Multidrug resistance-associated protein 1 (MRP1) is a member of the adenosine 5’-triphosphate (ATP)-binding cassette (ABC) transporters. MRP1 actively effluxes a variety of endogenous and exogenous substrates from cells, ultimately, working to remove these compounds from the body. MRP1 was initially discovered based on its ability to confer resistance against a variety of chemotherapeutics when overexpressed in cancer cells lines. MRP1 function is important for a number of physiological processes, including regulating cellular and extracellular levels of the anti-inflammatory leukotriene C4 (LTC4) and the antioxidant glutathione (GSH). Our studies have focused on the role of MRP1 in regulating hematopoietic stem cell (HSC) self-renewal and differentiation and the role of CK2 as a regulator of MRP1 function. Reactive Oxygen Species (ROS) cellular levels are tightly regulated and fluctuations in ROS levels affect many cellular processes, including the self-renewal and differentiation of hematopoietic stem cells and kinase signaling pathways. MRP1 regulates ROS through the transport of reduced and oxidized GSH. MRP1 is highly expressed in HSCs, therefore we hypothesized that MRP1 regulates ROS levels in HSCs via efflux of GSH. We have shown that MRP1 regulates HSC self-renewal by modulating cellular ROS via the efflux of GSH. The decrease in ROS results in downregulation of p38 activity and altered expression of a number of redox response genes. CK2 is a master regulator of the cell and controls cell growth, proliferation, death and survival. Yeast studies from our lab using Ycf1p (a homologue of MRP1) and Cka1p (a homologue of CK2) have found that Cka1p regulates Ycf1p function. This result suggests that CK2 regulates MRP1 function via phosphorylation. We have found that CK2 does regulate MRP1 function via phosphorylation of the N-terminal extension at Thr249. Using A549, H460, and HeLa cancer cell lines, we found that inhibition of CK2 with tetrabromobenzimidazole (TBBz) reduces MRP1 function and increases cellular toxicity to known MRP1 substrates.

Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for multiple sclerosis (MS) with now more than 600 documented cases in the medical literature. Long-term remission can be achieved with this therapy, but when is it justified to claim that a patient is cured from MS? In attempt to answer this question, the outcome of the Swedish patients is described, mechanisms behind the therapeutic effect are discussed and new tools for demonstration of absence of disease have been developed. In Swedish patients treated with HSCT for aggressive MS, disease free survival was 68 % at five years, and no patient progressed after three years of stable disease. Presence of gadolinium enhancing lesions prior to HSCT was associated with a favorable outcome (disease free survival 79 % vs 46 %, p=0.028). There was no mortality and no patient required intensive care. The immune system of twelve of these patients was investigated further. In most respects HSCT-treated patients were similar to healthy controls, demonstrating normalization. In the presence of a potential antigen, leukocytes from HSCT-treated patients ceased producing pro-inflammatory IL-17 and increased production of the inhibitory cytokine TGF-β1 suggesting restoration of tolerance. Cytokine levels and biomarkers of tissue damage were investigated in cerebrospinal fluid from a cohort of MS patients. The levels were related to clinical and imaging findings. A cytokine signature of patients with relapsing-remitting MS could be identified, characterized by increased levels of CCL22, CXCL10, sCD40L, CXCL1 and CCL5 as well as down-regulation of CCL2. Further, we could demonstrate that active inflammation in relapsing-remitting MS is a tissue damaging process, with increased levels of myelin basic protein and neurofilament light. Importantly, relapsing-remitting MS patients in remission displayed no tissue damage. In secondary progressive MS, moderate tissue damage was present without signs of active inflammation. From a clinical vantage point, it seems that we confidently can claim cure of relapsing-remitting MS patients after five years absence of disease activity. The new tools for evaluation of disease can strengthen this assertion and may enable earlier prediction of outcome.

