Lymph nodes (LNs) are highly organized structures containing adaptive and innate immune cells supported by a network of specialized stromal cells. These stromal cells provide the structural basis for immune cell migration, localization and specialized microenvironments for effector function through the production of specific chemokines. Crosstalk between stroma and haematopoietic cells is important in regulating the efficacy of the immune response in part through their plastic response to inflammation and capacity to generate specialized structures, including germinal centres (GCs). The mechanisms driving tissue remodelling and GC formation in LNs are unclear. Understanding the timing and molecular mechanisms leading to stromal cell reorganization will help generate novel vaccination strategies that can control and regulate immune responses. An adjuvant is a non-antigenic substance that when added to vaccines, enhances the immune response to inoculated antigens. TLR agonists have been shown to be potent second-generation adjuvants. TLR4 agonist adjuvants induce rapid LN remodelling through the loss of B cell follicles and the formation of a ring-like structure in the cortex; surprisingly this was not due to a loss of CXCL13 production by the stromal cells. After forming this ring, large numbers of new B cell follicles appear in the LN paracortex. The molecular mechanisms leading to this reorganization was investigated. TLR4 activation and signalling has to be tightly controlled to avoid uncontrolled inflammation and enable tissue repair. miRNAs constitute a key component in a negative feedback loop in innate immune responses. Deficiency in a TLR4-induced miRNA leads to an altered immune response and changes to adjuvant induced tissue remodelling. By using a simple antigen challenge model, it was possible to determine a novel molecular mechanism controlling LN remodelling and vaccine efficacy.

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In mammals, effective acquisition and handling of iron is critical for survival. Consequently, iron handling proteins are exquisitely responsive to changes in iron concentrations. Regulation occurs at both the transcriptional and translational levels. The work described in this thesis focuses on two iron handling proteins that are central to iron regulation and homeostasis, namely divalent metal transporter 1 (DMT1) and transferrin (Tf). Experiments were performed independently to test two aims and accordingly the outcomes of these experiments are described separately. 1. To determine the cellular location of heterologously expressed iron induced (DMT1) splice variants. Two novel DMT1 splice variants, lacking exon 5 (DMT1Δ5) and exons 12(DMT1Δ12) were induced by treating cells with desferrioxamine (DFO), a treatment that lowers the cellular iron concentration. Previously, DMT1Δ5 and DMT1Δ12 have been described in patients with DMT1 mutations, however,these studies did not establish a direct link between mutations, anaemia or splicing. Therefore, as a first step towards understanding their function we aimed to engineer DMT1Δ5 and DMT1Δ12 constructs tagged with mRFP and express them in non-polarised and polarised cell models. Heterologous expression of DMT1 in WKPT cells localised predominantly to structures resembling the late endosomes and lysosomes as previously shown. In contrast, mRFP-DMT1Δ5 localised to the nuclei and led to cell death after 16 hours post transfection in WKPT cells. However, no expression of mRFPDMT1Δ12 was detected in hamster embryonic kidney 293 or WKPT cells. These studies suggest that low-iron induced splicing events may differentially affect subcellular localisation of DMT1 in polarised and non-polarised cells. 2. To determine the mechanism of transferrin uptake into the WKPT renal epithelial cell line. Multi-ligand receptors, megalin/cubilin are suggested to be the mediators of Tf uptake in the proximal tubule. However in mice, the TfR1 has been shown to localise at the apical membrane of the proximal tubule epithelia. Therefore, we aimed to determine the contribution of megalin/cubilin to cellular Tf delivery in the PT. Experiments were performed in vitro, using a cell line (WKPT-0293Cl.2) derived from the proximal tubule of rats. Ligands of megalin/cubilin, receptor associated protein (RAP) and Tf were used for uptake studies. Uptake studies confirmed that when iron is replete, megalin/cubilin are the main mode of Tf (Tfmeg/cub) uptake in PT cells. However, when cellular iron was reduced, a switch to a RAP insensitive receptor (RIR) upregulated Tf (TfRIR)uptake, leading to an increase in intracellular Tf. Further experiments showed that Tf enters a common pathway irrespective of the receptor in which its uptake is mediated. However, TfRIR was retained for longer in comparison to Tfmeg/cub, highlighting a key difference between the two intracellular pathways of Tfmeg/cub and TfRIR. In summary, data suggested that megalin/cubilin are the dominant means by which Tf is endocytosed when iron was replete. However when cellular iron is restricted, TfR1 switches to become the principal receptor for Tf endocytosis.

Monocytes and macrophages are phagocytic cells of the innate arm of the immune system. Although initially they were considered just as ‘bug-eaters’ more recent studies suggest that they are effector cells, involved in the homeostasis of the tissues. Yet, more research is needed to understand their functions in vivo. In mice there are two subsets of blood monocytes: Ly6Chi and Ly6Clow monocytes. Ly6Clow monocytes have been studied less than Ly6Chi monocytes and their functions are still elusive. Although kidney macrophages were first described more than 30 years ago still nothing is known about their role in vivo. By exploited intravital imaging and flow cytometry this report showed that Ly6Clow monocytes are housekeepers of the endothelium, scavenging microparticles and cellular debris in the steady state and in inflammation. The results presented herein, further characterized kidney macrophages as effector cells per se and showed, for the first time, that kidney macrophages take up immune complexes in vivo, are activated by immune complexes and recruit monocytes in response to immune complexes. In a nutshell, this work sheds light on the in vivo functions of Ly6Clow monocytes and kidney macrophages, helping us to understand the role of these cells better.

