In this study, the interaction energies between aromatic organic molecules (AOMs) and cellulose are explored using density functional theory (DFT) through the software SIESTA and the exchange-correlational functional VDW-DRSLL. Three AOMs will be modelled: benzene, benzamide and benzoic acid. Firstly, the interaction energies of the dimers of the AOMs are determined. Then, the obtained interaction energies of the cellulose-AOM complexes are compared to the former in order to decide which interaction is stronger. It is found that the studied AOMs are more likely to interact with cellulose than with another identical monomer; benzamide has the highest propensity to interact, followed by benzoic acid and benzene. Furthermore, for all interaction energy calculations a counterpoise correction term will be introduced as an addition to the SIESTA optimisation and it will be shown that without this correction the acquired energy minima will deviate significantly from accepted values from previous studies.

Optimization of Affibody molecules would allow for high contrast imaging of cancer associated surface receptors using molecular imaging. The primary aim of the thesis was to develop Affibody-based PET imaging agents to provide the highest possible sensitivity of RTK detection in vivo. The thesis evaluates the effect of radiolabelling chemistry on biodistribution and targeting properties of Affibody molecules directed against HER2 and PDGFRβ. The thesis is based on five published papers (I-V). Paper I. The targeting properties of maleimido derivatives of DOTA and NODAGA for site-specific labelling of a recombinant HER2-binding Affibody molecule radiolabelled with 68Ga were compared in vivo. Favourable in vivo properties were seen for the Affibody molecule with the combination of 68Ga with NODAGA. Paper II. The aim was to compare the biodistribution of 68Ga- and 111In-labelled HER2-targeting Affibody molecules containing DOTA, NOTA and NODAGA at the N-terminus. This paper also demonstrated favourable in vivo properties for Affibody molecules in combination with 68Ga and NODAGA placed on the N-terminus. Paper III.  The influence of chelator positioning on the synthetic anti-HER2 affibody molecule labelled with 68Ga was investigated. The chelator DOTA was conjugated either at the N-terminus, the middle of helix-3 or at the C-terminus of the Affibody molecules. The N-terminus placement provided the highest tumour uptake and tumour-to-organ ratios. Paper IV. The aim of this study was to evaluate if the 68Ga labelled PDGFRβ-targeting Affibody would provide an imaging agent suitable for PDGFRβ visualization using PET. The 68Ga labelled conjugate provided high-contrast imaging of PDGFRβ-expressing tumours in vivo using microPET as early as 2h after injection. Paper V. This paper investigated if the replacement of IHPEM with IPEM as a linker molecule for radioiodination of Affibody molecules would reduce renal retention of radioactivity. Results showed that the use of the more lipophilic linker IPEM reduced the renal radioactivity retention for radioiodinated Affibody molecules. In conclusion, this thesis clearly demonstrates that the labelling strategy is of great importance with a substantial influence on the targeting properties of Affibody molecules and should be taken under serious considerations when developing new imaging agents.

