Calreticulin (CRT) is a multifunctional Ca2+ dependent chaperone protein, which is localized to the endoplasmic reticulum and plays many important biological roles. In addition to its critical role in cardiovascular development, CRT has been reported to be important for cell migration, adhesion and apoptosis. A few studies have also suggested different roles for exogenous CRT in angiogenesis and tumor growth however no direct evidence for the role of endogenous CRT in these processes is available. To study the in vivo role of CRT in angiogenesis and vascular development, we generated a transgenic mouse overexpressing CRT under the control of the Tie2 promoter (referred to as Tie2-CRT) which is active in both endothelial cells and hematopoietic stem cells (HSCs). The main phenotype of these mice is an increased incidence of lung tumors. These tumors have been characterized according to their histochemical properties as being adenocarcinoma with a Surfactant Protein-C positive (SP-CPos) and Clara Cell Protein negative (CC10Neg) phenotype suggesting an alveolar origin for these tumors. We observed that during the early stages of tumor formation, the lungs show signs of increased inflammation as evidenced by congestion, reddish discoloration and the accumulation of inflammatory cells. We have also identified that the early stage tumors contain cells that express exogenous CRT and HSC markers including CD133, Sca-1, and c-Kit. As the tumor progresses to a fully developed adenocarcinoma, these cells lose the expression of exogenous CRT and HSCs markers and gain an alveolar type II phenotype (SP-CPos). In vitro evaluation of tumor progression using lung tumor cells from Tie2-CRT mice demonstrated a differentiation dependent expression of HSC markers by tumor cells supporting the hypothesis that HSCs might be the cells of origin for the lung tumors observed in Tie2-CRT mice. In summary, the results from this study provide evidence that lung tumors from the Tie2-CRT mice are non-epithelial in origin and that the undifferentiated population of tumor cells have HSC characteristics. After differentiation, these cells lose their stem cell phenotype and acquire an epithelial phenotype. This study is the first to examine the potential link between CRT and lung cancer development.

Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19-day gestational age rat fetuses. Equivalent short-circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The ISC showed a fast dose-dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na+ channel) activity in experiments with permeabilized apical or basolateral membrane. 5-(N-Ethyl-N-isopropyl)amiloride inhibition of ISC was not affected, however, benzamil-sensitive ISC was increased in insulin-stimulated monolayers. The application of LY-294002 and Akti1/2 both completely blocked the stimulating effect of insulin on ISC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF-1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF-1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells.

Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.

Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.

Small Cell Lung Cancer (SCLC) is a very aggressive neuroendocrine tumour of the lung, which demonstrates a 5 year survival of only 10% for extensive stage disease (20-30% for limited stage), with only modest improvement over the last few decades. Identification of new molecular diagnostic and therapeutic targets is thus imperative. Previous efforts in identifying molecular changes in SCLC by gene expression profiling using microarrays have facilitated disease classification but yielded very limited information on SCLC biology. Previous DNA studies have been successful in identifying several loci important to SCLC. However the low resolution of conventional chromosomal Comparative Genomic Hybridization (CGH) has limited the findings to large chromosomal regions with only a few specific candidate genes discovered to date. Thus, to further understand the biological behaviour of SCLC, better methods for studying the genomic alterations in SCLC are necessary. This thesis highlights the development of array CGH technology for the high resolution dissection of aneuploidy in cancer genomes and the application of this new technology to the study of SCLC. I present the development of the first whole genome CGH array which offered unprecedented resolution in the profiling of cancer genomes allowing fine mapping of genes in a single experiment. Through application of DNA based analysis in conjunction with integrated expression analysis and comparison of SCLC to less aggressive non-small cell lung tumours I have identified novel patterns of pathway disruption specific to SCLC. This included alteration to Wnt pathway members and striking patterns of cell cycle activation through predominantly downstream disruption of signalling pathways including direct activation of the E2F transcription factors, which are normally repressed by the Rb gene. Analysis of targets of the E2F/Rb pathway identified EZH2 as being specifically hyper-activated in SCLC, compared to NSCLC. EZH2 is a polycomb group gene involved in the control of many cellular functions including targeted DNA methylation and escape from senescence in hematopoietic stem cells. Taken together these results suggest that in SCLC, downstream disruption may replace multiple upstream alterations leading to activation independent of a specific mitogenic pathway, and that EZH2 represents a potentially important therapeutic target.

