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Indiana University-Purdue University Indianapolis (IUPUI) / Omega-3 polyunsaturated fatty acids (n-3 PUFAs) relieve the symptoms of a wide variety of chronic inflammatory disorders. Typically, they must be obtained in the diet from sources such as fish oils. Docosahexaenoic acid (DHA) is one of these n-3 PUFAs. As yet the structural mechanism responsible for the health benefits within the body is not completely understood. One model that has emerged from biochemical and imaging studies of cells suggests that n-3 PUFAs are taken up into phospholipids in the plasma membrane. Thus the focus here is on the plasma membrane as a site of potential structural modification by DHA. Within cellular membranes, the huge variety of molecules (called lipids) which constitute the membrane suggest inhomogeneous mixing, thus domain formation. One potential domain of interest is called the lipid raft, which is primarily composed of sphingomyelin (SM) and cholesterol (chol). Here the molecular organization of [2H31]-N-palmitoylsphingomyelin (PSM-d31) mixed with 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), as a monounsaturated control, and cholesterol (chol) (1:1:1 mol) in a model membrane was examined by solid state 2H NMR spectroscopy. Solid state 2H NMR spectroscopy extracts details of molecular orientation and anisotropy of molecular reorientation by analysis of the lineshape. This essentially non-invasive technique allows for a direct measurement of dynamics in bulk materials which has been extensively applied to biological materials. It is a niche area of NMR for which standard software often lack necessary features. Two software programs, “EchoNMR processor” and “EchoNMR simulator”, collectively known as “EchoNMR tools”, that were developed to quickly process and analyze one-dimensional solid-state NMR data, will be described along with some theoretical background of the techniques used. EchoNMR tools has been designed with a focus on usability and the open-source mindset. This is achieved in the in the MATLAB® programming environment which allows for the development of the graphical user interfaces and runs as an interpreter which allows the code to be open-source. The research described here on model membranes demonstrates the utility of the software. The NMR spectra for PSM-d31 in mixtures with PDPC or POPC with cholesterol were interpreted in terms of the presence of nano-sized SM-rich/chol-rich (raft-like) and PC-rich/chol-poor (non-raft) domains that become larger when POPC was replaced by PDPC. An increase in the differential in order and/or thickness between the two types of domains is responsible. The observation of separate signals from PSM-d31, and correspondingly from [3α-2H1]cholesterol (chol-d1) and 1-[2H31]palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC-d31), attributed to the raft-like and non-raft domains enabled the determination of the composition of the domains. Most of the SM (84%) and cholesterol (88%) was found in the raft-like domain. There was also a substantial amount of PDPC (70%) in the raft-like domain that appears to have minimal effect on the order of SM. PDPC molecules sequestering into small groups to minimize the contact of DHA chains with cholesterol is one possible explanation that would also have implications on raft continuity. These results refine the understanding of how DHA may modulate the structure of raft domains in membranes.

ATP-Binding-Cassette (ABC) transporters are primary active pumps that typically couple the binding and hydrolysis of ATP to the translocation of compounds across cellular membranes. Some, like ABCB1, ABCC1 and ABCC3, are polyspecific and can efflux clinically important drugs which may contribute to their therapeutic failure. In this study I have investigated (1) the mechanism of ABC transporter function, (2) studied the potential for regulation by ubiquitin ligases (both using ABCB1 as a model), and (3) tested the involvement of ABCC1 and ABCC3 in autocrine signalling in cancer. (1) In 1966, Jardetzky et. al [1] proposed that membrane pumps function by exposing their ligand-binding pocket alternately on different sides of the membrane. For ABC transporters, this coupling of the aspect and affinity of the ligand-binding cavities of the two transmembrane domains (TMDs) to the ATP catalytic cycle of the two nucleotide-binding domains (NBDs) is fundamental to the transport mechanism but is poorly understood at the molecular level. Structure data suggest signals are transduced through intracellular loops of the TMDs which slot into grooves on the top surface of the NBDs. At the base of these grooves is the Q-loop. By analysing the function of Q-loop mutants in combination with ligand binding cavity mutants I have discovered that the Q-loops are crucial to the transport cycle and that they are required to couple ligand binding to conformational changes at the NBDs necessary to drive the transporter into an inward closed state. 4 (2) ABCB1 is known to be a key component of chemical barrier separating the circulation from the cerebrospinal fluid. It has also been reported to transport β-amyloid across the lumenal membrane and into the circulation. Loss of ABCB1 from the barrier with age has therefore been suggested to play a role in Alzheimer’s Disease. The ubiquitin ligase Nedd4-1 has been implicated in the post-translational regulation of ABCB1 abundance in cells. Here, I report that ABCB1 can be ubiquitinated by Nedd4-1 in vitro and identify the residues modified (by mass spectrometry). (3) Lysophosphatidylinositol (LPI) is an autocrine metabolite produced by cancer cells that binds to the G-protein coupled transmembrane receptor GPR55 on the surface of cells. Stimulation of GPR55 activates a signalling cascade that induces proliferation and metastases of the cancer cells. How LPI is released from the cells was not known. In this study I show that ABCC1 and ABCC3, which are known to be expressed in ovarian and pancreatic cancers, can transport LPI into inside-out vesicles suggesting a new role for these “drug resistance” transporters in cancer biology.

