Specific binding of the mammalian high mobility group protein AT-hook 2 to the minor groove of AT -rich DNAs: Thermodynamic and specificity studies

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small transcriptional factor involved in cell development and oncogenesis. It contains three "AT-hook" DNA binding domains, which specifically recognize the minor groove of AT-rich DNA sequences. It also has an acidic C-terminal motif. Previous studies showed that HMGA2 mediates all its biological effects through interactions with AT-rich DNA sequences in the promoter regions. In this dissertation, I used a variety of biochemical and biophysical methods to examine the physical properties of HMGA2 and to further investigate HMGA2's interactions with AT-rich DNA sequences. The following are three avenues perused in this study: (1) due to the asymmetrical charge distribution of HMGA2, I have developed a rapid procedure to purify HMGA2 in the milligram range. Preparation of large amounts of HMGA2 makes biophysical studies possible; (2) Since HMGA2 binds to different AT-rich sequences in the promoter regions, I used a combination of isothermal titration calorimetry (ITC) and DNA UV melting experiment to characterize interactions of HMGA2 with poly(dA-dT) 2 and poly(dA)poly(dT). My results demonstrated that (i) each HMGA2 molecule binds to 15 AT bp; (ii) HMGA2 binds to both AT DNAs with very high affinity. However, the binding reaction of HMGA2 to poly(dA-dT) 2 is enthalpy-driven and the binding reaction of HMGA2 with poly(dA)poly(dT) is entropy-driven; (iii) the binding reactions are strongly depended on salt concentrations; (3) Previous studies showed that HMGA2 may have sequence specificity. In this study, I used a PCR-based SELEX procedure to examine the DNA binding specificity of HMGA2. Two consensus sequences for HMGA2 have been identified: 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These consensus sequences have a unique feature: the first five base pairs are AT-rich, the middle four to five base pairs are GC-rich, and the last five to six base pairs are AT-rich. All three segments are critical for high affinity binding. Replacing either one of the AT-rich sequences to a non-AT-rich sequence causes at least 100-fold decrease in the binding affinity. Intriguingly, if the GC-segment is substituted by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step towards finding its binding sites within the genome.

Summertime Influences of Tidal Energy Advection on the Surface Energy Balance in a Mangrove Forest

Mangrove forests are ecosystems susceptible to changing water levels and temperatures due to climate change as well as perturbations resulting from tropical storms. Numerical models can be used to project mangrove forest responses to regional and global environmental changes, and the reliability of these models depends on surface energy balance closure. However, for tidal ecosystems, the surface energy balance is complex because the energy transport associated with tidal activity remains poorly understood. This study aimed to quantify impacts of tidal flows on energy dynamics within a mangrove ecosystem. To address the research objective, an intensive 10-day study was conducted in a mangrove forest located along the Shark River in the Everglades National Park, FL, USA. Forest–atmosphere turbulent exchanges of energy were quantified with an eddy covariance system installed on a 30-m-tall flux tower. Energy transport associated with tidal activity was calculated based on a coupled mass and energy balance approach. The mass balance included tidal flows and accumulation of water on the forest floor. The energy balance included temporal changes in enthalpy, resulting from tidal flows and temperature changes in the water column. By serving as a net sink or a source of available energy, flood waters reduced the impact of high radiational loads on the mangrove forest. Also, the regression slope of available energy versus sink terms increased from 0.730 to 0.754 and from 0.798 to 0.857, including total enthalpy change in the water column in the surface energy balance for 30-min periods and daily daytime sums, respectively. Results indicated that tidal inundation provides an important mechanism for heat removal and that tidal exchange should be considered in surface energy budgets of coastal ecosystems. Results also demonstrated the importance of including tidal energy advection in mangrove biophysical models that are used for predicting ecosystem response to changing climate and regional freshwater management practices.

Conformational dynamics associated with ligand binding to vertebrate hexa-coordinate hemoglobins

Neuroglobin (Ngb) and cytoglobin (Cygb) are two new additions to the globin family, exhibiting heme iron hexa-coordination, a disulfide bond and large internal cavities. These proteins are implicated in cytoprotection under hypoxic-ischemic conditions, but the molecular basis of their cytoprotective function is unclear. Herein, a photothermal and spectroscopic study of the interactions of diatomic ligands with Ngb, Cygb, myoglobin and hemoglobin is presented. The impact of the disulfide bond in Ngb and Cygb and role of conserved residues in Ngb His64, Val68, Cys55, Cys120 and Tyr44 on conformational dynamics associated with ligand binding/dissociation were investigated. Transient absorption and photoacoustic calorimetry studies indicate that CO photo-dissociation from Ngb leads to a volume expansion (13.4±0.9 mL mol-1), whereas a smaller volume change was determined for Ngb with reduced Cys (ΔV=4.6±0.3 mL mol-1). Furthermore, Val68 side chain regulates ligand migration between the distal pocket and internal hydrophobic cavities since Val68Phe geminate quantum yield is ∼2.7 times larger than that of WT Ngb. His64Gln and Tyr44Phe mutations alter the thermodynamic parameters associated with CO photo-release indicating that electrostatic/hydrogen binding network that includes heme propionate groups, Lys 67, His64, and Tyr 44 in Ngb modulates the energetics of CO photo-dissociation. In Cygb, CO escape from the protein matrix is fast (< 40 ns) with a ΔH of 18±2 kcal mol-1 in Cygbred, whereas disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4 kcal mol-1. Therefore, the disulfide bond modulates conformational dynamics in Ngb and Cygb. I propose that in Cygb with reduced Cys the photo-dissociated ligand escapes through the hydrophobic tunnel as occurs in Ngb, whereas the CO preferentially migrates through the His64 gate in Cygbox. To characterize Cygb surface 1,8-ANS interactions with Cygb were investigated employing fluorescence spectroscopy, ITC and docking simulations. Two 1,8-ANS binding sites were identified. One binding site is located close to the extended N-terminus of Cygb and was also identified as a binding site for oleate. Furthermore, guanidinium hydrochloride-induced unfolding studies of Cygb reveal that the disulfide bond does not impact Cygb stability, whereas binding of cyanide slightly increases the protein stability.

