Glycosaminoglycans (GAGs) are long chains of negatively charged sulfated polysaccharides. They are often found to be covalently attached to proteins and form proteoglycans in the extracellular matrix (ECM). Many proteins bind GAGs through electrostatic interactions. GAG-binding proteins (GBPs) are involved in diverse physiological activities ranging from bacterial infections to cell-cell/cell-ECM contacts. This thesis is devoted to understanding how interactions between GBPs and their receptors modulate biological phenomena. Bacteria express GBPs on surface that facilitate dissemination and colonization by attaching to host ECM. The first GBP investigated in this thesis is decorin binding protein (DBP) found on the surface of Borrelia burgdorferi, causative pathogens in Lyme disease. DBPs bind GAGs of decorin, a proteoglycan in ECM. Of the two isoforms, DBPB is less studied than DBPA. In current work, structure of DBPB from B. burgdorferi and its GAG interactions were investigated using solution NMR techniques. DBPB adopts a five-helical structure, similar to DBPA. Despite similar GAG affinities, DBPB has its primary GAG-binding site on the lysine-rich C terminus, which is different from DBPA. Besides GAGs, GBPs in ECM also interact with cell surface receptors, such as integrins. Integrins belong to a big family of heterodimeric transmembrane proteins that receive extracellular cues and transmit signals bidirectionally to regulate cell adhesion, migration, growth and survival. The second part of this thesis focuses on αM I-domain of the promiscuous integrin αMβ2 (Mac-1 or CD11b/CD18) and explores the structural mechanism of αM I-domain interactions with pleiotrophin (PTN) and platelet factor 4 (PF4), which are cationic proteins with high GAG affinities. After completing the backbone assignment of αM I-domain, paramagnetic relaxation enhancement (PRE) experiments were performed to show that both PTN and PF4 bind αM I-domain using metal ion dependent adhesion site (MIDAS) in an Mg2+ independent way, which differs from the classical Mg2+ dependent mechanism used by all known integrin ligands thus far. In addition, NMR relaxation dispersion analysis revealed unique inherent conformational dynamics in αM I-domain centered around MIDAS and the crucial C-terminal helix. These dynamic motions are potentially functionally relevant and may explain the ligand promiscuity of the receptor, but requires further studies.

The oxygen sensitivity of hydrogenase is a large barrier in maximizing the efficiency of algal hydrogen production, despite recent efforts aimed at rewiring photosynthesis. This project focuses on the role of photosystem II (PSII) in extended hydrogen production by cells expressing the PSI-HydA1 chimera, with the goal of optimizing continuous production of photobiohydrogen in the green alga, Chlamydomonas reinhardtii. Experiments utilizing an artificial PSII electron
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.

Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve CVN's glycan-binding affinity by conjugating a boronic acid functional group to the N-terminus via N-terminal specific reductive alkylation by way of a benzaldehyde handle. However, large discrepancies were observed when attempting to confirm a successful conjugation, and further work is necessary to identify the causes and solutions for these issues.

Predicting the binding sites of proteins has historically relied on the determination of protein structural data. However, the ability to utilize binding data obtained from a simple assay and computationally make the same predictions using only sequence information would be more efficient, both in time and resources. The purpose of this study was to evaluate the effectiveness of an algorithm developed to predict regions of high-binding on proteins as it applies to determining the regions of interaction between binding partners. This approach was applied to tumor necrosis factor alpha (TNFα), its receptor TNFR2, programmed cell death protein-1 (PD-1), and one of its ligand PD-L1. The algorithms applied accurately predicted the binding region between TNFα and TNFR2 in which the interacting residues are sequential on TNFα, however failed to predict discontinuous regions of binding as accurately. The interface of PD-1 and PD-L1 contained continuous residues interacting with each other, however this region was predicted to bind weaker than the regions on the external portions of the molecules. Limitations of this approach include use of a linear search window (resulting in inability to predict discontinuous binding residues), and the use of proteins with unnaturally exposed regions, in the case of PD-1 and PD-L1 (resulting in observed interactions which would not occur normally). However, this method was overall very effective in utilizing the available information to make accurate predictions. The use of the microarray to obtain binding information and a computer algorithm to analyze is a versatile tool capable of being adapted to refine accuracy.

Since the discovery of graphene, two dimensional materials (2D materials) have become a focus of interest for material research due to their many unique physical properties embedded in their 2D structure. While they host many exciting potential applications, some of these 2D materials are subject to environmental instability issues induced by interaction between material and gas molecules in air, which poses a barrier to further application and manufacture. To overcome this, it is necessary to understand the origin of material instability and interaction with molecules commonly found in air, as well as developing a reproducible and manufacturing compatible method to post-process these materials to extend their lifetime. In this work, the very first investigation on environmental stability on Te containing anisotropic 2D materials such as GaTe and ZrTe3 is reported. Experimental results have demonstrated that freshly exfoliated GaTe quickly deteriorate in air, during which the Raman spectrum, surface morphology, and surface chemistry undergo drastic changes. Environmental Raman spectroscopy and XPS measurements demonstrate that H2O molecules in air interact strongly on the surface while O2, N2, and inert gases don't show any detrimental effects on GaTe surface. Moreover, the anisotropic properties of GaTe slowly disappear during the aging process. To prevent this gas/material interaction based surface transformation, diazonium based surface functionalization is adopted on these Te based 2D materials. Environmental Raman spectroscopy results demonstrate that the stability of functionalized Te based 2D materials exhibit much higher stability both in ambient and extreme conditions. Meanwhile, PL spectroscopy, angle resolved Raman spectroscopy, atomic force microscopy measurements confirm that many attractive physical properties of the material are not affected by surface functionalization. Overall, these findings unveil the degradation mechanism of Te based 2D materials as well as provide a way to significantly enhance their environmental stability through an inexpensive and reproducible surface chemical functionalization route.

