Dissertations / Theses on the topic 'Analytical biochemistry'

Cell membranes have been shown to be able to change the conformation of proteins/peptides. However, the structure of the cell membrane is complicated and has been divided to three regions: the hydrophobic region containing alkyl chains, the hydrophilic head group, and the hydration layer, or lipid-water interface, which exists between the hydrophilic head group and the bulk water solution, but with lower dielectric constant compared with fully hydrated water. The air-water interface has been used to mimic the structure of the hydration layer because of their similar dielectric constant.1,2 Some proteins were found to form a stable Langmuir monolayer and accumulate at the air-water interface. For example, ?-synclein, a membrane protein containing 140 amino acids, is unstructured in aqueous solution but changes its conformation to α-helix at the air-water interface. This incites interest to investigate short motifs of α-helix to form a stable Langmuir monolayer at the air-water interface. In this thesis, a peptide with sequence of YAAAA(KAAAA)4 (referred as Pep25 hereafter) was used as a model peptide of α-helix to spread at the air-water interface, because our group has determined the conformation of Pep25 in residue level by the 13C isotope-edited FTIR. Langmuir monolayer technique together with IRRAS showed that Pep25 does not form a typical Langmuir monolayer at the interface. Potential plans to make Pep25 to form a stable monolayer are also discussed in this thesis.

Bovine heparin is characteristically different than porcine intestinal heparin. These differences include sulfation, molecular weight properties, activity, structure, and shape. Bovine lung heparin has a higher amount of GlcNY6S (where Y can represent Ac or S) while the amount of GlcNY6S is much lower in bovine intestinal heparin. All heparins have high amounts of trisulfated TriS disaccharide but the level of TriS is in lower in bovine intestinal heparin. The amount of NS2S is much higher in bovine intestinal heparin than in bovine lung and porcine intestinal heparins. The average molecular weight of bovine intestinal heparin is similar to porcine intestinal heparin but the molecular weight of bovine lung heparin is much lower. The activities of bovine tissue heparins were comparable to, but lower than, the activity of porcine intestinal heparin.

The differences in heparins from different sources become much more subtle as they are depolymerized into low molecular weight heparin (LMWH). These differences are often sufficiently small so that they require principle component analysis (PCA) to determine. Differences in the reducing end and the non-reducing end structures in heparin determined by mass spectrometry (MS) as well as differences in the glucosamine and uronic acid residues determined by nuclear magnetic resonance spectroscopy (NMR) were selected for PCA. Using PCA it was possible to link parent heparin starting material to its daughter LMWH. This analysis demonstrated the lower variation between LMW bovine and LMW porcine heparins than between bovine and porcine heparins. This lower variation afforded the LMW bovine heparin similar anti-Xa and anti-IIa activity comparable to commercial heparin.

The harsh purification process used to prepare heparin leaves the heparin product largely unaffected except for its reducing end tetrasaccharide linkage region. GlyserineAc is present in porcine and bovine heparin but is absent in LMW heparin. We hypothesized that the peracetic acid bleaching adds an O-acetyl group that is selectively lost during β-elimination in the LMWH production process. The tetrasaccharide composition of bovine and porcine heparin is similar as are different batches from the same supplier. This emphasizes how similar processing results in similar heparin regardless of whether bovine or porcine is used.

Small differences in counterfeit heparin, i.e. blended porcine and bovine heparins were next examined. Porcine heparin and bovine heparin of similar molecular weights to obtain a bovine heparin counterfeit drug of enhanced activity. Such counterfeit blends are undetectable by current methods of analysis. Diffusion ordered spectroscopy (DOSY) method for analysis was developed. DOSY exploits differences in the molecular shape of bovine heparin and porcine heparin and achieved partial separation in the diffusion dimension. Additional spectra for component resolution (SCORE) analysis were used to demonstrate detection and identification of blend mixture.