Study of Ammonia Borane and its Derivatives: Influence of Nanoconfinements and Pressures

Recently, ammonia borane has increasingly attracted researchers’ attention because of its merging applications, such as organic synthesis, boron nitride compounds synthesis, and hydrogen storage. This dissertation presents the results from several studies related to ammonia borane. The pressure-induced tetragonal to orthorhombic phase transition in ammonia borane was studied in a diamond anvil cell using in situ Raman spectroscopy. We found a positive Clapeyron-slope for this phase transformation in the experiment, which implies that the phase transition from tetragonal to orthorhombic is exothermic. The result of this study indicates that the rehydrogenation of the high pressure orthorhombic phase is expected to be easier than that of the ambient pressure tetragonal phase due to its lower enthalpy. The high pressure behavior of ammonia borane after thermal decomposition was studied by in situ Raman spectroscopy at high pressures up to 10 GPa. The sample of ammonia borane was first decomposed at ~140 degree Celcius and ~0.7 GPa and then compessed step wise in an isolated sample chamber of a diamond anvil cell for Raman spectroscopy measurement. We did not observe the characteristic shift of Raman mode under high pressure due to dihydrogen bonding, indicating that the dihydrogen bonding disappears in the decomposed ammonia borane. Although no chemical rehydrogenation was detected in this study, the decomposed ammonia borane could store extra hydrogen by physical absorption. The effect of nanoconfinement on ammonia borane at high pressures and different temperatures was studied. Ammonia borane was mixed with a type of mesoporous silica, SBA-15, and restricted within a small space of nanometer scale. The nano-scale ammonia borane was decomposed at ~125 degree Celcius in a diamond anvil cell and rehydrogenated after applying high pressures up to ~13 GPa at room temperature. The successful rehydrogenation of decomposed nano-scale ammonia borane gives guidance to further investigations on hydrogen storage. In addition, the high pressure behavior of lithium amidoborane, one derivative of ammonia borane, was studied at different temperatures. Lithium amidoborane (LAB) was decomposed and recompressed in a diamond anvil cell. After applying high pressures on the decomposed lithium amidoborane, its recovery peaks were discovered by Raman spectroscopy. This result suggests that the decomposition of LAB is reversible at high pressures.

High-performance liquid chromatography measurement of carotenoids in human serum, etoposide in pig cerebrospinal fluid and measurement of the aggregation of xanthophylls in aqueous solutions

The present study measured a chemotherapy drug, etoposide, in pig cerebrospinal fluid after intraventricular administrations were made directly into the fourth ventricle of the brain; cytotoxic concentrations for a twenty-four hour period after infusions. The analytical method developed validates the potential treatment of malignant brain tumors. The increase in serum carotenoid concentration in 30 healthy individuals was measured after supplementation with lutein. HPLC analysis of serum levels of carotenoids showed an increase in the concentration of lutein and a constant concentration of other major serum carotenoids. An initial attempt to measure the enthalpy of aggregation of xanthophylls was conducted by using ultraviolet-visible spectroscopy. The enthalpy of lutein aggregation and AH range of zeaxanthin disordering of aggregation are reported. Monomethyl ether of lutein did not aggregate in any of the aqueous solutions.

Multi-objective optimization of the molecular structure of refrigerants

The aim of this work was to develop a new methodology, which can be used to design new refrigerants that are better than the currently used refrigerants. The methodology draws some parallels with the general approach of computer aided molecular design. However, the mathematical way of representing the molecular structure of an organic compound and the use of meta models during the optimization process make it different. In essence, this approach aimed to generate molecules that conform to various property requirements that are known and specified a priori. A modified way of mathematically representing the molecular structure of an organic compound having up to four carbon atoms, along with atoms of other elements such as hydrogen, oxygen, fluorine, chlorine and bromine, was developed. The normal boiling temperature, enthalpy of vaporization, vapor pressure, tropospheric lifetime and biodegradability of 295 different organic compounds, were collected from open literature and data bases or estimated. Surrogate models linking the previously mentioned quantities with the molecular structure were developed. Constraints ensuring the generation of structurally feasible molecules were formulated and used in commercially available optimization algorithms to generate molecular structures of promising new refrigerants. This study was intended to serve as a proof-of-concept of designing refrigerants using the newly developed methodology.

