Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS). In the current thesis, we have preformed an immunological investigation of patients with MS and developed an immunosuppressive cell therapy that could be beneficial for these patients. MS has been considered to be driven by T helper type1 (Th1) lymphocytes but new data indicate the involvement of Th17 responses. T cells from patients with MS that were evaluated for immunological status secreted both interferon-γ and interleukin-17 upon stimulation. However, T cells from patients with MS in remission, in contrast to relapse, had poor proliferative capacity suggesting that they are controlled and kept in anergy. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4+CD25+FoxP3+ Tregs in autoimmunity has been extensively investigated. We analyzed Tregs from patients with MS in relapse and remission by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3 and the IL7R (CD127). Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls, indicating that Tregs might attempt to restrain immune activity during relapse. In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, therapy with suppressive cells such as Tregs or mesenchymal stromal cells (MSCs) has proven beneficial. However, systemic administration of such cells may immunologically compromise the recipient and promote infections due to general immunosuppression. We hypothesized that suppressive cells can be equipped with a CNS-targeting receptor and be delivered intra-nasally to avoid systemic exposure. CD4+ T cells were modified with a lentiviral vector system to express a myelin oligodendrocyte (MOG)-targeting receptor in trans with the FoxP3 gene that drives Treg differentiation. Genetically engineered Tregs demonstrated suppressive capacity in vitro and localized to the brain and suppressed ongoing encephalomyelitis in vivo. Cured mice were rechallenged with an EAE-inducing inoculum but remained healthy. MSCs are a heterogeneous population of stromal cells residing in most connective tissues and have the capacity to suppress effector cells of the immune system. MSCs were engineered to express MOG-targeting receptors using lentiviral vectors. Genetically engineered MSCs retained their suppressive capacity in vitro and successfully targeted the brain upon intranasal delivery. Engineered MSCs cured mice from disease symptoms and these mice were resistant to further EAE challenge. Encephalitic T cells isolated from cured mice displayed an anergic profile while peripheral T cells were still responsive to stimuli. In conclusion, MS patients have peripheral CNS-reactive T cells of both Th1 and Th17 type that, while in remission, are kept in anergy. Also, MS patients in relapse exhibit increased levels of CD25 negative Tregs indicating an attempt to restrain immune activity. Finally, immunosuppressive cells can be genetically engineered to target CNS and efficiently suppress encephalomyelitis in an active EAE model upon intranasal delivery.

Broad distribution and activity limit the utility of anti-cancer compounds by causing unacceptable systemic toxicity and narrow therapeutic indices. To improve tumour accumulation, drug-loaded macromolecular assemblies have been designed to replace conventional surfactant-based formulations. Their nanoscale size enhances tumour accumulation via hyperpermeable vasculature and reduced lymphatic drainage. Incorporating targeting ligands introduces cell specificity through receptor-specific binding and uptake, enabling drugs to reach intracellular targets. In this work, the targeting properties of polymer nanoparticle micelles of poly(2-methyl-2-carboxytrimethylene carbonate-co-D,L-lactide)-graft-poly(ethylene glycol)-furan (poly(TMCC-co-LA)-g-PEG) were verified using in vitro and in vivo models of breast cancer. To select a relevant mouse model, the vascular and lymphovascular properties of two tumour xenograft models were compared. Greater accumulation of a model nanocarrier was observed in orthotopic mammary fat pad (MFP) tumours than size matched ectopic subcutaneous tumours, suggesting that the organ environment influenced the underlying pathophysiology. Immunostaining revealed greater vascular thickness, density and size, and thinner basement membranes in MFP tumours, likely contributing to greater blood perfusion and vascular permeability. Based on these observations, MFP tumour-bearing mice were used to characterize the pharmacokinetics and biodistribution of a taxol drug, docetaxel, encapsulated in poly(TMCC-co-LA)-g-PEG nanoparticles. The nanoparticle formulation demonstrated longer docetaxel circulation in plasma compared to the conventional surfactant-based formulation. As a result, greater docetaxel retention was uniquely measured in tumour tissue, extending exposure of tumour cells to the active compound and suggesting potential for increased anti-cancer efficacy. Furthermore, active targeting of antibody-modified nanoparticles to live cells was shown to be selective and receptor-specific. Binding isotherms were used to quantify the impact of antibody density on binding strength. The equilibrium binding constant increased linearly with the average number of antibodies per particle, which is consistent with a single antibody-antigen interaction per particle. This mechanistic understanding enables binding behaviour to be adjusted in a predictive manner and guides rational nanoparticle design. These studies validate poly(TMCC-co-LA)-g-PEG nanoparticles as a platform for targeted delivery to cancer on both a tissue and cellular level, forming a compelling justification for further pre-clinical evaluation of this system for safety and efficacy in vivo.