This dissertation details original work focused on the DNA adduct N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG). This DNA adduct results from the spontaneous reaction of DNA with the endogenous and exogenous formed, carbohydrate-derived, reactive carbonyl species, methylglyoxal. Using in vitro steady state kinetics, we have shown that CEdG in template DNA leads to DNA miscoding effects when the model replicative polymerase, exonuclease-free Klenow fragment (KF-) is used. The development, validation and application of a novel stable isotope dilution, triple quadrupole mass spectrometric method for the quantitation of CEdG is also detailed. This method was used to quantitate CEdG in urine from diabetic rats, urine from human patients, human tumor and adjacent biopsy tissue, diabetic animal tissue and DNA treated with methylglyoxal. Finally, we detail the adaptation, validation and application of a novel, commercially-available microfluidic HPLC-chip for increased sensitivity in the quantitation of CEdG and also apply it to the quantitation of the RNA analogue, CEG. Combined, these studies establish CEdG as a potential biomarker for glycation and point to a viable avenue for connecting chronic glycolytic flux with genetic instability.

Many recent efforts in the field of microfluidics have been focused on reducing the size and the complexity of devices and on simplifying the methods of analysis performed with them. Gradient elution moving boundary electrophoresis (GEMBE) is a recently described counterflow electrophoresis method that was developed to simplify the analysis of ions in complex matrices. In this thesis, the improvement of the limit of detection of GEMBE and reduction of the GEMBE channel length is investigated.

Integration of simple and robust device components required for the successful adaptation of many analytical methods to multiplexed and field-portable devices often has negative effects on detection sensitivity, such as in the optical detection components in a capillary electrophoresis (CE) system. One of the simplest methods to improve sensitivity in the CE field is known as sample stacking. This method involves preparing the sample in a buffer with a different concentration (and conductivity) than that of the run buffer, such that when an electric field is applied the analyte concentration is increased at the boundary between the two different buffer concentrations. A method in which the sample is prepared in a buffer at a lower concentration than the run buffer has been implemented. This method achieves a significantly greater signal enhancement than expected for sample stacking. The concentration enhancement ability of this method is demonstrated utilizing GEMBE with channel current detection.

Current GEMBE device construction methods impose limitations on the minimum length of the separation channel. One technique well suited for minimizing the size of the GEMBE separation channel is multiphoton absorption polymerization (MAP). Because MAP is a non-linear optical fabrication method, polymerization is limited to a small region near the focal point of a laser beam. As a result, three-dimensional structures with small feature sizes can be easily created. The 3D capabilities of MAP have been exploited to create channels with circular cross sections and ∼300 &mgr;m lengths for GEMBE. The integration of device components fabricated with MAP and molded with PMDS allows visualization of the GEMBE separations, and provides insights into the effect of channel length on GEMBE step width.

Bloodstain evidence can be obscured and lost when deposited on dark surfaces where there is no contrast between the bloodstain and the surface. This can also occur when criminals attempt to conceal bloodstains by painting over them. This research investigated the detection and visualization of bloodstains deposited on dark surfaces and concealed under paint with the use of an infrared (IR) alternate light source produced by the Foster and Freeman company under the name Crime-lite^® 82S Infrared.

The results show that the Crime-lite^® 82S Infrared in conjunction with an IR sensitive camera can aid in the detection and visualization of bloodstains best on porous surfaces such as indoor carpets and most clothing as well as on and under red-tinged paints with the more flat or matte finishes. The results also suggest the component within blood responsible for absorbing IR light is hemoglobin present in red blood cells. Further, the findings indicate that when hemoglobin is present in too low of a quantity, it falls below the detection threshold to absorb IR light and bloodstains will not be visualized. Given its ease-of-use and portability, combined with the support of the findings from this collaborative study with the Los Angeles Police Department (LAPD) Field Investigation Unit (FIU), the Crime-lite^® 82S Infrared and camera is recommended as an additional tool in the search for bloodstain evidence that may otherwise go undetected.

The challenges to forensic body fluid analysis have placed limitations on the type of information that investigators can acquire and how that information can be collected. In recent years, Raman spectroscopy has proven itself useful for characterizing body fluids. In 2008, a large-scale investigation was undertaken to explore the use of Raman spectroscopy as a means of identifying body fluids. This work resulted in multidimensional Raman spectroscopic signatures for the five main body fluids: semen, peripheral blood, saliva, vaginal fluid, and sweat. These studies were incredibly successful and created the foundation for years of continued research. Accordingly, the studies included in this thesis have been specifically chosen to frame the previous research projects. They include a suite of projects aimed to advance and validate the developed method.