 In rat testes, at stage VIII of the epithelial cycle of spermatogenesis, two cellular events, namely blood-testis barrier (BTB) restructuring and spermiation, take place simultaneously but at the opposite ends of the seminiferous epithelium. BTB is constituted by tight junctions (TJs), basal ectoplasmic specializations (ES), gap junctions and desmosomes, which must disassemble intermittently at stage VIII to facilitate preleptotene spermatocyte migration across the barrier. Synchronously, spermiation occurs at the luminal edge of the tubule lumen, involving the disruption of the apical ES, the only anchoring device there, and the release of sperm. The mechanism coordinating these events is not well understood. In this dissertation, I provide evidence that intercellular adhesion molecule (ICAM)-1 and -2, are working in concert with sarcoma (Src) family kinases to regulate these events. ICAMs comprise an immunoglobulin subfamily of cell adhesion proteins expressed by hematopoietic, endothelial and epithelial cells. They are known to function in the transendothelial migration of leukocytes. In the rat testis, ICAM-1 was shown to localize to both BTB and apical ES stage-specifically, with its immunoreactivity highest at stage VIII at the BTB. Besides co-immunoprecipitation and co-localization with BTB proteins, such as occludin and N-cadherin, ICAM-1 was found to promote BTB integrity in that its over-expression (O-E) in Sertoli cells in vitro increased transepithelial electrical resistance (TER). However, O-E of a truncated form of ICAM-1 (sICAM-1) that only consisted of the extracellular domain resulted in decreased TER and down-regulation of several BTB constituent proteins, possibly via the Src/Pyk2 signaling pathway. O-E of sICAM-1 in vivo also compromised the BTB integrity. These findings illustrate that ICAM-1 is an important regulator of the BTB. On the other hand, the localization of ICAM-2 was restricted to the Sertoli-germ cell interface and absent from the BTB, and associated with β1-integrin, nectin-3 and F-actin at the apical ES. Further, ICAM-2 was shown to interact with Src and Pyk2, as well as annexin II, a phospholipid-binding protein. Intriguingly, ICAM-2, Src and annexin II were specifically up-regulated during CdCl2-induced germ cell loss. These results reveal that ICAM-2 actively participates in the restructuring of apical ES based on studies using the cadmium model. The function of c-Yes, a member of the Src family, was also investigated. It was found to be stage-specifically expressed at the BTB and the apical ES, and it structurally associated with BTB components (e.g., occludin and N-cadherin) and with the apical ES proteins (e.g., β1-integrin, laminin β3 and γ3). In the study, the knockdown of c-Yes by RNAi in vitro and in vivo affected BTB and apical ES function, causing changes in the distribution/localization of adhesion proteins at the BTB and the apical ES, inducing germ cell loss from the seminiferous epithelium, possibly via an interference with the F-actin network. These findings implicate that ICAMs and c-Yes are regulatory molecules of cell adhesion at the BTB and the apical ES, and are biomarkers for male contraceptive development. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Doctoral / Doctor of Philosophy

Nowadays, RNA is extensively acknowledged an important role in the functions of information transfer, structural components, gene regulation and etc. The secondary structure of RNA becomes a key to understand structure-function relationship. Computational prediction of RNA secondary structure does not only provide possible structures, but also elucidates the mechanism of RNA folding. Conventional prediction programs are either derived from evolutionary perspective, or aimed to achieve minimum free energy. In vivo, RNA folds during transcription, which indicates that native RNA structure is a result from both thermodynamics and kinetics. In this thesis, I first reviewed the current leading kinetic folding programs and demonstrate that these programs are not able to predict secondary structure accurately. Upon that, I proposed a new sequential folding program called GTkinetics. Given an RNA sequence, GTkinetics predicts a secondary structure and a series of RNA folding trajectories. It treats the RNA as a growing chain, and adds stable local structures sequentially. It is featured with a Z-score to evaluate stability of local structures, which is able to locate native local structures with high confidence. Since all stable local structures are captured in GTkinetics, it results in some false positives, which prevents the native structure to form as the chain grows. This suggests a refolding model to melt the false positive hairpins, probable intermediate structures, and to fold the RNA into a new structure with reliable long-range helices. By analyzing suboptimal ensemble along the folding pathway, I suggested a refolding mechanism, with which refolding can be evaluated whether or not to take place. Another way to favor local structures over long-distance structures, we introduced a distance penalty function into the free energy calculation. I used a sigmoidal function to compute the energy penalty according to the distance in the primary sequence between two nucleotides of a base pair. For both the training dataset and the test dataset, the distance function improves the prediction to some extent. In order to characterize the differences between local and long-range helices, I carried out analysis of standardized local nucleotide composition and base pair composition according to the two groups. The results show that adenine accumulates on the 5' side of local structure, but not on that of long-range helices. GU base pairs occur significantly more frequent in the local helices than that in the long-range helices. These indicate that the mechanisms to form local and long range helices are different, which is encoded in the sequence itself. Based on all the results, I will draw conclusions and suggest future directions to enhance the current sequential folding program.