Circulating tumor DNA analysis has several potential applications in cancer diagnostics. However, results in literature vary considerably, often due to different blood collection methods and protocols. Several new products to address pre-analytical variables of cfDNA processing have recently become available, and little is understood about their effects on DNA quality and downstream applications. We evaluated the effects of blood collection protocols and DNA extraction kits on cfDNA yield, quality, and fragment size using droplet-digital PCR (ddPCR) and targeted deep sequencing. To quantify cfDNA yield and size distribution, we developed a multiplexed ddPCR assay with FAM-labeled short amplicons and TET-labeled long amplicons targeting housekeeping genes. After assay validation, we compared the performance of several commercially-available cfDNA extraction kits using control plasma samples and different blood collection protocols using paired plasma samples from healthy volunteers. To assess whether cell-stabilizing preservative in Streck tubes may induce low-abundance noise in cfDNA, we performed molecularly-tagged targeted deep sequencing and developed an informatics approach for enumeration and variant calling from uniquely tagged DNA fragments. We found a significant difference between extraction methods but no significant difference across blood collection protocols in cfDNA yield or size distribution. Sequencing results showed no significant evidence of preservative-induced cfDNA damage across tested blood collection protocols. In summary, the multiplexed ddPCR assay enabled quantitative assessment of cfDNA extraction methods and blood collection protocols, and allowed for normalization of input to create reproducible sequencing libraries. Our results suggest that plasma samples processed up to 72 hours following venipuncture in Streck Cell-free DNA tubes may be used for downstream sequencing of circulating tumor DNA in patients with cancer.

As prices for fuel along with the demand for renewable resources grow, it becomes of paramount importance to develop new ways of obtaining the energy needed to carry out the tasks we face daily. Costs of production due to energy and time constraints impose severe limitations on what is viable. Biological systems, on the other hand, are innately efficient both in terms of time and energy by handling tasks at the molecular level. Utilizing this efficiency is at the core of this research. Proper manipulation of even common proteins can render complexes functionalized for specific tasks. In this case, the coupling of a rhenium-based organometallic ligand to a modified myoglobin containing a zinc porphyrin, allow for efficient reduction of carbon dioxide, resulting in energy that can be harnessed and byproducts which can be used for further processing. Additionally, a rhenium based ligand functionalized via biotin is tested in conjunction with streptavidin and ruthenium-bipyridine.

Over the past two decades, a significant amount of research has been conducted investigating cyanovirin-N (CVN), which has been shown to be an effective antiviral agent by inhibiting entry of HIV into the cell. The virucidal activity of CVN is attributed to the tight binding interactions with the glycosylated surfaces of the envelope protein gp120. In this study we investigated how the incorporation of various single point mutations in the glycan binding site would ultimately affect the overall binding affinity of the protein with the glycan. These mutations were predicted through computational methods. Using a BP-docking program and molecular dynamics (MD) simulation, the free energy change upon the ligand binding to the each protein was determined. Experimental work and Isothermal Titration Calorimetry (ITC) was used to determine the Kd values for each protein mutant. A total of three different CVN mutants, T57S, S52T, and a double mutant T57S-S52T, or simply TS, were investigated on the background of P51G-m4-CVN. After conducting the experimental work, it was concluded that the overall fold and stability of the protein was conserved for each mutant. ITC data showed that T57S displayed the lowest dissociation constant valued in the micromolar range. In fact, T57S had a much lower Kd value in comparison to P51G-m4. In contrast, the double mutant TS, showed poor binding affinity for the glycan. When comparing experimental data with the data provided by MD simulation and BP-docking, the results were fairly correlated for all mutants, except for that of the double mutant, TS. According to information provided by MD simulation and BP docking, the binding of the sugar to TS is a very exergonic reaction, which is indicative of very negative free energy change (ΔG). However, the experimental Kd, which was very high, contradicts this data and is thus indicative of lower binding affinity for the glycan. This contradiction is currently being investigated.

Hydrogen has the potential to be a highly efficient fuel source. Its current production via steam reformation of natural gas, however, consumes a large amount of energy and gives off carbon dioxide. A newer method has since surfaced: using a microorganism's metabolism to drive hydrogen production. In this study, the conditions for maximum hydrogen production in Heliobacterium modesticaldum were identified and assessed. The cells were grown under varying conditions and their headspaces were sampled using a gas chromatogram to measure the amount of accumulated hydrogen during each condition. Two cell batches were grown under nitrogen-fixing conditions (-NH4+), while the other two cell batches were grown under non-nitrogen-fixing conditions (+NH4+). The headspaces were then exchanged with either nitrogen (N2) or argon (Ar2). It was found that the condition for which the most hydrogen was produced was when the cells were grown under nitrogen-fixing conditions and the headspace was exchanged with argon. These results suggest that most of Heliobacteria modesticaldum's hydrogen production is due to nitrogenase activity rather than hydrogenase activity. Further research is recommended to quantify the roles of nitrogenase, [NiFe] hydrogenase, and [FeFe] hydrogenase.