The development of effective methods to cryopreserve precious cell types has had tremendous impact on regenerative and transfusion medicine. Hematopoietic stem cell (HSC) transplants from cryopreserved umbilical cord blood (UCB) have been used for regenerative medicine therapies to treat conditions including hematological cancers and immodeficiencies. Red blood cell (RBC) cryopreservation in blood banks extends RBC storage time from 42 days (for hypothermic storage) to 10 years and can overcome shortages in blood supplies from the high demand of RBC transfusions. Currently, the most commonly utilized cryoprotectants are 10% dimethyl sulfoxide (DMSO) for UCB and 40% glycerol for RBCs. DMSO is significantly toxic both to cells and patients upon its infusion. Glycerol must be removed to <1% post-thaw using complicated, time consuming and expensive deglycerolization procedures prior to transfusion to prevent intravascular hemolysis. Thus, there is an urgent need for improvements in cryopreservation processes to reduce/eliminate the use of DMSO and glycerol. Ice recrystallization during cryopreservation is a significant contributor to cellular injury and reduced cell viability. Compounds capable of inhibiting this process are thus highly desirable as novel cryoprotectants to mitigate this damage. The first compounds discovered that were ice recrystallization inhibitors were the biological antifreezes (BAs), consisting of antifreeze proteins and glycoproteins (AFPs and AFGPs). As such, BAs have been explored as potential cryoprotectants, however this has been met with limited success. The thermal hysteresis (TH)activity and ice binding capabilities associated with these compounds can facilitate cellular damage, especially at the temperatures associated with cryopreservation. Consequently, compounds that possess “custom-tailored” antifreeze activity, meaning they exhibit the potent ice recrystallization inhibition (IRI) activity without the ability to bind to ice or exhibit TH activity,are highly desirable for potential use in cryopreservation. This thesis focuses on the rational design of potent ice recrystallization inhibitors and on elucidating important key structural motifs that are essential for potent IRI activity. While particular emphasis in on the development of small molecule IRIs, exploration into structural features that influence the IRI of natural and synthetic BAs and BA analogues is also described as these are some of the most potent inhibitors known to date. Furthermore, this thesis also investigates the use of small molecule IRIs for the cryopreservation of various different cell types to ascertain their potential as novel cryoprotectants to improve upon current cryopreservation protocols, in particular those used for the long-term storage of blood and blood products. Through structure-function studies the influence of (glyco)peptide length, glycosylation and solution structure for the IRI activity of synthetic AFGPs and their analogues is described. This thesis also explores the relationship between IRI, TH and cryopreservation ability of natural AFGPs, AFPs and mutants of AFPs. While these results further demonstrated that BAs are ineffective as cryoprotectants, it revealed the potential influence of ice crystal shape and growth progression on cell survival during cryopreservation. One of the most significant results of this thesis is the discovery of alkyl- and phenolicglycosides as the first small molecule ice recrystallization inhibitors. Prior to this discovery, all reported small molecules exhibited only a weak to moderate ability to inhibit ice recrystallization. To understand how these novel small molecules inhibit this process, structure-function studies were conducted on highly IRI active molecules. These results indicated that key structural features, including the configuration of carbons bearing hydroxyl groups and the configuration of the anomeric center bearing the aglycone, are crucial for potent activity. Furthermore, studies on the phenolic-glycosides determined that the presence of specific substituents and their position on the aryl ring could result in potent activity. Moreover, these studies underscored the sensitivity of IRI activity to structural modifications as simply altering a single atom or functional group on this substituent could be detrimental for activity. Finally, various IRI active small molecules were explored for their cryopreservation potential with different cell types including a human liver cell line (HepG2), HSCs obtained from human UCB, and RBCs obtained from human peripheral blood. A number of phenolic-glycosides were found to be effective cryo-additives for RBC freezing with significantly reduced glycerol concentrations (less than 15%). This is highly significant as it could drastically decrease the deglycerolization processing times that are required when RBCs are cryopreserved with 40% glycerol. Furthermore, it demonstrates the potential for IRI active small molecules as novel cryoprotectants that can improve upon current cryopreservation protocols that are limited in terms of the commonly used cryoprotectants, DMSO and glycerol.

Self-renewal is central to the expansion of normal and cancerous stem cells. Its understanding is therefore critical for future advances in transplantation-based therapies and cancer treatment. Although the molecular machinery controlling stem cell self-renewal remains poorly defined, a number of genes important to this process have recently been identified. Two prominent genes in this group are Hoxb4 and Bmi1. Members of our group led the way to demonstrate important regulatory functions of these genes in hematopoietic stem cell (HSC) self-renewal and expansion. / The major goal of my thesis project is to dissect mechanisms that regulate self-renewal of HSCs. Our starting hypothesis was that HSC activity is regulated by complementary and independent self-renewal mechanisms: self-renewal of expansion and self-renewal of maintenance (Chapters 1-2). In order to further verify this theory, we have analyzed the genetic interaction between Hoxb4 and Bmi1. While Hoxb4 overexpression triggers HSC expansion, Bmi1 proper expression is essential to sustain long-term stem cell activity. We have also demonstrated that Hoxb4 and Bmi1 regulate distinct gene targets, likely suggesting a complementary and independent function for these two regulators in HSC activity (Chapter 3). / The second part of this thesis highlights efforts that were made in order to get a better understanding of self-renewal mechanisms. We have identified potential new regulators of stem cell activity by characterizing a stem cell leukemia population (Chapter 4) and by assessing the expression of asymmetrical distributed factors (Chapter 5) and selected nuclear factors of the Hematopoietic Stem Cell Nuclear Factor Database (Chapter 6) in stem cell-enriched sub-fractions. / This project will lead to a better understanding of the cellular basis regulating self-renewal of both normal and cancer stem cells and potentially to the future identification of new self-renewal determinants.

Errata inserted on back end papers. / Includes bibliographical references. / 99 leaves, [9] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Identifies a novel site of action for IL-3, and suggests that it can influence immune and inflammatory responses and hemopoiesis by acting not only on hemopoietic cells but also on vascular endothelium. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1996?

Addendum pasted on front end papers. / Includes bibliographies. / 109, [99] leaves, [5] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the investigation as to whether the mouse granulocyte-macrophage colony-stimulating factor and interleukin-3 genes are regulated in a similar manner as those of the human, focussing on regulation through an enhancer. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1996

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?

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 132-142.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 3 uppsatser.