Degradation of mRNA is a highly regulated step important for proper gene expression. Degradation of eukaryotic mRNA is initiated by shortening of the 3’ end located poly(A) tail. Poly(A)-specific ribonuclease (PARN) is an oligomeric enzyme that degrades the poly(A) tail with high processivity. A unique property of PARN is its ability to interact not only with the poly(A) tail but also with the 5’ end located mRNA cap structure. A regulatory role in protein synthesis has been proposed for PARN based on its ability to bind the cap that is required for efficient initiation of eukaryotic mRNA translation. Here we have investigated how the cap structure influences PARN activity and how PARN binds the cap. We show that the cap activates PARN and enhances the processivity of PARN. Further we show that the cap binding complex (CBC) inhibits PARN activity through a protein-protein interaction. To investigate the cap binding property of PARN, we identified the cap binding site at the molecular level using site-directed mutagenesis and fluorescence spectroscopy. We identified tryptophan 475, located within the RNA recognition motif (RRM) of PARN, as crucial for cap binding. A crystal structure of PARN bound to cap revealed that cap binding is mediated by the nuclease domain and the RRM of PARN. Tryptophan 475 binds the inverted 7-Me-guanosine residue through a stacking interaction. Involvement of the nuclease domain in cap binding suggests that the cap site and the active site overlap. Mutational analysis showed that indeed amino acids involved in cap binding are crucial for hydrolytic activity of PARN. Taken together, we show that PARN is an allosteric enzyme that is activated by the cap structure and that the allosteric cap binding site in one PARN subunit corresponds to the active site in the other PARN subunit.

There are three novel 2-trifluoromethyl-activated bisfluoro monomers have been successfully synthesized in this study, and the nomenclatures are shown as follows: 4,4¡¦¡¦¡¦¡¦-Difluore-3,3¡¦¡¦¡¦¡¦-bis(trifluoromethyl)-2¡¦¡¦,3¡¦¡¦,5¡¦¡¦,6¡¦¡¦-triphenyl(M4), 4,4¡¦¡¦¡¦¡¦-Difluore-3,3¡¦¡¦¡¦¡¦-bis(trifluoromethyl)-2¡¦¡¦,3¡¦¡¦,5¡¦¡¦-triphenyl(M3), 4,4¡¦¡¦¡¦¡¦-Difluore-3,3¡¦¡¦¡¦¡¦-bis(trifluoromethyl)-2¡¦¡¦,3¡¦¡¦-triphenyl(M2). Through polymerization with 1,1-dihydroxydiphenyl cyclododecane the monomers M2, M3 and M4 were accordingly converted into poly(arylene ether)s P2-1,1C, P3-1,1C and P4-1,1C, respectively. These polymers exhibit weight-average molecular weight up to 2.25¡Ñ105g/mol. The molecular weight were investigated and confirmed by MASS and GPC. The molecular structures were investigated and confirmed by NMR and FTIR. The UV-VIS absorption and photoluminescence spectra measurement of all the monomers and polymers in dilute solutions and in solid state were conducted. The results show that all monomers and polymers in dilute solutions have no absorption in the vision light region of spectrum. The absorption spectra of polymer thin films showed high optical transparency up to 90%. The photoluminescence spectra of all monomers and polymers in dilute solutions and thin film emits light with high intensity and wavelength in region of 350~380nm. Thermal analysis studies were conducted with TGA, DSC, TMA and crystal property study was performed by XRD. The results show that these polymers did not show melting but showed ultrahigh Tg values ranging from 270~330¢XC in DSC and TMA measurements, so it indicated that three polymers were not crystalline materials. Outstanding thermal stability is over then 440~ 460¢XC for 5% weight loss in TGA under nitrogen atmosphere. So it could make manufacture in higher temperature and have higher thermal stability. With optical properties of polymer thin films, we utilized Ellipsometer to measure refractive index and the results showed no birefringence for these polymers. The polymer thin films show low polarity and high hydrophobicity could be attested by the measured results of contact angle and surface energy. The HOMO and LUMO energy level of monomers are both measured by Cyclic Voltammetry and theoretical calculation. The absorption spectra of polymer thin films showed no absorption in the visible light region of the spectrum i.e., having a high optical transparency. All above stated material properties are good for doing as a plastic substrate of devices or panel display.

Hematopoietic stem cells (HSCs) are commonly used in the treatment of blood cancers, like leukemia, and other cancers where radiation or chemotherapy damages the native HSC population. The development of a novel system to study and maintain HSCs ex vivo would give researchers and clinicians the ability to investigate the basic biological processes of HSCs, improve current treatment regimens, and explore their use in new therapies. The work in this thesis focuses on the development of a synthetic PEG hydrogel scaffold that accurately mimics aspects of the HSC microenvironment and can expand clinically relevant HSC populations. PEG hydrogel well surfaces were covalently functionalized with bioactive factors known to be critical in controlling HSC fate in vivo. In initial studies, 32D cells, a myeloid progenitor, were cultured in the wells for 6 days. On surfaces with the adhesive RGDS peptide sequence, 32D cell adhesion increased concurrently with RGDS surface concentrations. With the immobilization of two niche cytokines, SCF and SDF1α, onto hydrogel surfaces, 32D cells demonstrated significant increases in adhesion and spreading. These results confirmed that hematopoietic cell behavior could be controlled through the design of the bioactive PEG scaffold. In studies with a primary hematopoietic cell population (c-kit + , lin - ), the effects of bioactive molecules on cell expansion and differentiation were investigated after 2 weeks in culture. The adhesive peptides sequences, RGDS and CS1, and four niche proteins, SCF, SDF1α, JAG1, and IFNγ, were covalently tethered to hydrogel well surfaces. Primary cells proliferated significantly on gels containing SCF and IFNγ though only SCF was capable of preventing HSC differentiation. Cells cultured on surfaces functionalized with JAG1 and SDF1α did not proliferate extensively, but they were able to maintain primitive HSC populations. Primary c-kit + cells were also encapsulated within biodegradable PEG hydrogels and cultured for 2-5 weeks. Cells remained viable for 5 weeks in culture, and preliminary results indicated minimal cell differentiation. In this work, biomimetic PEG hydrogels were successfully employed to expand HSC populations in both two and three dimensions. The ability to generate large populations of primitive HSCs ex vivo has broad clinical and research implications.