Hepcidin is a peptide hormone involved in the control of iron homeostasis. It has 25 amino acids with an antiparallel beta-sheet structure stabilized by four disulfide bonds. Hepcidin binds to the sole known iron exporter, ferroportin, leading to its internalization and degradation by a mechanism not fully understood. Hepcidin has an important role in iron metabolism disorders, such as hemochromatosis and anaemia. However, hepcidin analogues are currently not available for clinical use. The aims of this project are twofold: - to design and synthesise a fluorescent hepcidin analogue that can be used as a biological tool to further investigate hepcidin-ferroportin interactions; - to design and synthesise small peptide-like hepcidin analogues which can bind and internalize ferroportin. At the end of the thesis there is a pullout which summarises the structures of the peptides synthesised in this project. Chapter 2 describes the synthesis of eleven linear peptides, synthesised to facilitate a structure-activity relationship study on the N-terminus of hepcidin, which is the most active part of the peptide. Chapter 3 describes the synthesis of two hepcidin analogues (15 and 19) containing intramolecular disulfides. Peptide 15 represents the N-terminus of hepcidin constrained by one intramolecular disulfide. Peptide 19 sequence contains amino acids from the N-terminus of hepcidin and from the C-terminus, which we considered to be also relevant for the binding to ferroportin. The cysteines in peptide 19 were oxidised to disulfides. Chapter 4 focuses on the synthesis of two hepcidin analogues (27 and 35) with intermolecular disulfides: peptide 27 containing one intermolecular disulfide and peptide 35 containing two. Intermolecular disulfide formation is more challenging to achieve, as oxidation of the cysteines needs to be selective. A successful approach was developed by carefully selecting the protecting groups for the thiol group of the cysteines, which were removed stepwise in order to achieve selectivity. Chapter 5 discusses the synthesis and folding of [Lys21] 6-carboxy tetramethylrhodamine (TMR) labelled hepcidin and N13 6-carboxy uorescein (CF) labelled hepcidin 20. Hepcidin sequence presents a methionine in position 21, near the C-terminus, which was replaced, in the synthesis of [Lys21] TMR hepcidin, with a lysine protected at the N with 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde). This approach provides a site where TMR can be selectively attached. In N13 CF hepcidin 20 the last ve amino acids of hepcidin were not included in the synthesis and the peptide was labelled at the lysine in position 13. This peptide is not active and was synthesised as a negative control for biological evaluation purposes. Chapter 6 is divided in two sections. The first section describes the structural analysis by circular dichroism of the analogues synthesised in this project. The second section illustrates the biological evaluation results. Biological assays were performed at Vifor Pharma laboratories in Zurich. The [Lys21] TMR hepcidin was found to possess appreciable biological activity, being able to bind and internalize ferroportin with a potency only 4 fold lower than that of synthetic hepcidin. The structure-activity relationship study, conducted with peptides mimicking the N-terminus of hepcidin, suggests that a disul de exchange may be involved in the binding between the N-terminus and ferroportin. Furthermore, between these analogues, peptides 5 and 8 were found to be able to bind ferroportin without leading to its internalisation, suggesting an interesting antagonist activity. Peptides 19 and 27 show some activity, being 189 and 13 fold less active than hepcidin 25, respectively.

Limited availability of adult stem cells is a major draw back to their use both in regenerative medicine and in the treatment of malignant disease. A rapidly growing body of evidence has shown that enforced expression of small panel of genes, including Oct-3/4, KLF4, Sox2 and c-Myc, can induce the reprogramming of previously differentiated cells, to generate pluripotent stem (iPS) cells. However, the generation of iPS by genetic modification is highly desirable. Using GFP and Apoptin as model proteins, we have recently described a strategy for the generation of cell lines that secrete proteins that carry a modified HIV-TAT based secretable protein transduction domain. The presence of secreted Oct-3/4 and KLF4 in the culture medium of the producer 293T cell, we have not been able to demonstrate the uptake of this protrein by target cells. The biollogical consequences, and the reversibility of the induced effects of Oct-3/4 protein transduction into JURKAT and FDCP-1 cells are now under investigation. With a similar objective for the transient, non-genetic modification of target cells with induced pluripotency factors, we have also started the generation of non-integrating lentivirus vectors for the transient expression of the induced pluripotency factors. In preliminary studies we have already shown the efficient expression of Oct-3/4, KLF4 and Sox-2 in CD34+ primary human cells infected with integrating lentivirus vectors encoding each of these factors. The combined enforced expression of all three factors appear to generate a surface adherent, embryonic stem cell-like pheotype.

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No description available.

PECAM-I is a 130kDa member ofthe immunoglobulin superfamily, expressed on the surface ofhaematopoietic cells. The cytoplasmic tail contains two immullOreceptor tyrosine based inhibition motifs which can mediate both stimulatory and inhibitory signals. The aim of this thesis is to investigate the function of PECAM-I in the regulation of platelet activation and fonnation. Using PECAM-Io/' mice and cross-linking of PECAM-I using specific antibodies, I demonstrate a minor inhibitory role on platelet responses to the collagen receptor GPVI, the integrin allb~3 and the C-type lectin receptor CLEC-2. Furthennore, the degree of inhibition is considerably less than that produced by P0I2Ã?Â? This weak inhibitory effect ofPECAM-1 on platelet activation indicates that PECAM-I is not a major regulator of platelet activation. Further research reveals an important role in thrombopoiesis. Following induced thrombocytopenia, recovery . . of the peripheral platelet count is impaired in PECAM-l'' mice. A migration defect in PECAM-l'' megakaryocytesis identified in response to a gradient of SDFla. These observations are confinned in vivo with the demonstration of altered spatial localisation of megakaryocytes within the bone marrow in PECAM-Io/- mice. A further chapter considers the various approaches that could be used to establish the molecular basis ofthis effect.

No description available.