Magnetic nanoparticles (MNPs) hold great promise in the fields of biology and medicine. The synthesis of functional MNPs with precisely controlled crystallographic, physicochemical, and magnetic properties on a large scale still remains the challenge today. This thesis reports the exploration of liquid-fed flame spray pyrolysis (FSP) in the synthesis of functional MNPs, their surface modifications, and potential bio-applications. Superparamagnetic and ferromagnetic maghemite (γ-Fe2O3) nanoparticles, and silica-coated maghemite (SiO2/γ-Fe2O3) nanocomposites were synthesised using FSP. The size of γ-Fe2O3 was controllable from 6 to 53 nm, with morphology evolving from a disordered near-spherical shape to fully ordered 2-D hexagonal/octagonal platelet. The saturation magnetisation (Ms) increased from 21 to 74 emu/g with increasing particle size, up to 13 nm when Ms approached the bulk γ-Fe2O3 characteristics. In the case of SiO2/γ-Fe2O3, three distinct morphologies, namely the single segregated γ-Fe2O3 core- SiO2 shell, transitional mixed morphologies, and multi γ-Fe2O3 cores embedded in submicron SiO2 shell, were obtained. The core size, composite size, and morphology of γ- Fe2O3 were tunable by varying %SiO2 loading and the use of a quartz tube enclosure during flame synthesis. The magnetic behaviour correlated well with the crystal microstructure. Following the core particle design, protein adsorption-desorption behaviour on FSP-madeMNPs was studied. Bovine serum albumin (BSA) adsorption was found to follow the Langmuir isotherm, with high binding capacities (150−348 mg BSA/g particle) and fast association constants. Electrostatically governed BSA orientations were proposed for different particle-buffer systems. The adsorbed BSA was effectively recovered by pH-shift using K2HPO4. Subsequently, terminal amine, aldehyde, carboxylic, epoxy, mercapto and maleimide functionality were anchored onto the FSP-made γ-Fe2O3 particles. These versatile functional groups led to conjugation of active trypsin. The immobilised trypsin exhibited superior durability with >60% residual activity after one week, and excellent reusability for >5 cycles. The trypsin-conjugated MNPs are promising carriers in proteomics, demonstrating good substrate specificity with equivalent or better sequence coverage compared to free trypsin in insulin and BSA digestion. In another application, a refined silanisation procedure simultaneously reduced γ-Fe2O3 to Fe3O4, and generated thiol enriched surface for matrix metalloproteinase-2 (MMP-2) conjugation. The highly active MMP-2-conjugated MNPs could potentially enhance the interstitial transport of macromolecule/nanoparticles in drug delivery.

<p>Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release <i>in vivo</i> and <i>ex vivo</i> during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure.</p><p>In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO.</p><p>The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM.</p><p>ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.</p>

Serum levels of eosinophil granule proteins namely ECP, EPO and EPX, which are stored in the matrix of specific granules, were shown to correlate with the course of disease in disorders involving eosinophils. The concentration of eosinophil proteins in serum is the result of their release in vivo and ex vivo during the sampling procedure. Generally, eosinophils release the content of their specific granules in three ways: exocytosis, piecemeal degranulation (PM) or cytolysis. Which of them is operating in circulating eosinophils has not yet been defined. The aim of this thesis was to study the mechanisms of granule protein release from blood eosinophils in respect of protein subcellular localization and cell ultrastructure. In patients with bacterial infections, serum levels of ECP but not EPO increased, while in patients with viral infections both proteins remained within the range of healthy controls. G-CSF is a cytokine involved in the response mechanism to bacterial but not viral infections. Administration of G-CSF to healthy subjects induced an elevation of eosinophil numbers and a preferential increase of serum EPX and ECP in comparison to EPO. The model of PM consists of the stepwise transportation of specific granule contents from the granules towards the plasma membrane. We observed that administration of G-CSF to healthy subjects and the allergen exposure of allergic subjects during the pollen season, caused changes in the ultrastructure of eosinophil specific granules such as loosening of the matrix, granule matrix lucency and ragged losses of their core. Similar alterations of morphology had been previously described for eosinophils undergoing PM. ECP, EPX and EPO were localized not only in the specific granules but also in extra-granular compartments as shown both by immuno electron microscopy and subcelular fractionations, An extra-granular EPX compartment was present in healthy as well as in allergic and in hypereosinophilic subjects, and there were no significant differences in its size between the groups. The size of the extra-granular compartments of ECP and EPO was increased in allergics during the season, and these compartments were clearly separate from that of EPX. Results of this show the differential mobilization ofgranule proteins in blood stream eosinophils serum and indicates PM as its mechanism.

