Acute Lung Injury, characterised clinically as the Acute Respiratory Distress Syndrome is a catastrophic response to a range of pulmonary and non-pulmonary insults. Despite much work the key mechanisms involved in generating the exaggerated immune response that results in lung injury are not completely understood. Hypoxia-inducible factor-1 has been shown to be a key transcription factor in the myeloid cell response to inflammatory signals. The aims of this thesis were to develop a model of acute lung injury and to study the role of Hif-1 in the generation of lung injury in this model. A model of direct pulmonary injury as a result of intratracheal instillation of endotoxin is described. Using this model the role of myeloid cell Hif-1α was characterised using a myeloid cell specific conditional knockout system. The injury in Hif-1α deficient mice was quantitatively similar to the injury seen in wild type animals over a range of time points. However, the quality of the injury, assessed by a measure of nitric oxide mediated damage was reduced. The in vivo data were supported by in vitro studies using a murine macrophage cell line which showed that manipulation of the cellular oxygen tension in the presence of endotoxin alters the ability of the cell to generate nitric oxide. Furthermore, pharmacological manipulation of cellular Hif-1 levels by Dimethyloxallyl Glycine (DMOG) in the macrophage cell increased the generation of nitric oxide in response to endotoxin by altering the expression of a number of the isoforms of Nitric Oxide Synthase. In a final set of experiments the response to intratracheal endotoxin was modulated in mice by the concurrent administration of DMOG. As expected the qualitative response to endotoxin was similar but the NO mediated damage was enhanced in the animals administered DMOG. Manipulation of Hif-1 may have a role in the therapy of lung injury by altering the characteristics of the response.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / In this study we examine the utility of using CD8+ T cell hybridomas to measure the ability of bone marrow dendritic cells (BMDCs) to internalize cancer proteins and display them to cytotoxic T cells, a process termed cross‐presentation. We test the ability of a newly generated T cell hybridoma called BUSA14 to detect cross‐presentation of the melanoma antigen gp100. BUSA14 produces a dose‐dependent response to human and mouse gp100 peptides. However, cross‐presentation of gp100 by BMDCs using SK‐MEL‐28 human melanoma cell lysates or direct MHC class I‐restricted presentation by B16 murine melanoma cells was not detected. Both SKMEL‐28 and B16 cells express gp100 protein by immunoblot, and gp100 as a membrane bound protein may be concentrated by cell fractionation techniques. We validated our crosspresentation assay with another T cell hybridoma B3Z to detect cross‐presentation of the model antigen ovalbumin. Lastly, we determined that although BUSA14 expresses the coreceptor CD8, BUSA14 lacks CD3 expression, which likely impairs the ability of this hybridoma to respond to engagement of the T cell receptor and contributes to the inability to detect presentation of native gp100 protein. To resolve these issues, we plan to use primary gp100‐specific T cells from pmel mice expressing the same T cell receptor as the BUSA14 hybridoma to detect presentation of gp100 protein. Ultimately, we plan to evaluate the requirements for cross‐presentation of gp100, including a role for gamma‐interferon‐inducible lysosomal thiol reductase (GILT), a disulfide bond reducing enzyme.

Nasopharyngeal carcinoma is a commonly occuring tumor in Southern China. However, the genetic basis underlying its tumorigenicity is still unclear. In eukaryotic cells, progression of the cell cycle is regulated by interactions of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors (CDKIs). These cell cycle-regulator proteins play important roles in growth of both normal and tumor cells. Many human tumors exhibit deregulation of one or more genes which involved in regulation of cell cycle progression. p27Kip1, a member of the Cip/Kip family, inhibits both cyclin D-CDK4, and cyclin E-CDK2 complexes and regulates progression of the cell cycle from G1 to S phase. Although p27Kip1 gene mutations are rare in human tumors, low expression of p27Kip1 are observed in several cancers, such as colon, breast and esophagus. In our previous study, p27Kip1 shown lower expression in two NPC cell lines compared with NNE and 293 (HEK). A doxycycline inducible construct, pBIG2r/p27Kip1, included a full length of human p27Kip1 cDNA was transfected into two NPC cell lines. Expression of several cell cycle-related genes were analyzed. By increasing p27Kip1 in NPC cell lines, we found that the G1 phase and the doubling time were lengthened. Protein expression of cyclin E and CDK2 were up-regulated. These data suggest that the overexpression of p27Kip1 might be cause NPC cells to arrest at G1 phase and might lead to apoptosis.