Because the gel-spinning process has the potential to yield fibers of high strength and high modulus, this technique was employed to process continuous filaments of PVA/CNT, having CNTs at ¡Ü1 weight percent of polymer. A gel aging technique was employed with the goal of increasing the draw ratio for composite fibers and for promoting the development of crystalline PVA. Since residual solvent can lower the mechanical properties of drawn fibers, solvent phases of water and dimethyl sulfoxide (DMSO) within the drawn fibers were also characterized. As embedded SWNTs were uniaxially aligned along the drawn fiber axis, they were found to induce preferential alignment in the PVA side groups as well as for the residual solvent. This was attributed to charge transfer between SWNT and the respective functional groups. This orientation behavior has been characterized using Raman spectroscopy and infra-red dichroism. The behaviors of gel crystallization and solvent freezing within PVA/CNT dispersions were studied using thermal analysis and rheology. Carbon nanotubes were found to nucleate PVA crystallization in the gel state. PVA/CNT gel aging behavior was characterized by structural, thermal, and mechanical, and dynamic mechanical means. Gel aging was shown to increase the draw ratio of PVA/CNT fibers, and the development of the higher temperature melting peak was attributed to the draw induced ordering of PVA along CNTs. The scanning electron micrographs of fractured PVA/CNT fibers showed fibrils having an average diameter of about 22 nm. The storage modulus of aged gel was a function of solvent diffusion, which changed with aging time. CNTs were shown to have stabilized the gel network, as characterized by the dynamic mechanical properties, and to provide nucleation sites for the ordering of PVA chains, as characterized by WAXD.

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.

Nonequilibrium work theorems, such as the Jarzynski equality and the Crooks fluctuation theorem, allow one to use nonequilibrium measurements to determine equilibrium free energies. For example, it has been demonstrated that the Crooks fluctuation theorem can be used to determine RNA folding energies. We used single-molecule manipulation with an atomic force microscope to measure the work done on poly(dA) as it was stretched and relaxed. This single-stranded nucleic acid exhibits unique base-stacking transitions in its force-extension curve due to the strong interactions among A bases, as well as multiple pathways. Here we showed that free energy curves can be determined by using the Crooks fluctuation theorem. The nonequilibrium work theorem can be used to determine free energy curves even when there are multiple pathways.

In the recent years, development of various organic and inorganic nano-sized systems has gained great interests especially for cancer diagnosis and treatment and intense researches are carried out in this area. Regarding to the recent trends for drug delivery system design, the novel approaches for drug carriers are mainly based on development of smart and nano-size drug carriers which are targeted to cancer cells. Hence, for an effective tumor-targeted delivery device, besides its chemical structure further criteria such as detection of tumor site and sensitivity to the higher temperature and lower pH of the tumor compare to rest of the body gains importance. The aim of this study is to design and prepare polysebacic anhydride (PSA) based nanocapsules (NCs) loaded with Doxorubicin (DOX) which is an anti cancer drug. In order to obtain an intelligent delivery system, drug-loaded nanocapsules were coated with pH sensitive poly (L-histidine). PSA nano-carriers were firstly loaded with DOX and then in order to introduce pH sensitivity, they were coated with poly (L-histidine). PLH-coated NCs were modified with polyethylene glycol (PEG) to prevent their macrophage uptake. Drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH 4) and physiological (pH 7.4) media. The physical and chemical properties of the nano particles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were performed on MDA-MB-231 breast cancer cells by using WST Kit 8 cell viability test. In order to obtained results, pH sensitive nanocapsules with size 230 nm exhibited cellular uptake and promising intracellular release of drug.

The biological function of a protein is determined by its amino acid sequence, structure, and internal dynamics. In turn the prediction of a protein structure from its folding pathway involves the characterization of the dynamics of the polypeptide backbone. This study addresses how the internal dynamics of arborescent polypeptides are affected by increased crowding of the interior of these branched polymer molecules. Linear, comb-branched, and arborescent poly(L-glutamic acid) (PGA) samples were analyzed by 1H NMR spectroscopy to determine their chain conformation. The PGA chains of these constructs were shown to adopt α-helical and random coil conformations in N,N-dimethylformamide (DMF) and in dimethyl sulfoxide (DMSO), respectively. The hydrodynamic diameter (Dh) of the arborescent PGAs, determined using dynamic light scattering measurements, increased with increasing generation number and when the side-chains adopted random coil instead of α-helical conformations. The PGA samples were labelled with 1-pyrenemethylamine to determine how their structure affected the internal dynamics of the arborescent polymers in solution, from the analysis of their fluorescence spectra and decays. For each pyrene-labelled polymeric construct excimer formation increased with increasing pyrene content, and the efficiency of excimer formation increased with the generation number due to the increased density of the macromolecules. Comparison of the time-resolved fluorescence results acquired in DMF and in DMSO demonstrated that the helical conformation led to slower chain dynamics in DMF and that despite the higher viscosity of DMSO, the polypeptide side-chains were more mobile as a consequence of the random coil conformation of the linear PGA segments. These results suggest that the formation of structural motives inside a polypeptide slows down its internal dynamics.