First, a statistical model was developed to automatically identify and differentiate body fluids based on their Raman spectra. The multidimensional spectroscopic signatures mentioned above are very effective at identification, but they are body fluid-specific. In other words, they individually evaluate whether or not an unknown spectrum is from a particular body fluid, such as blood. Additionally, each signature was built on spectra from a limited number of donors. To improve on this capability, a single classification model was built on the Raman spectra from 60 donors (12 for each body fluid). This model was externally validated with an additional 15 donors in order to objectively assess the model’s performance. All of the external validation donors were correctly identified, illustrating how accurate and robust the model is.

Second, the limit of detection (LOD) for the classification model was explored as a form of validation. It is vitally important that a method’s limits be established before deploying it into use. The LOD of peripheral blood was investigated. Peripheral blood is unique from other body fluids because its Raman spectrum has been attributed almost entirely to one molecule- hemoglobin. Because hemoglobin is only found in red blood cells (RBCs), the Raman spectrum of peripheral blood essentially results purely from RBCs. Given this, we chose to start with a single RBC, and then increase the volume until identification was successful. We found that we were able to conclusively and confidently identify peripheral blood using a single red blood cell. This limit is 5000X smaller than the amount of blood required for DNA analysis, demonstrating the sensitivity of the developed method.

Finally, the method was further advanced by incorporating donor characterization into the process. Besides identifying body fluids, the method can now extract “phenotypic” information about the donor. Raman spectroscopy and multivariate data analysis were used to determine the biological sex of saliva donors, and the race of semen donors. These studies will help forensic investigators extract incredibly useful information about a potential suspect or victim, and can be performed directly at a crime scene for instant results.

Altogether, these studies combine to strengthen the method previously developed by our research group. More importantly, they help to bridge the gap between research and application. Creating a universal method to differentiate and identify body fluids, investigating the method’s LOD, and developing additional techniques to characterize body fluids represents a significant contribution to the field of forensic chemistry. The universal method created within this thesis will be adapted to perform on-site analysis of physical evidence at crime scenes. The methods’ incredible sensitivity has been demonstrated by determining that it can identify peripheral blood based on a single RBC. Finally, by developing models to characterize body fluid donors, investigators will be able to extract useful information about individuals that may have been present at a crime scene. Additional studies are already being conducted to make further improvements, and our method is poised to make a significant contribution to the field of crime scene investigation.

This Thesis describes applications of standing-waveultrasonic traps for sensitive biomedical analysis. Two majorapproaches have been investigated where functionalizedmicroparticles are employed in bioaffinity assays. In the firstapproach, a longitudinal flow-through capillary ultrasonic trapis used for size selective separation and retention ofdifferently sized microparticles. This device may be used fordetection of particle pairs, which are formed during theinitial stage of microparticle immunoagglutination. Theperformance of the capillary ultrasonic trap for enrichment andcounting of particle pairs is characterized by a model systemof differently sized homogeneous fluorescent microparticles.The selectivity of this detection method relies on thecharacteristics of the force field inside the narrow borecapillary, which is formed by the competition between acousticradiation forces and viscous drag forces from the fluidflow.

The second approach is an investigation of the potential forsensitive protein quantification by combining ultrasonicenrichment and confocal laser-scanning fluore-scence detection.Here, the design of the ultrasonic trap is tailor-made for theimaging properties of a confocal microscope, resulting inrearrangement and concentration of suspended microparticlesinto single, dense layers that is scanned by a focused laserbeam. The bioaffinity assay employed is based on detecting thetarget molecules via fluorescent tracer antibodies immobilizedon the surface of each single particle.

The final part of the work presented in this Thesis is athorough investigation of both the biochemical and the physicalproperties that determine the performance and potentialsensitivity of the particle doublet assay. In thisinvestigation, a novel approach is presented for doubletdetection, namely fluorescence-microscopy-based classificationof doublets and singlets by a pattern recognition algorithm.The experimental results are also compared with the resultsfrom flow cytometry analysis. Furthermore, the initial stage ofimmuno-agglutination is theoretically investigated by a modelbased on diffusion-limited agglutination combined with a stericfactor determined by the geometry of the bio-molecules and theamount of specific and non-specific binding that is present inthe particular assay.

To conclude, the Thesis presents several approaches wherestanding-wave ultrasonic fields may be used for sensitiveparticle-based biomedical analysis. The best prospect for highsensitivity was found for the confocal laser-scanningfluorescence detection system, with a detection limit of theorder of 10^-14M. On the other hand, the agglutination-basedassay may give sensitivity of the order of 10^-11-10^-10M with very simple and inexpensiveequipment.