Phenotypic plasticity is an environmentally based change in phenotype and can be adaptive. Often, the change in an organism's phenotype is induced by the presence of a predator and serves as a defense against that predator. Defensive phenotypes are induced in freshwater physid snails in response to both crayfish and molluscivorous fish. Alternative morphologies are produced depending on which of these two predators snails are raised with, thus protecting them from each of these predators' unique mode of predation. Snails and other mollusks have been shown to produce thicker, differently shaped shells when found with predators relative to those found without predators. This production of thicker, differently shaped shells offers better protection against predators because of increased predator resistance. The first study in this thesis explores costs and limits to plasticity using the snailfish- crayfish system. I exposed juvenile physid snails (using a family structure) to either early or late shifts in predation regimes to assess whether developmental flexibility is equally possible early and late in development. Physid snails were observed to produce alternative defensive morphologies when raised in the presence of each of the two predators. All families responded similarly to the environment in which they were raised. Morphology was found to be heritable, but plasticity itself was not heritable. Morphology was found to become less flexible as snails progressed along their respective developmental pathways. In the second study, I raised physid snails with and without shell-crushing sunfish and examined the differences in shell thickness, shell mass, shell size and shell microstructural properties between the two treatment groups. Shells of snails raised with predators were found to be larger, thicker and more massive than those raised without predators, but differences in microstructure were found to be insignificant. I conclude that the observed shell thickening is accomplished by the snails' depositing more of the same material into their shells and not by producing a more complex shell composition.

This study explored the possibility that upregulation of Hypoxia Inducible Factor-1 (Hif-1)-responsive genes in Human Umbilical Vein Endothelial Cells (HUVEC) would promote and stabilize HUVEC formation into inchoate vascular beds within artificial collagen gels. This experiment was designed to explore the above possibility by sub-cloning Hif-1[alpha], the related chimeric construct Hif-1[alpha]/VP16, and the marker gene dsRed into retroviral expression vectors, producing retroviral vectors containing these genes, and stably transducing HUVEC using these retroviruses. Transduced HUVEC were to be observed in cell culture as well as after implantation into artificial collagen gels that have previously supported vascular bed formation by HUVEC. Our results show, preliminarily, that HUVEC transduced with Hif-1[alpha]/VP16 go into cell-cycle arrest. Attempts to transduce HUVEC with Hif-1[alpha] failed to achieve high enough transduction efficiency to determine the cells angiogenic potential. This study concluded that more experiments need to be conducted to better characterize the effects of hypoxia-responsive gene upregulation in controlling HUVEC angiogenesis and cell-cycle signaling and that straightforward transduction of HUVEC by Hif-1[alpha]/VP16 is probably not sufficient, in itself, to induce in vitro vascular bed formation.

Model-based radiostereometric analysis (MBRSA) of a total ankle arthroplasty (TAA) prosthesis was studied for the first time. The TAA MBRSA system precision was determined from the double exams of 20 patients implanted with the Mobility(TM). The MTE for any direction was 0.07mm for the tibial component. The MTE was 0.09mm and the MRE was 0.51° for the talar component. The MTPM detection limits were 0.22mm and 0.85mm for the tibial and talar components. Both components followed the typical subsidence-stabilization pattern. There was little detectable continuous migration at one to two years. The median(range) MTPM at two years was 0.96mm(0.17-2.28mm) and 1.23mm(0.39-1.9 mm) for the tibial and talar components. There was no detectable inducible displacement observed for any components at two years, except one talar component. The median(range) MTPM induced by the loading at two years was 0.08mm(0.03-0.18mm) and 0.39mm(0.27-1.06mm) for the tibial and talar components.

Knee osteoarthritis is a debilitating disease causing pain and disability in adults. Biomechanical factors including obesity, abnormal magnitude and load distribution have been cited to play a role in its initiation and progression with its definite cause being multi-factorial. Total knee arthroplasty has become the treatment of choice for knee osteoarthritis and although the procedure is mostly successful, there are some patients who experience implant failures which necessitates revision surgery. Revision surgery is more complicated and thus there is the need to monitor patients who have undergone TKA so as ensure better outcomes and also address problems much earlier. Objective methods like Radiostereometric Analysis (RSA) has proven to be a good tool at diagnosing these implant failures. Inducible displacement with RSA has the potential to serve as a one-time measure to diagnose implant failures. Previous studies have applied loads to induce motion to the knee in various ways- squatting, exercising and weight-bearing on the affected limb. This was not standardized and caused wide variations in the data. This work looked at refining a device used to apply standardized loads to the knee resulting in a more portable and faster way of applying load to the joint. Gait analysis is used to assess implant function pre and post surgery. Some gait patterns have also been related to implant failure. Previous works have focussed primarily on associations between well-working implants (non-revised patients) and these gait patterns (adduction moments and flexion angles). This work focussed on any differences in the gait patterns between patients who did not undergo revision surgery and those that did. Although most parameter differences did not reach statistical differences, they point to important trends that may explain the causative factors (adduction moments) whiles others may point to the effects of disease progression (external rotation).