The aim of the present thesis was to further increase our understanding of mechanisms contributing to and maintaining cellular insulin resistance in type 2 diabetes (T2D). For this reason, the effects of high glucose and insulin levels on glucose transport capacity and insulin signaling, with emphasis on insulin receptor substrate 1 (IRS-1) were assessed in fat cells. Altered levels of IRS-1 have previously been observed in adipose tissue from insulin-resistant and T2D subjects. A high glucose level (≥15 mM) for 24 h exerted only a minor impairment on glucose transport capacity in human adipocytes, as opposed to rat adipocytes. However, when combined with a high insulin level (104 µU/ml), basal and insulin-stimulated glucose transport was significantly impaired in both human and rat adipocytes. This was associated with a depletion of IRS-1 and IRS-2 protein levels in rat adipocytes, as a result of post-translational changes and altered gene transcription, respectively. In human adipocytes was only IRS-1 protein levels reduced. The high glucose/high insulin setting achieved maximal impairment of glucose transport within 6 h. Subsequent incubations of rat adipocytes under physiological conditions could partially restore insulin sensitivity. Interestingly, in both human and rat fat cells, decreased levels of IRSs occurred after the establishment of impaired glucose transport, suggesting that the observed depletion of IRSs is a consequence rather than a cause of insulin resistance. Nonetheless, IRS depletion is likely to further aggravate insulin resistance. Tyrosine phosphorylation of IRS-1 upon insulin stimulation activates the signaling pathway that mediates glucose transport. Pre-treatment of human adipocytes with high glucose and insulin levels was not associated with any alterations in the total IRS-1 Tyr612 phosphorylation following 10 min insulin stimulation. However, a significant increase in basal Tyr612 phosphorylation was observed. Furthermore, a rise in basal IRS-1 Ser312 phosphorylation was found. This is associated with reduced IRS-1 function and is considered to target IRS-1 to degradation pathways, and thus could potentially explain the observed decrease in IRS-1 protein levels. Our results imply an enhanced activation of insulin’s negative-feedback control mechanism that inhibit IRS-1 function. This could potentially have contributed to the observed impairment of insulin action on glucose transport in these cells. Accordingly, we have also shown that the downstream activation of protein kinase B upon insulin-stimulation is significantly impaired in human adipocytes exposed to the high glucose/high insulin setting, indicating a defect in the signaling pathway mediating glucose transport. We also investigated whether there are humoral factors in the circulation of T2D patients that contribute to peripheral insulin resistance. Human adipocytes cultured for 24 h in medium supplemented with 25% serum from T2D subjects, as compared to serum from non-diabetic subjects, displayed significantly reduced insulin-stimulated glucose uptake capacity. The effect could neither be attributed to glucose, insulin, FFA, TNF-α or IL-6 levels in the serum, but other circulating factor(s) seem to be of importance. In conclusion, chronic conditions of elevated glucose and/or insulin levels all impair insulin action on glucose turnover, but to different extents. A clear distinction between rat and human fat cells in the response to these different milieus was also observed. Alterations in the function of the key insulin signaling protein IRS-1 might be involved in the mechanisms underlying the impaired glucose uptake capacity. IRS-1 reduction however, occurs after but probably aggravates the existing insulin resistance. The effects of high glucose and/or insulin levels may be of importance in T2D, but additional novel factors present in the circulation of T2D patients seem to contribute to cellular insulin resistance.

Fibrotic diseases have a poor prognosis with no FDA approved therapies. Monocyte-derived, fibroblast-like cells called fibrocytes participate in the formation of fibrotic lesions. The conserved pentraxin protein serum amyloid P (SAP) inhibits fibrocyte differentiation in cell culture, and injections of SAP significantly reduce fibrosis in several animal models. SAP binds to the receptors for the Fc portion of immunoglobulin G (FcγR), and has been crystallized bound to FcγRIIa. The in vivo activity of SAP appears to be dependent on the common γ chain (FcγR) of activating Fc receptors. The goal of my project is to elucidate the functional domains of SAP and the receptor responsible for SAP bioactivity, which could lead to refinements for SAP as a therapeutic agent and additional drug targets. I found that mutagenesis of the residues critical for SAP binding to FcγRIIa only moderately decreases SAP's ability to inhibit fibrocyte differentiation. In murine cells, deletion of FcγR or FcγRI significantly reduced sensitivity to SAP. Deletion of the combination of FcγRIIb/FcγRIIIa/FcγRIV did not significantly affect sensitivity to SAP, while deletion of just the inhibitory receptor FcγRIIb increased sensitivity to SAP. In human cells, siRNA-mediated reduction of FcγR or FcγRI levels significantly decreased sensitivity to SAP, while reduction of FcγRIIb levels increased sensitivity to SAP. These observations suggest that SAP, at least in part, uses FcγRI and FcγR to inhibit fibrocyte differentiation. I am also interested in how SAP functions in various disease states. SAP is known to be elevated in Alzheimer's disease (AD) and binds to amyloid plaques in the brain, a key hallmark of AD. There is a significant population of individuals that have key hallmarks of AD but show no signs of cognitive impairment, termed non-demented with AD neuropathology (NDAN). I evaluated SAP levels in post mortem samples of hippocampus and frontal cortex in age-matched controls, AD, and NDAN individuals. AD individuals had significantly increased SAP levels, while NDAN samples had no significant difference in SAP levels compared to controls. These results suggest that low levels of SAP in plaques marks the brains of individuals that escape dementia despite the presence of beta amyloid plaques.