The single-particle momentum is studied as a tool for the visualization of the electronic regions in atoms and molecules. The limiting values of this function correctly obey two fundamental theorems: Kato's cusp condition and the Hoffmann-Ostenhof and Hoffmann-Ostenhof exponential decay. The local momentum also depicts the electron shell structure in atoms as given by its local maxima and inflection points. The integration of the electron density in a shell gives electron populations that are in agreement with the ones expected from the periodic table of the elements. The shell structure obtained is in agreement with higher level of theory computations. The average of the local kinetic energy associated with the local momentum is the Weizsäcker kinetic energy. It is shown that this quantity provides an estimate of steric interactions in molecules. The single-particle momentum is a practical tool for the exploration of new stabilizing interactions for all kinds of molecular systems. It provides a three-dimensional representation of the molecular structure and depicts the polarizability regions, a feature not available with other continuous analyses. A general definition of the radius of an atom in terms of its ionization energy is found. A relationship between these two fundamental properties is derived from the radial distribution function and the local momentum for the valence electrons. Strong correlations with well-known atomic radii suggest that this is a universally valid definition of the atomic radius. The stability of peptides in the alpha-helix conformation upon replacement of the central amino acid is studied. These systems were optimized with a continuous solvent model and a recently developed DFT functional with empirical terms accounting for dispersion interactions. Both, the dispersion terms and the solvent model are directly related to the polarizability of the involved atoms. A new formula for an ab initio computation of the polarizability is introduced and tested for the amino acids.

No description available.

E2A-PBX1 is expressed as a consequence of a recurring chromosomal translocation seen in 5% of acute lymphoblastic leukemia cases. We recently reported that substitution of a leucine residue (L20A) within the N-terminal transcriptional activation domain (AD1) of E2A-PBX1 markedly impairs binding to the KIX domain of CBP/p300 and, importantly, leukemia induction in a mouse bone marrow transplantation model. Since both the protein-protein interaction and consequent leukemogenesis rely on a focal contact point and might therefore be susceptible to antagonism by small molecules, we devised a cell-free assay based on fluorescence anisotropy (FA) to detect binding of a fluorescently labeled peptide derived from AD1 of E2A-PBX1 (FITC-E2A) with recombinantly expressed KIX domain. The optimized FA assay reveals a dissociation constant of 2 µM for the wild-type interaction and correctly detects disruption of the complex by naphthol AS-E phosphate, a compound previously shown to antagonize KIX binding. The optimized FA assay was used to screen the Prestwick, Spectrum and Chembridge libraries containing 12400 compounds in total. Of the initial 43 positive hits from the libraries, 10 caused a reproducible decrease in FA. Since intrinsic small molecule fluorescence can produce false positive results in the FA-based screen, intrinsically fluorescent compounds were excluded from further analysis unless they could be shown to bind to KIX. Two hits, L1 and C2, were intrinsically fluorescent but demonstrated KIX interactions and one hit, P9, was not intrinsically fluorescent. These three compounds were tested for their ability to inhibit binding of a larger portion of E2A (residues 1 to 483) to full length CBP in a pull down assay with only compound P9 demonstrating efficacy. Further characterization of P9 by NMR showed no binding to KIX, however evaluation by FA showed binding to FITC-E2A with a 20 µM affinity. A cell-based cytotoxicity assay demonstrated that compound P9 was slightly more toxic on leukemic cells that express E2A-PBX1, compared to leukemic cells lacking E2A-PBX1 expression. Mammalian two-hybrid analysis did not provide details of the effects of P9 on the E2A:KIX interaction. We expect the identification of a novel compound, P9, capable of disrupting the oncogenic E2A-PBX1:CBP interaction, to guide the development of effective, less toxic leukemia drugs and provide new tools for elucidating the molecular mechanisms of leukemia induction by E2A-PBX1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-08-31 11:13:19.517

No description available.

No description available.