Cytochrome P450s (P450s) are a superfamily of enzymes that catalyze oxidation of unactivated hydrocarbons. However, the means by which P450s control (1) regioselectivity of their activity and (2) specificity in their molecular recognition remain largely elusive. Toward investigation of the role of dynamics in the regioselectivity of the archetypal cytochrome P450cam (P450cam), two-dimensional infrared spectroscopy has been applied with heme-bound carbon monoxide (CO) as an infrared probe of the active site. The data support a model for P450cam regioselectivity in which binding of different substrates to P450cam variably stabilizes the active site into two distinct states, each associated with different dynamics linked to different levels of regioselectivity. To investigate the role of conformational heterogeneity in P450cam substrate specificity, infrared spectoscopy was combined with the site-specific incorporation of nitrile probes at distinct P450cam microenvironments. This approach enabled differentiation of changes experienced at each of those environments when d-camphor and/or CO binds to the active site. Finally, the impact of conformational heterogeneity on the affinity of substrate molecular recognition by wild-type and mutant P450cam was evaluated using both CO and nitrile probes. This study suggests that the nature of the conformations populated in the unbound states influences the affinity for different substrates. Collectively, these studies provide new insight into the roles of conformational heterogeneity and dynamics in P450cam activity. Furthermore, these studies help to lay the foundation for efforts toward understanding the roles of conformational heterogeneity and dynamics in the function of human P450s, for which unraveling the mechanisms involved in Phase I metabolism is a topic of great pharmacological concern.

This dissertation describes metabolic engineering of cyanobacterium Synechococcus elongatus PCC7942 as a photosynthetic host for the conversion of CO₂ into 2,3-butanediol. Current advances in pathway design, genetic tool development, and yield improvement are described (Chapter 1). A pathway for the synthesis of 2,3-butanediol is designed based on collective concepts of pathway strength, robustness, and irreversibility, and extensively tested through the generation of mutants (Chapter 2). This pathway is then optimized through modulation of translation by combinatorial mixing of ribosome binding sites (Chapter 3). Finally, photosynthetic productivity is investigated through expression of an exogenous pathway targeting every step between fixation and product (Chapter 4). All materials and methods are given separately for easy reference (Chapter 5).

Heavy metal pollution is a growing concern as growing worldwide population and industrial processes increase pollution levels in most environments. High metal concentrations throughout ecosystems pose a serious threat to wild-life and human health. Methods to monitor rising threat levels of metals are a primary concern for monitoring overall ecosystem health. Mechanisms which spread pollution must be intimately understood because of the persistence of heavy metals. Heavy metal contamination in the Tar Creek superfund site provides a great case study to selectively observe differences in heavy metals concentrations both upstream and downstream of mining activity. Thus, research is able to identify natural and man-made point sources of pollution.

The abilities of bivalves to filter-feed and sediment-feed provide a unique monitoring tool for analyzing heavy metals. Mussels are constantly filtering the environment around them. A mussel's seasonal and annual growth layers provide an excellent sample media for obtaining historical records of environmental data. Many species of mussels are found in most freshwater ecosystems throughout the United States. Mussels have low migration rates, live for a suitable amount of time, and leave relic shells. These features make mussels very practical for monitoring heavy metal pollution.

Various studies were conducted to obtain insight into developing methods for using LA-ICP-MS as a tool for monitoring heavy metals in mussel shells. Surface laser ablations, compared at additional depths, resulted in a more than 20% increase in signal intensity. Theoretical and experimental designs show signal changes as a function of depth. Mussel tissue and shell digestions were found to be best when using approximately 1.0 mL of hydrogen peroxide and 1.0 mL of nitric acid for each 0.1 grams of sample. Mussel tissue was found to have greater heavy metal concentrations than shells. Shells were found to average a 96% weight of calcium carbonate; however, the organic layers contained the greatest concentrations of heavy metals per weight.

This thesis includes many different production and research projects that can help make many positive advances in the pharmaceutical and biomedical area. The first part of my thesis describes my practical experience at Anatrace Products, LLC. Included in this chapter is the large scale synthesis of amphiphiles for biological uses. The second part of the thesis describes my attempt to build an analytical profile of polar extracts from mastic gum for the potential treatment of chronic obstructive pulmonary disease.