No description available.

Pharmacodynamic studies are important for the optimal use of antimicrobial agents. Combination antifungal therapy may be one method to improve outcome in invasive Candida infections. An in vitro kinetic model to study the pharmacodynamic effects of a combination of two antifungal agents with different elimination rates was developed and the pharmacodynamics of amphotericin B (AMB), voriconazole (VRC) or the combination was evaluated. Exposure to VRC inhibited the fungicidal activity of sequential doses of AMB against VRC-susceptible strains of C. albicans. The interaction was VRC dose-dependent. AMB activity was regained once VRC was removed or it increased gradually when the concentration of VRC had fallen below the minimum inhibitory concentration (MIC). The VRC-AMB interaction, however, was also present against strains of C. albicans, C. glabrata and C. krusei despite reduced VRC susceptibility. Against these strains the interaction was not predicted by the MIC value, suggesting that mechanisms of resistance may be of importance. Until more data are available, a reasonable recommendation is probably to avoid the sequential use of VRC followed by AMB and to use the combination of VRC and AMB for the treatment of Candida infections with caution. Only the unbound fraction of a drug is generally accepted as pharmacologically active. The activity of posaconazole (POS) with a protein binding of 98-99% was tested in serum against Candida species and compared with the calculated unbound serum concentration in protein-free media. Significant differences emerged at clinically relevant POS serum concentrations of 1.0 and 0.10 mg/l compared with the serum control regimen against one strain of C. lusitaniae. In RPMI 1640 the corresponding calculated unbound concentrations resulted in no effect for the low dose regimen compared with the RPMI 1640 control regimen. Further, against seven additional Candida strains tested, the effect of POS was greater in serum than in RPMI 1640. A flux from serum protein bound to fungal lanosterol 14α-demethylase bound POS may be the explanatory mechanism.

The diagnosis and monitoring of AML relies predominantly on the identification of blast cells in the bone marrow and peripheral blood. While at the time of diagnosis the identification of leukemic cells is relatively easy, during remission the identification of small numbers of blasts is problematic. This is most evident by the fact that patients who achieve complete remission frequently relapse, despite pathologic examination indicating a marked reduction in leukemic cell burden. In this thesis I have explored the potential of using serum proteins secreted by leukemic cells as a means of monitoring disease in patients. To identify proteins that might be useful for monitoring, I took advantage of published gene expression arrays and looked into online bioinformatics databases. Using specific characteristics, I was able to identify approximately 107 candidate proteins secreted by AML cells. RT-PCR analysis and ELISA assays were performed to evaluate the variability of expressions and serum level differences of twelve different proteins in the list.

The diagnosis and monitoring of AML relies predominantly on the identification of blast cells in the bone marrow and peripheral blood. While at the time of diagnosis the identification of leukemic cells is relatively easy, during remission the identification of small numbers of blasts is problematic. This is most evident by the fact that patients who achieve complete remission frequently relapse, despite pathologic examination indicating a marked reduction in leukemic cell burden. In this thesis I have explored the potential of using serum proteins secreted by leukemic cells as a means of monitoring disease in patients. To identify proteins that might be useful for monitoring, I took advantage of published gene expression arrays and looked into online bioinformatics databases. Using specific characteristics, I was able to identify approximately 107 candidate proteins secreted by AML cells. RT-PCR analysis and ELISA assays were performed to evaluate the variability of expressions and serum level differences of twelve different proteins in the list.

In this thesis, a direct method of Atomic Force Microscopy (AFM) technique has been developed to measure the adhesion forces between BSA and two different surfaces: mica (a hydrophilic surface); and polystyrene (a hydrophobic surface); in PBS solution. We have shown possible to measure interactions between proteins and substrate surface directly without any modification to the substrate and the AFM tip; this means protein molecules can keep the natural elastic property within the force measurements. The average measured value of adhesion forces between BSA and mica is 0.036 ± 0.002 nN, and between BSA and polystyrene is 0.066 ± 0.003 nN. The polystyrene surface is more adhesive to BSA than the mica surface. This is consistent with previous research, which assessed that hydrophobic surfaces enhance protein adhesion but hydrophilic surfaces do not.