| Advisor : | DR. PAUL A. PRICE | ||
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| Abstract Title : | Microbial Calcification: A New Method to Combat Microbial Infections at Wound Sites? | ||
| Abstract : | The Mineralization by Inhibitor Exclusion (MIE) mechanism is the selective mineralization of collagen using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Previous studies have shown that the cell wall of bacteria is also a target for calcification by the MIE mechanism, and that calcification kills the bacteria. Our goals were to improve the lethality of the MIE calcification procedure by determining the optimal conditions at which to conduct the procedure and to determine whether the cell wall of other pathogenic microorganisms is a target of calcification by this method. Currently, we are able to consistently kill 99.7% of live Staphylococcus aureus bacteria via calcification at physiological pH and 37°C. With the success of our calcification procedure, this method could potentially serve as an alternative treatment to combat pathogens that have become antibiotic resistant. | ||
| Advisor : | PARTHO GHOSH | ||
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| Abstract Title : | Studies of Burkholderia pseudomallei's Type VI Secretion System Protein, VgrG1 | ||
| Abstract : | Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, a disease common to third world countries. Infection by this bacterium leads to cytopathic effects due to the formation of large multinucleated cells, resulting from cell-cell membrane fusion. Membrane fusion is a highly regulated process that requires specific proteins and conditions. Evidence suggests that Burkholderia pseudomallei uses the VgrG protein of its type VI secretion system, which resembles the T4 bacteriophage tail, to cause cell-cell membrane fusion. The VgrG family of proteins is well conserved in type VI secretions systems. In particular, these proteins have a conserved N-terminal domain that has similarities to the gp5 and gp27 proteins of the T4 bacteriophage tail spike. However, Burkholderia pseudomallei contains an evolved VgrG1, which has an extra C-terminal domain that does not align with any known sequence or structure. As the N-terminal domain of VgrG1 is known to interact with the host cell membrane, we hypothesize that the additional C-terminal domain of VgrG1 is involved in facilitating membrane fusion. The goal of this project is to express and purify this C-terminal domain in order to crystallize the protein and determine its structure. | ||
| Advisor : | PAUL PRICE | ||
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| Abstract Title : | Studies on the Mechanisms of Collagen Calcification in Bone | ||
| Abstract : | Type 1 collagen is the most abundant protein in tendon, skin, bone, and dentine. The collagen in these tissues has the same amino acid sequence, and is organized into fibers with the same structure. The only difference is the presence of mineral within the collagen fibers of bone and dentine and the presence of water within the collagen fibers of tendon, skin, and other non-calcified tissues. In the formation of bones and teeth, mineral replaces the water within the collagen fiber to create a structure that combines the high tensile strength of collagen with the resistance to compression of mineral. We have developed a novel strategy to recapitulate in vitro the normal process of collagen fiber mineralization that occurs in the formation of bone and dentine. This strategy is based on the unique size exclusion characteristics of the collagen fiber, and is called the Mineralization by Inhibitor Exclusion (MIE) mechanism. This mechanism works via fetuin, a 48 kDa inhibitor of apatite growth. Fetuin works to localize mineralization within collagen while inhibiting mineralization outside. In our study, we have used the MIE mechanism to introduce a minute amount of apatite mineral crystals within the collagen fibers of tendon. We have then examined the ability of these mineral crystal seeds to grow and harden the tissue when the tendon is subsequently incubated in bovine and human serum at physiological conditions (pH 7.4). Qualitative analysis has shown that flexible collagen remains flexible after the pre-seeding step, but becomes significantly stiff after incubation in serum at physiological conditions. Furthermore, flexible pre-seeded collagen, when manipulated into certain structures and incubated in serum, has shown to harden and stiffen into desired structures. This ability to calcify collagen may have significant utility in the ability to calcify some collagen implants, as they could potentially provide structural support in areas in the body experiencing high forces and impact. | ||
| Advisor : | DR. VIRGIL WOODS JR. / DR. TIMOTHY MORRIS | ||
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| Abstract Title : | Comparison between the Structural Dynamics of Wild-Type and Mutant Fibrinogen Protein Using Amide Hydrogen/Deuterium Mass Spectrometry | ||
