PHD, Kent State University, 2014, College of Arts and Sciences / Department of Chemistry

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is an important signaling lipid in the cell plasma membrane, playing an important role in many diverse signaling processes. It is important to gain an understanding of how PI(4,5)P2's role in these signaling processes is regulated. The headgroup of PI(4,5)P2 is highly negatively charged and electrostatics play a significant role in interactions between PI(4,5)P2 and proteins. In this thesis we have extensively studied the ionization properties of PI(4,5)P2 and its interactions with common components of the plasma membrane inner leaflet and the cytosol. Using a new fitting procedure we have developed a model for the ionization of the phosphatidylinositol polyphosphates within the membrane system. While this ionization had been described before in a qualitative manner, this new fitting model has allowed us to quantitatively describe the ionization and measure pKa values for each ionization step. The ionization of PI(4,5)P2, was also examined in complex ternary lipid systems. We investigated PI(4,5)P2 ionization with each of the plasma membrane inner leaflet lipids, PE, PI, and PS. We found evidence for a significant interaction between PE and PI(4,5)P2. PE forms a hydrogen-bond with the PI(4,5)P2 headgroup, leading to a shift in the ionization of PI(4,5)P2 to lower pKa values. PI was also found to interact with PI(4,5)P2, resulting in the formation of large bulge shaped PI(4,5)P2 rich domains. It has been proposed that lateral localization of PI(4,5)P2 within the plasma membrane is critical for signaling, with multiple pools of PI(4,5)P2 used for different signaling purposes. In vitro studies have indicated that both Ca2+ and cholesterol have the capacity to promote formation of PI(4,5)P2 clusters in model membranes. To shed light on this we have examined the interaction of PI(4,5)P2 with Ca2+, Mg2+, and cholesterol. Cholesterol was found to have a small but significant effect on the ionization of PI(4,5)P2. Th (open full item for complete abstract)

Committee: Arne Gericke Dr. rer. nat. (Advisor); Edgar Kooijman Ph.D. (Advisor); Anatoly Khitrin Ph.D. (Committee Member); Roger Gregory Ph.D. (Committee Member); Derek Damron Ph.D. (Committee Member); Elizabeth Mann Ph.D. (Other) Subjects: Biochemistry

PHD, Kent State University, 2011, College of Arts and Sciences / Department of Chemistry

Membrane lipids are asymmetrically distributed between the inner and the outer leaflet of the plasma membrane, and this asymmetric distribution of lipids is an important factor for many signaling events. The lipids investigated in this study, phosphoinositides, are found in the inner leaflet of the plasma membrane and have been shown to mediate a wide variety of important physiological processes by affecting the activity and/or localization of membrane associated proteins. Phosphoinositide properties are largely determined by the characteristics of their headgroup, which at physiological pH is highly charged but is also capable of hydrogen bond formation. For phosphoinositide mediated signaling events to occur, it requires the local enrichment of phosphoinositides, which depend on the interchange between attractive and repulsive forces. Factors expected to affect mutual phosphoinositide interaction are pH as well as the presence of cations or positively charged proteins. The primary goal of this study was to gain more insight about the unique physiochemical properties of phosphoinositides and how they organize laterally in the membrane. We hypothesized that the type and concentration of salt in the subphase affect the phase behavior of phosphatidylinositol and phosphatidylinositol monophosphate monolayers at the air/water interface. Additionally, we hypothesized that the position of the phosphate group at the inositol ring of phosphatidylinositol monophosphates has an effect on how the phosphoinositide molecules interact with each other and other molecular entities embedded in the biomembrane. Using surface pressure/area isotherms, Infrared Reflection Absorption Spectroscopy (IRRAS) and epifluorescence microscopy we have shown that: 1) the presence of monovalent and divalent salt affect the phase behavior, acyl chain conformational order, and domain morphology of phosphatidylinositol monolayers. 2) The monovalent salt concentration affects the phase behavior, acyl c (open full item for complete abstract)

Committee: Arne Gericke PhD (Advisor); Michael Tubergen PhD (Committee Member); Roger Gregory PhD (Committee Member); Elizabeth Mann PhD (Committee Member); James Blank PhD (Committee Member) Subjects: Chemistry

MS, Kent State University, 2010, College of Arts and Sciences / Department of Chemistry

Phosphatidyl inositides (PIs) are important regulators of cell signaling. Phosphoinositide 3-kinase (PI3-K)-activated signaling plays a key role in the development of cancer. Therefore various anticancer treatments target this pathway. The aim of this work was to develop an optimized method to separate fluorescent PIPs quickly and efficiently at room temperature using CE-LIF. The reason for the development of a method capable of separating the PIPs at room temperature was that, it could be used for single cell studies. In this regard, the effect of various cations on the separation of the PIs and their seven PIP derivatives was investigated. The effect of pH, temperature, voltage, as well as other buffer mixtures were also studied. Based on this developed method, the activity of PI3-K and PTEN enzymes in NIH 3T3 cells and MDA-MB 231 cells was also studied. The bioactivity of PIPs in these cells was investigated to ascertain whether preliminary CE studies would prove that they were able to be converted inside the cells into other phosphorylated derivatives. Lipid kinases and phosphatases play active roles in cell signaling. These have serious implications in many disease states. For example, accumulation of PIP3 leads to metastatic cancers. Therefore, inhibition studies were also performed to find out whether PI3-K inhibitors were able to block the conversion of PIP2 to PIP3. This is of particular interest in cell-based assays and research involving cancer drug development.

Committee: Simon M Mwongela PhD (Advisor); Songping Huang PhD (Committee Member); Bansidhar Datta PhD (Committee Member) Subjects: Biochemistry

PHD, Kent State University, 2008, College of Arts and Sciences / Department of Chemistry

PTEN, phosphatase and tensin homologue deleted on chromosome 10, is a tumor suppressor that is commonly lost or mutated in many different diseases, including cancer, Bannayan Zonana syndrome, heart disease and cancer. The mechanism of the effects of the loss or mutation of PTEN is not known, other than the effects on the regulation of downstream effector molecules. While many other research groups have studied the effects of PTEN loss or mutation on tumor formation in mouse models, no group has undergone the daunting task of characterizing the protein biophysically, in order to determine how these mutations affect the actual action of the protein. In this study, we have shown that PTEN binds specifically to PI(4,5)P2 over all other phosphoinositides, including its substrate, PI(3,4,5)P3. This binding event is also associated with a conformational change, which is believed to activate the protein and increase its rate of turnover of PI(3,4,5)P3. Additionally, we shown in these studies that the binding specificity of PTEN is dependent on the presence and identity of the N-terminus, which contains a PI(4,5)P2 binding domain. Removal or mutation of the protein's N-terminus abrogates the binding and thus activity of the protein. One particular amino acid which is often mutated in many forms of cancer, the lysine in position 13, has been shown to be extremely important in the interaction of PTEN with PI(4,5)P2 containing membranes. We show that even while maintaining overall charge of the N-terminus and even overall identity, mutation of the lysine in this position results in a loss of binding specificity. We have also studied the binding of an autism related mutation of PTEN, which results in an increased binding to phosphatidylserine, which may play a role in the ability of the protein to turnover its substrate in vivo. We have also studied the effects of cholesterol on the binding of proteins to phosphoinositide containing membranes, which has proven to have different (open full item for complete abstract)

Committee: Arne Gericke (Advisor) Subjects: Biochemistry