Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.

Examining the growth and evolution of magnetic fields in clusters of galaxies: a numerical perspective

Magnetic fields are ubiquitous in galaxy cluster atmospheres and have a variety of astrophysical and cosmological consequences. Magnetic fields can contribute to the pressure support of clusters, affect thermal conduction, and modify the evolution of bubbles driven by active galactic nuclei. However, we currently do not fully understand the origin and evolution of these fields throughout cosmic time. Furthermore, we do not have a general understanding of the relationship between magnetic field strength and topology and other cluster properties, such as mass and X-ray luminosity. We can now begin to answer some of these questions using large-scale cosmological magnetohydrodynamic (MHD) simulations of the formation of galaxy clusters including the seeding and growth of magnetic fields. Using large-scale cosmological simulations with the FLASH code combined with a simplified model of the acceleration of cosmic rays responsible for the generation of radio halos, we find that the galaxy cluster frequency distribution and expected number counts of radio halos from upcoming low-frequency sur- veys are strongly dependent on the strength of magnetic fields. Thus, a more complete understanding of the origin and evolution of magnetic fields is necessary to understand and constrain models of diffuse synchrotron emission from clusters. One favored model for generating magnetic fields is through the amplification of weak seed fields in active galactic nuclei (AGN) accretion disks and their subsequent injection into cluster atmospheres via AGN-driven jets and bubbles. However, current large-scale cosmological simulations cannot directly include the physical processes associated with the accretion and feedback processes of AGN or the seeding and merging of the associated SMBHs. Thus, we must include these effects as subgrid models. In order to carefully study the growth of magnetic fields in clusters via AGN-driven outflows, we present a systematic study of SMBH and AGN subgrid models. Using dark-matter only cosmological simulations, we find that many important quantities, such as the relationship between SMBH mass and galactic bulge velocity dispersion and the merger rate of black holes, are highly sensitive to the subgrid model assumptions of SMBHs. In addition, using MHD calculations of an isolated cluster, we find that magnetic field strengths, extent, topology, and relationship to other gas quantities such as temperature and density are also highly dependent on the chosen model of accretion and feedback. We use these systematic studies of SMBHs and AGN inform and constrain our choice of subgrid models, and we use those results to outline a fully cosmological MHD simulation to study the injection and growth of magnetic fields in clusters of galaxies. This simulation will be the first to study the birth and evolution of magnetic fields using a fully closed accretion-feedback cycle, with as few assumptions as possible and a clearer understanding of the effects of the various parameter choices.

A pilot study on the productivity and behavior of the gestating sow housed in a flex stall

Animal welfare is a controversial topic in modern animal agriculture, partly because it generates interest from both the scientific community and the general public. The housing of gestating sows, particularly individual housing, is one of the most critical concerns in farm animal welfare. We hypothesize that the physical size of the standard gestation stall may limit movement and evoke demands and challenges on the sow to affect the physiological and psychological well-being of the individually housed sow. Thus, improvements in the design of the individual gestation stall system that allow more freedom to move, such as increasing stall width or designing a stall that could accommodate the changing size of the pregnant sow, may improve sow welfare. The objective of this pilot study was to evaluate the effects of a width adjustable stall (FLEX) on productivity and behavior of dry sows. The experiment consisted of 3 replications (block 1, n=4 sows; block 2, n=4 sows; block 3, n=8 sows), and multi-parious sows were allotted to either a FLEX stall or standard gestation stall for 1 gestation period. Sow mid-girth (top of the back to bottom of the udder) was measured 5-6 times throughout gestation to determine the best time points for FLEX stall width expansions. FLEX stall width was adjusted according to mid-girth measurements, and expanded to achieve an additional 2 cm of space between the bottom of the sow’s udder and floor of the stall so that sows could lie in full lateral recumbency without touching the sides of the stall. Productivity data recorded included: sow body weight (BW) and BW gain, number of piglets born and born alive, proportions of piglets stillborn, mummified, lost between birth and weaning, and weaned, and litter and mean piglet birth BW, weaning BW, and average BW gain from birth-to-weaning. Lesions were recorded on d 21 and d 111 of gestation. Sub-pilot behavior data were observed and registered for replicate 1 sows using continuous video-records for the l2 hour lights on period (period 1, 0600-1000; period 2, 1000-1400; period 3, 1400-1800) prior FLEX stall adjustment and 12 hour lights on period post adjustment on d 21, 22, 23, 43, 44, 45, 93, 94, 95. A randomized complete block design with a 2 × 2 factorial arrangement for treatments was used to analyze sow productivity and performance traits. Data were analyzed using the Mixed Models procedure of SAS. A preliminary analysis of data means and numerical trends was used to analyze sow behavior measurements. Sows housed in a FLEX stall had more (P < 0.05) total born and a tendency for more piglets born alive (P = 0.06) than sows housed in a standard stall. Sow body weight also tended to be higher (P = 0.06) for sows housed in a FLEX stall compared to sows housed in a standard stall. There were numerical trends for mean durations of sit, lay, lay (OUT), and eat behaviors to be greater for sows housed in a FLEX stall compared with sows housed in a standard stall. The mean duration of lay (IN) behavior tended to be numerically less for sows housed in a FLEX stall compared with sows housed in a standard stall. There were numerical trends for the mean durations of stand and drink behaviors to be greater for sows housed in a standard stall compared with sows housed in a FLEX stall. The mean frequencies of postural changes and mean durations of oral-nasal-facial and sham-chew behaviors were numerically similar between types of gestation stall. Mean durations and numerical trends indicate that time of day influenced all of the behaviors assessed in this study. The results of this pilot study indicate that the adjustable FLEX stall may affect sow productivity and behavior differently than the standard gestation stall, and thus potentially improve sow well-being. Future research should continue to compare the new FLEX stall design to current housing systems in use and examine physiological traits and immune status in addition to behavioral and productivity traits to assess the effects that this housing system has on the overall welfare of the gestating sow.