Unique among the intracellular lipid binding proteins, acyl CoA binding protein (ACBP) exclusively binds long chain fatty acyl CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not result in any live homozygous deficient (null) ACBP^(-/-) pups. Heterozygous and wild type embryos were detected at all postimplantation stages, but no homozygous ACBP null embryos were obtained– suggesting that an embryonic lethality occurred at a preimplantation stage of development, or that embryos never formed. While ACBP null embryos were not detected at any blastocyst stage, ACBP null embryos were detected at the morula (8- cell), cleavage (2-cell), and zygote (1-cell) preimplantation stages. Two other LCFACoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCPx) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins only at the earliest preimplantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.

Controlled drug delivery systems deliver drugs at predetermined rates for extended periods. Although there are various types such as capsules, tablets etc, micro and nano spheres are the most commonly used systems. In this study, a set of chitosan-polyvinylpyrrolidone (CH-PVP) microspheres containing different amounts of polyvinylpyrrolidone as semi inter penetrating networks (semi-IPN) were prepared as controlled release systems. Emulsification method was applied for the preparation of microspheres and some of them were conjugated with a monoclonal antibody which is immunoglobulin G (IgG). CH-PVP films were also prepared by solvent casting method with the same composition as in the microspheres and, mechanical and surface properties of the films were examined. Prepared microspheres were characterized by SEM, stereo and confocal microscopes. Some microspheres were loaded with a model chemotherapeutic drug, 5-Fluorouracil (5-FU), and in-vitro release of 5-FU were examined in phosphate buffer solutions (pH 7.4, 0.01 M.) It was shown that for semi-IPN samples release was faster compared to pure CH samples and the total release was achived 30 days for CH:PVP-2:1, CH:PVP-3:1 semi-IPNs and CH microspheres and 27 days for CH:PVP-1:1 semi-IPN microspheres. The antibody conjugated microspheres were targeted to MDA-MB (human causasian breast carcinoma cancer cells and coculture cells in culture medium. For the CH-PVP films, it was obtained that as the amount of PVP increased, hydrophobicity as well as mechanical strength of the system was decreased.

This study investigates the implications of financial dollarization for inflation targeting framework. To this end, monetary policy rule, inflation targeting performance and international reserves equations are estimated for twenty four inflation targeting countries with different levels of financial dollarization. The results based on the panel data estimations indicate that monetary policy rule and behavior of accumulating international reserves are affected by the degree of financial dollarization. Furthermore, the study finds that inflation targeting performance is not affected by the level of financial dollarization. Consequently, the empirical analysis suggests that even if financial dollarization does not affect the inflation targeting performance, it does affect the monetary policy rule and the variables to be taken into account to reach the target.

The primary objective of this dissertation is to introduce several algebraic procedures to the targeting of material recycle networks. The problem involves the allocation of process streams and fresh sources to process units (sinks) with the objective of minimizing fresh purchase and waste discharge. In the case of composition-limited sinks, allocation to process sinks is governed by feasibility constraints on flowrates and compositions. A systematic non-iterative algebraic approach is developed to identify rigorous targets for minimum usage of fresh resources, maximum recycle of process resources and minimum discharge of waste. These targets are identified a priori and without commitment to the detailed design of the recycle/reuse network. The approach is valid for both pure and impure fresh resources. The devised procedures also identifies the location of the material recycle pinch point and addresses its significance in managing process sources, fresh usage, and waste discharge. The dissertation also addresses the targeting of material-recycle networks when the constraints on the process units are described through flowrates and properties. This property-integration problem is solvedusing a non-iterative cascade-based algebraic procedure. Finally, for more complex cases with multiple fresh sources and with interception networks, a mathematical-programming approach is developed. Because of the nonlinear non-convex characteristics of the problem, the mathematical model is reformulated to enable the global solution of the problem. Several case studies are solved to illustrate the ease, rigor, and applicability of the developed targeting technique.

Correct localization of lipidated cytosolic proteins to the plasma membrane (PM) is mediated by interactions between lipid anchors of proteins and cell membranes. Previously, dietary fish oil and its major n-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), have been shown to decrease Ras membrane association, concomitantly reducing rat colon tumor incidence and Ras signaling, compared with corn oil and linoleic acid (LA), a highly prevalent vegetable fat and dietary PUFA in the U.S. diet. In order to explore the potential regulatory role of the cellular lipid environment in PM targeting of lipidated proteins, young adult mouse colon (YAMC) cells were treated with 50 µM DHA, LA, or oleic acid (OA) 24 h prior to and 36-48 h after transfection with green fluorescent protein (GFP) fusion constructs of various lipidated cytosolic proteins. Relative expression of each GFP fusion protein at the PM and the Golgi in living cells was quantified using z-serial confocal microscopy and digital image processing. DHA differentially altered the subcellular localization of Ras isoforms and Src-related tyrosine kinases in a reversible manner. DHA significantly decreased the PM localization and increased the endomembrane association of H-Ras, N-Ras, and Lck, which are targeted to the PM via the exocytic pathway, regardless of their functional state. In contrast, the subcellular distribution of K-Ras and Fyn, of which transport is independent of the vesicular transport pathway, was unaffected by DHA. Moreover, DHA selectively inhibited lipidated cytosolic protein targeting since the PM delivery of transmembrane protein cargo was unaffected, indicating that DHA does not alter the bulk flow of secretory vesicular traffic. Overall, the present study presents compelling evidence that select dietary constituents with membrane lipid-modifying properties can differentially modulate subcellular localization of important lipidated signaling proteins depending on their intracellular trafficking route to the PM.

