuweisen, Mutationsanalysen in histologischem Material durchgeführt und dieses mit den zytologischen Proben derselben Patienten verglichen. Weiterhin wurden bestimmte Korrelationen des k-ras-Mutationsstatus der Patienten mit individuellen und klinischen Parametern analysiert und mit aus der Literatur bekannten Aussagen verglichen. Es ist bekannt, dass k-ras-Mutationen im Tumor bei Patienten mit fortgeschrittenen nichtkleinzelligen Lungenkarzinomen negative Prädiktoren für einen möglichen Erfolg einer Rezeptortyrosinkinaseinhibitor-Therapie darstellen und die Prognose der Erkrankung verschlechtern. Neuartige Therapiekonzepte scheinen aber auch eine Möglichkeit zu bieten, Patienten mit nachgewiesener k-ras-Mutation eine Therapiealternative ermöglichen zu können. Die vorliegende Arbeit liefert einen wichtigen Beitrag um eine einfache Methode zur Analyse von k-ras-Mutationen bei Patienten mit nichtkleinzelligen Lungenkarzinomen zu finden und damit eine Entscheidung über etwaige weitere Therapieoptionen zu erleichtern.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmaceutics. Title from title-page of electronic thesis. Bibliography: leaves 207-235.

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This thesis examines the challenges of decellularizing porcine lungs. The thesis identifies hydrostatic pressure and the role it plays in perfusate distribution within the lung as a potential roadblock for the decellularization of porcine lungs. The thesis examines methods for mitigating the affect of hydrostatic pressure on the decellularization of porcine lungs. Specifically, the thesis details the design and construction of a vessel that rotates lungs during decellularization. This rotating vessel is evaluated in comparison to two other, non-rotation based, decellularization methods. The rotation device is found to more extensively decellularize the lung than either of the other two options. The thesis concludes by examining what other areas of work could be conducted in this field.