The development of intratumoral hypoxia is associated with resistance to therapy in many forms of human cancer, and pre-exposure of tumor cells to hypoxia confers multidrug resistance. Research over the last several years has led to considerable advances in the understanding of the cellular response to oxygen deprivation, however the hypoxia-induced mechanisms that contribute to the chemoresistance phenotype are still not well understood. Recent studies have identified hypoxia-inducible factor 1 (HIF-1), a master transcriptional regulator of oxygen homeostasis, as an important mediator of hypoxia-induced chemoresistance in cancer cells. The research described in this thesis confirms these findings and demonstrates HIF-1 is required for hypoxia-induced resistance to doxorubicin and etoposide in human tumor cells. In addition, novel findings revealed that hypoxia-induced drug resistance occurred independently of changes in the apoptotic fraction and was associated with decreased drug-induced senescence. DNA damage measured at the single-cell level revealed that the increase in survival correlated well with a HIF-1-dependent decrease in etoposide-induced DNA strand breaks, providing direct evidence that exposure of tumor cells to hypoxia leads to protection against some forms of drug-induced DNA damage. Characterization of several classical mechanisms of drug resistance upstream of DNA damage identified multiple determinants of cellular resistance to anticancer agents. The relative contributions of each varied depending on the particular drug and cancer cell line studied. Together, the findings presented here support a model in which hypoxia-induced chemoresistance is a multifactorial phenomenon that is regulated, at least in part, through HIF-1-dependent mechanisms. / Thesis (Ph.D, Anatomy & Cell Biology) -- Queen's University, 2008-08-29 12:35:36.219

Rhodotorula (Rhodosporidium) toruloides is an oleaginous basidiomycete yeast with great biotechnological potential. Capable of accumulating lipid up to 76 % of its dry biomass and well suited to the metabolism of lignocellulosic hydrolysate, it is a good candidate for production of advanced biofuels as well as a host of other potential roles in industry. However, molecular genetic tools for manipulation of this yeast are lacking and its high genomic GC content can make routine cloning difficult. Agrobacterium tumefaciens-mediated transformation of R. toruloides CBS 14 was demonstrated, and plasmid vectors were developed for transformation of R. toruloides, including elements for Saccharomyces cerevisiae in-yeast assembly. In-yeast assembly is robust to the manipulation of GC-rich DNA and of large plasmids. Using these vectors and an EGFP reporter, a screen to identify inducible promoters was performed, and promoters from the genes NAR1, ICL1, CTR3, and MET16 identified. These promoters have independent induction/repression conditions and different levels and rates of induction. Minimal inducible promoters were determined, which are as small as 200 bp. As well as showing tight regulation of the EGFP marker, the NAR1 promoter was able to drive conditional rescue of a leu2 mutant strain. In parallel, as a proof of principle for production of advanced biofuels, hydrocarbon biosynthesis pathways were expressed in R. toruloides and analysed by GC-MS. After co-expression of Synechococcus elongatus fatty acyl-ACP reductase and fatty aldehyde decarbonylase, and E. coli ferredoxin and ferredoxin reductase, production of the alkane heptadecane was observed. To increase the availability of free fatty acids (FFA) for production of hydrocarbons by other pathways, Thermomyces lanuginosus lipase 2 was expressed, resulting in a 1.3-fold increase in the concentration of FFAs.

This thesis is focused on the ARP2/3 complex, which is a de novo actin cytoskeleton nucleator. This highly conserved complex is composed of seven subunits and regulates branching of actin filaments at a constant angle of 70 degrees. In plant and animal cells ARP2/3 is involved in various processes, which are connected with the initiation of actin polymerization; for example it participates in determining the direction and speed of cell growth and the movement of vesicles and organelles within the cell. The mutation of individual subunits is lethal for animal cells, but in plants, these mutants have only mild symptoms such as distorted trichomes or changes in epidermal cells. The aim of the presented work was to study the function of the ARP2/3 complex by the method of partial silencing of subunits using RNA interference. Specifically, it was the ARPC1 subunit of Arabidopsis thaliana and the ARPC2 subunit studied on the cellular model, the tobacco BY-2 cell line. Experimental work involved the creation of DNA constructs for induction of silencing, transformation of plant material, silencing rate analysis, and phenotype tracking in selected lines. Although lines with reduced transcript levels of the given ARP2/3 complex subunit were found, no phenotypic changes were observed in these lines. Key words...