Mobile group II introns are retroelements that insert site-specifically into double-stranded DNA sites by a process called retrohoming. Retrohoming activity rests in a ribonucleoprotein (RNP) complex that contains an intron-encoded protein (IEP) and the excised intron RNA. The intron RNA uses its ribozyme activity to reverse splice into the top strand of the DNA target site, while the IEP cleaves the bottom DNA strand and reverse transcribes the inserted intron. My dissertation focuses on the Lactococcus lactis Ll.LtrB group II intron and its IEP, denoted LtrA. First, I investigated the ability of microinjected Ll.LtrB RNPs to retrohome into plasmid target sites in Drosophila melanogaster precellular blastoderm stage embryos. I found that injection of extra Mg2+ into the embryo was crucial for efficient retrohoming. Next, I compared retrohoming of linear and lariat forms of the intron RNP. Unlike lariat RNPs, retrohoming products of linear intron RNPs displayed heterogeneity at the 5’-intron insertion junction, including 5’-exon resection, intron truncation, and/or repair at regions of microhomology. To investigate whether these junctions result from cDNA ligation by non-homologous end-joining (NHEJ), I analyzed retrohoming of linear and lariat intron RNPs in D. melanogaster embryos with null mutations in the NHEJ genes lig4 and ku70, as well as the DNA repair polymerase polQ. I found that null mutations in each gene decreased retrohoming of linear compared to lariat intron RNPs. To determine whether novel activities of the LtrA protein contributed to the linear intron retrohoming 5’ junctions, I assayed the polymerase, non-templated nucleotide addition and template-switching activities of LtrA on oligonucleotide substrates mimicking the 5’-intron insertion junction in vitro. Although LtrA efficiently template switched to 5’-exon DNA substrates, the junctions produced differed from those observed in vivo, indicating that template switching is not a significant alternative to NHEJ in vivo. Finally, I designed and constructed retargeted Ll.LtrB RNPs to site-specifically insert into endogenous chromosomal DNA sites in D. melanogaster. I obtained intron integration efficiencies into chromosomal targets up to 0.4% in embryos and 0.021% in adult flies. These studies expand the utility of group II intron RNPs as gene targeting tools in model eukaryotic organisms. / text

Mobile group II introns insert site-specifically into DNA target sites through a mechanism ("retrohoming") that involves reverse splicing of the intron RNA into the DNA and its subsequent reverse transcription by an intron-encoded protein (IEP) that is associated with the RNA in a ribonucleoprotein (RNP) complex. Characterization of this RNP complex and its retrohoming activities have enabled the development of programmable mobile group II intron gene targeting vectors routinely used in prokaryotic organisms. Building upon recent research by our lab to develop gene targeting in Xenopus laevis and Drosophila melanogaster using the group II intron Ll.LtrB from Lactococcus lactis, I describe work to extend this system to mammalian cells. I demonstrate that group II intron RNPs can be delivered to mammalian cells efficiently and produced in vivo via a CMV/T7 hybrid expression system. Using a robust single-strand annealing assay to detect homologous recombination induced by double-strand breaks (DSBs), I found that group II intron-mediated DSBs are efficiently repaired by mammalian cells. Despite varied approaches, I failed to detect endogenous group II intron-mediated gene targeting in human and mouse cells in culture. Gene expression microarray analysis and in vivo imaging of RNP molecules indicated that group II intron RNPs are sequestered away from the genome and induce host innate immune responses. I also investigated how the C-terminal DNA-binding domain of the Ll.LtrB IEP contributes to DNA target site recognition. Building upon previous mass spectrophotometric analysis of site-specific UV-crosslinking, I used genetic and biochemical analyses to identify potential protein contacts for key target site residues T-23 and T+5. Genetic selection of mutants in a region contacting T+5 led to identification of LtrA variants with increased retrohoming efficiency. My results provide evidence that the DNA-binding domain of a group II intron reverse transcriptase functions in DNA target site recognition and suggest new methods for changing its DNA target specificity and targeting efficiency. / text

published_or_final_version / Anatomy / Master / Master of Philosophy