Obstructive sleep apnoea (OSA) is a common respiratory disorder characterised by repetitive periods of upper airway (UA) collapse during sleep. OSA is more common in males and the obese but the reasons why remain poorly understood. Abdominal obesity, particularly common in males, is likely to indirectly modulate the amount of tension (tracheal traction) exerted on the UA by the trachea and other intrathoracic structures, potentially leading to increased UA collapsibility. Other factors such as lung volume changes with obesity, altered drive to UA muscles and exaggerated arousal responses are also likely to contribute to UA instability. An investigation of these potential contributing factors forms the basis of this thesis. In the first study, the effect of external abdominal compression on UA collapsibility during sleep was investigated in a group of obese male OSA patients. A large pneumatic cuff wrapped around the abdomen was inflated to increase intra-abdominal pressure, aiming to produce an upward force on the diaphragm, designed to reduce axial tension on the UA. Abdominal compression increased end-expiratory gastric (PGA) and end-expiratory transdiaphragmatic (PDI) pressure by ~50% and produced a significant rise in UA collapsibility compared to the cuff deflated condition. These data support that increased intra-abdominal pressure has a negative effect on UA function during sleep. This effect may help explain why obesity is the leading risk factor for OSA and why OSA affects men more than women, given that abdominal obesity is particularly common in obese males. In the second study, differences in minimum expiratory (tonic) diaphragm activity during wakefulness were compared between 8 obese OSA patients and 8 healthyweight controls. Changes in tonic diaphragm activity and lung volume following sleep onset were also compared between the two groups. There was no evidence of increased tonic diaphragmatic activity during wakefulness in obese OSA patients to support significant diaphragmatic compensation for abdominal compressive effects of obesity. There were small decrements in lung volume following sleep onset in both groups (<70 ml), with significantly greater lung volume and diaphragmatic EMG decrements when sleep onsets were immediately followed by respiratory events. While lung volume decrements at sleep onset were relatively small, this does not discount that UA function is not more sensitive to effects of reduced lung volume in obese OSA patients. To more closely investigate the potential interactive effects of obesity on physiological variables likely influencing UA function, the third study investigated the temporal relationships between a comprehensive range of relevant physiological variables leading into and following the termination of obstructive apnoeas during sleep in 6 obese OSA patients. Prior to UA obstruction, diaphragm and genioglossus muscle activity decreased, while UA resistance increased. Lung volume and end-expiratory PGA and end-expiratory PDI also fell during this period, consistent with diaphragm ascent. There was a substantial increase in ventilation, muscle activity and lung volume immediately following the termination of obstructive events. Respiratory events and arousals occurred in close temporal proximity prior to and following obstructive apnoeas, supporting that cyclical respiratory events and arousals may both help to perpetuate further events. The results from this study support that there is a ‘global’ loss in respiratory drive to UA dilator and pump muscles precipitating obstructive respiratory events. The associated decreases in UA dilator muscle activity and lung volume may therefore both contribute to the propensity for the UA to obstruct. In summary, increased intra-abdominal pressure was shown to negatively impact UA airway collapsibility during sleep. A decrease in lung volume at sleep onset and prior to UA obstruction further support that lung volume decrement, coincident with a decline in overall respiratory drive, potentially contributes to the propensity for airway obstruction. Further studies are needed to elucidate the relative contribution of relatively small changes in lung volume versus changes in respiratory and UA muscle activity per se on UA patency in OSA patients. / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2010

The purpose of this study was to examine the relationships between inflammation, infection and lung function in cystic fibrosis during the evolution of lung disease in childhood and early adolescence. The developmental stages of childhood and the progression of lung disease together affected the methods and techniques used in the study, with the consequence that the work for this thesis fell naturally into two parts. The first part concerned the study of early lung disease in infants and young children who were unable to expectorate or to cooperate with lung function testing. In the second part, the inflammatory processes in both stable lung disease and during clinical exacerbations in older children and adolescents were studied non-invasively using sputum. The absence of a recognised definition of pulmonary exacerbation lead to further investigation into clinical heterogeneity in the diagnosis and management of an exacerbation. In early lung disease, inflammation was not found to be independent of infection, with pathogens in the lower airways found to correlate with levels of inflammation, respiratory system compliance and degree of air trapping (a relationship not previously shown). This suggested that infection remains the key target to minimizing lung damage in cystic fibrosis. The relationship between sputum markers of inflammation and lung pathology in established disease was found to be less clear, with high inflammation levels in both stability and during exacerbation. Reduction in sputum inflammatory levels following treatment of an exacerbation was found to be greater in those with lower pre-treatment levels. The definition and management of an exacerbation was found to be an area lacking consensus among clinicians, with likely consequent heterogeneity of clinical care and therefore inhomogeneity of hospitalization as a surrogate measure of exacerbation in a research setting. The work from this thesis, and the ensuing publications, has contributed to the understanding of the interactions between the inflammatory and infectious processes involved in CF lungdisease, in both early and more established lung disease in childhood.

Licentiatavhandling (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 2 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.