Effect of pH and temperature on the carbonate promoted dissolution of sodium meta-autunite

Release of uranium from Na-autunite, an artificial mineral created as a result of polyphosphate injection in the subsurface at the DOE Hanford Site, takes place during slow dissolution of the mineral structure. Stability information of the uranyl-phosphate phases is limited to conditions involving pH, temperature, and a few aqueous organic materials. The carbonate ion, which creates very strong complexes with uranium, is the predominant ion in the groundwater composition. The polyphosphate technology with the formation of autunite was identified as the most feasible remediation strategy to sequester uranium in contaminated groundwater and soil in situ. The objectives of the experimental work were (i) to quantify the effect of bicarbonate on the stability of synthetic sodium meta-autunite created as a result of uranium stabilization through polyphosphate injection, (ii) calculate the kinetic rate law parameters of the uranium release from Na-autunite during dissolution, and (iii) to compare the process parameters with those obtained for natural calcium meta-autunite. Experiments were conducted using SPTF apparatus, which consists of syringe pumps for controlling flow rate, Teflon reactors and a heating/cooling system. 0.25 grams of synthetic Na-autunite was placed in the reactor and buffer solutions with varying bicarbonate concentrations (0.0005 to 0.003 M) at different pH (6 - 11) were pumped through the reactors. Experiments were conducted at four different temperatures in the range of 5 - 60oC. It was concluded that the rate of release of uranium from synthetic Na-autunite is directly correlated to the bicarbonate concentration. The rate of release of uranium increased from 1.90 x 10-12 at pH 6 to 2.64 x 10-10 (mol m-2 s-1) at pH 11 at 23oC over the bicarbonate concentration range tested. The activation energy values were invariant with the change in the bicarbonate concentration; however, pH is shown to influence the activation energy values. Uranyl hydroxides and uranyl carbonates complexes helped accelerate the dissolution of autunite mineral.

An Investigation of Vertical Turbulent Transport Processes in Coastal Regions Using Tower Observations

High-resolution tower observations of turbulent transport processes in the coastal atmospheric surface layer show that the exchange coefficients for momentum, enthalpy, and moisture behave differently for different environmental and atmospheric conditions. The drag coefficient is closely tied to wind speed and turbulent intensity. The exchange coefficient for enthalpy shows a dependence on stability. Analysis of the turbulent kinetic energy budget yields a new parameterization framework that well explains the observed variation of the drag coefficient, particularly at low wind speeds.

Study of ammonia borane and its derivatives: Influence of nanoconfinements and pressures

Recently, ammonia borane has increasingly attracted researchers’ attention because of its merging applications, such as organic synthesis, boron nitride compounds synthesis, and hydrogen storage. This dissertation presents the results from several studies related to ammonia borane. ^ The pressure-induced tetragonal to orthorhombic phase transition in ammonia borane was studied in a diamond anvil cell using in situ Raman spectroscopy. We found a positive Clapeyron-slope for this phase transformation in the experiment, which implies that the phase transition from tetragonal to orthorhombic is exothermic. The result of this study indicates that the rehydrogenation of the high pressure orthorhombic phase is expected to be easier than that of the ambient pressure tetragonal phase due to its lower enthalpy. ^ The high pressure behavior of ammonia borane after thermal decomposition was studied by in situ Raman spectroscopy at high pressures up to 10 GPa. The sample of ammonia borane was first decomposed at ∼140 degree Celcius and ∼0.7 GPa and then compessed step wise in an isolated sample chamber of a diamond anvil cell for Raman spectroscopy measurement. We did not observe the characteristic shift of Raman mode under high pressure due to dihydrogen bonding, indicating that the dihydrogen bonding disappears in the decomposed ammonia borane. Although no chemical rehydrogenation was detected in this study, the decomposed ammonia borane could store extra hydrogen by physical absorption. ^ The effect of nanoconfinement on ammonia borane at high pressures and different temperatures was studied. Ammonia borane was mixed with a type of mesoporous silica, SBA-15, and restricted within a small space of nanometer scale. The nano-scale ammonia borane was decomposed at ∼125 degree Celcius in a diamond anvil cell and rehydrogenated after applying high pressures up to ∼13 GPa at room temperature. The successful rehydrogenation of decomposed nano-scale ammonia borane gives guidance to further investigations on hydrogen storage. ^ In addition, the high pressure behavior of lithium amidoborane, one derivative of ammonia borane, was studied at different temperatures. Lithium amidoborane (LAB) was decomposed and recompressed in a diamond anvil cell. After applying high pressures on the decomposed lithium amidoborane, its recovery peaks were discovered by Raman spectroscopy. This result suggests that the decomposition of LAB is reversible at high pressures.^