A simulação computacional de membranas biológicas, em particular membranas formadas por bicamadas lipídicas, é uma área de grande interesse na atualidade. Enquanto simulações moleculares são bastante populares, a simulação na escala de uma célula inteira requer modelos baseados na mecânica dos meios contínuos. Essas membranas apresentam um comportamento de fluido viscoso incompressível bidimensional. Além disso, as formas de equilíbrio são bem explicadas pela energia de Canham-Helfrich, que depende da curvatura da membrana. Neste trabalho, um novo método de simulação de membranas viscosas, baseado em elementos finitos, é apresentado. Ele se inspira no conceito de James Clerk Maxwell de elasticidade fugaz, o qual é usado para adaptar técnicas bem estabelecidas de simulação de membranas elásticas. Trata-se do primeiro método a levar em conta, de maneira rigorosa, o aspecto viscoso da membrana, que é dominante na escala de tamanho de uma célula biológica, além da sua característica de fluido incompressível / The computational simulation of biological membranes, in particular of those made of lipid bilayers, is currently an area of great interest. While molecular simulations are quite popular, the simulation on the scale of a whole cell requires models based on continuum mechanics. Those membranes behave like a bidimensional incompressible viscous fluid. Furthermore, the equilibrium shapes are well explained by means of the Canham-Helfrich energy, which depends on the curvature of the membrane. In this work, a novel finite element based method for the simulation of viscous membranes is presented. It is inspired by James Clerk Maxwells concept of fugitive elasticity, which is used to adapt well established simulation techniques for elastic membranes. This is the first method to take into account, in a rigorous fashion, the viscous aspect of the membrane, which is dominant at the length scale of a biological cell, in addition to its characteristics as an incompressible fluid

A simulação computacional de membranas biológicas, em particular membranas formadas por bicamadas lipídicas, é uma área de grande interesse na atualidade. Enquanto simulações moleculares são bastante populares, a simulação na escala de uma célula inteira requer modelos baseados na mecânica dos meios contínuos. Essas membranas apresentam um comportamento de fluido viscoso incompressível bidimensional. Além disso, as formas de equilíbrio são bem explicadas pela energia de Canham-Helfrich, que depende da curvatura da membrana. Neste trabalho, um novo método de simulação de membranas viscosas, baseado em elementos finitos, é apresentado. Ele se inspira no conceito de James Clerk Maxwell de elasticidade fugaz, o qual é usado para adaptar técnicas bem estabelecidas de simulação de membranas elásticas. Trata-se do primeiro método a levar em conta, de maneira rigorosa, o aspecto viscoso da membrana, que é dominante na escala de tamanho de uma célula biológica, além da sua característica de fluido incompressível / The computational simulation of biological membranes, in particular of those made of lipid bilayers, is currently an area of great interest. While molecular simulations are quite popular, the simulation on the scale of a whole cell requires models based on continuum mechanics. Those membranes behave like a bidimensional incompressible viscous fluid. Furthermore, the equilibrium shapes are well explained by means of the Canham-Helfrich energy, which depends on the curvature of the membrane. In this work, a novel finite element based method for the simulation of viscous membranes is presented. It is inspired by James Clerk Maxwells concept of fugitive elasticity, which is used to adapt well established simulation techniques for elastic membranes. This is the first method to take into account, in a rigorous fashion, the viscous aspect of the membrane, which is dominant at the length scale of a biological cell, in addition to its characteristics as an incompressible fluid