| Abstract : | Fibrinogen, a large protein complex synthesized in hepatocytes of the liver for blood clot formation, consists of two identical disulfide linked subunits, each containing three homozygous polypeptide chains Aα, Bβ and γ. Under normal circumstances these function together to play a vital role in the wound healing process. However with a few changes in the right places, their malfunction can lead to an inability to clot or resolve when one needs it where one needs it and is a phenomena that has not yet been fully elucidated. Using Deuterium Exchange Mass Spectrometry (DXMS), we were able to resolve over 90 percent of Fibrinogen's primary sequence including previously unidentified regions in X-ray crystal structures, and provide vital information towards understanding the changes imposed against the clotting mechanism. Through multiple experimental trials the deuterium exchange profiles of both wild-type Fibrinogen and mutant/wild-type Fibrinogen mixture consistently indicate a substantial increase in structural flexibility of the gamma chain due to two key amino acid modifications to the beta chain which likely disrupt favorable arrangement of secondary structure between the two. Comprehending how these conformational changes in mutant fibrinogen exactly influence the clotting process is of great importance in furthering our understanding the pathology of human blood clot diseases such as Chronic Thromboembolic Pulmonary Hypertension due to such dysfibrinogenemias. | ||
| Advisor : | VIRGIL WOODS | ||
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| Abstract Title : | Structural Dynamic Changes in PrP during Prion Formation as Characterized by Deuterium Exchange Mass Spectrometry | ||
| Abstract : | Prions are the infectious agents responsible for transmissible spongiform encephalopathies. These diseases are associated with the conversion of a normal host-encoded cellular prion protein (PrPC) into an abnormal, self-propagating conformation known as PrPSc. Both the structure of PrPSc and its mechanism of propagation remain unclear. Pathological, protease-resistant, self-perpetuating PrPSc can be generated in vitro from recombinant PrPC (α-PrP) through protein misfolding cyclic amplification (PMCA) with the addition of phospholipids and RNA polyanions. Before conversion into PrPSc, conformations of PrP with distinct properties can be isolated and purified, suggestive of structural intermediates in the transformation process. The dynamic changes that occur during the transition from PrPC to PrPSc were characterized by deuterium exchange mass spectrometry (DXMS). Phosphatidylglycerol (POPG) and RNA were consecutively added to α-PrP, creating intermediates designated PrP-POPG and PrP-POPG-RNA, respectively. α-PrP, PrPSc, and the intermediates were examined by DXMS. Two regions of conformational rearrangement were observed when POPG is added to PrPC: one area of decreased protection (148-167) and one area of increased protection (187-206). The addition of RNA to PrP-POPG increases solvent-accessibility in the N-terminus region. When PrP-POPG-RNA complexes are converted to PrPSc, major structural features of the intermediate are retained, but with a global increase in protection. These findings indicate that POPG and RNA cofactors play major roles in PrP conformational change during the formation of PrPSc. Knowing the conformational changes that occur during prion misfolding is essential for understanding prion self-replication and provides specific drug targets for hindering prion disease progression by inhibiting pathologic PrP conversion. | ||
| Advisor : | PARTHO GHOSH | ||
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| Abstract Title : | Biochemical Studies of YscO and YscP, two Components of the Type III Secretion System in Yersinia pseudotuberculosis | ||
| Abstract : | A constant health care problem in the world is the existence of disease-causing bacteria. A group of these pathogenic bacteria are gram negative bacteria that possess a needle-like protein appendage called the type three secretion (T3S) system. This system is responsible for translocating bacterial proteins called effectors from the bacterial cytosol directly into the cytosol of host cells. Once in host cells, these effectors are able to combat the host's immune response, enabling the bacteria to propagate. The focus of this project is on two proteins, YscO and YscP, which are important for the proper function of the T3S system in the bacterial pathogen Yersinia pseudotuberculosis. Previous studies have suggested that proper translocation of effectors fails to occur when the bacteria do not possess YscO. Other studies have shown that YscP sets the length of the needle, and that without YscP, the bacteria fail to set the appropriate length of their needles. Furthermore, it has been suggested that a certain region of YscP called the T3S4 (type three secretion substrate specificity switch) region is involved in the proper export of effectors. Also, studies have shown that YscO and YscP interact with each other. The goal of the project is to determine the atomic and molecular structure of these proteins in complex. To investigate the structure of these proteins, this project will pursue the crystallization of the two proteins in complex with each other. Once these proteins are crystallized, x-ray crystallography techniques will be performed in an attempt to determine the atomic-resolution structure of the proteins. Previous attempts to produce the proteins on their own have not succeeded, as these proteins are unstable by themselves when overexpressed in E. coli. By expressing both proteins together, perhaps both proteins will be produced in stable form such that crystallization trials can be pursued. | ||