Development of methods to monitor maturation and trafficking of Carboxypeptidase Y (CPY) and its G255R mutant (CPY*)

Protein folding is a vital biological process which underpins many cellular functions in both eukaryotic and prokaryotic cells. This mechanism is a prime example of macromolecular self-assembly which leads to important biological function, such as molecular trafficking to specific cellular parts and cellular differentiation. However, whilst for the majority of cases proteins fold into their correct 3D structure with long-term stability, there is a propensity for proteins to misfold due to insufficient molecular interactions between the amino acids within the polypeptide chain. Once formed, these misfolded proteins have the potential to aggregate and cause pathological or even neurological diseases. Thus, it is of importance to probe the mechanism(s) of protein misfolding to uncover its molecular origins. The yeast species known as baker’s yeast (S. Cerevisiae) is a model organism to probe this mechanism, and carboxypeptidase Y (CPY) has been proposed as a suitable model protein, due to its high abundance within the yeast endoplasmic reticulum (ER), to understand this process. Literature has shown that CPY is a widely used model protein in understanding protein sorting events within the ER of S. Cerevisiae. The advantages, and subsequent choice of this protein, are based upon its structure and role. It plays a part in the C-terminal chemistry of polypeptides, and thus may inform on mis-interactions which contribute to misfolding. Furthermore, CPY trafficking from the ER to the Golgi to the vacuole has provided information on sorting signal events which are like mammalian cellular signals, and thus share similar features with other organisms. It is also a preferable model protein due to its unique catalytic triad (active site). Although classified as a serine protease, it has a much greater pH and temperature range than other proteases, and can thus maintain high activity across environmental changes. Its mutated analogue, carboxypeptidase Y* (CPY*), has also been chosen as a model protein to compare its molecular sorting mechanism with CPY. It is characterised by a glycine-arginine mutation at the 255 amino acid position. The purpose of this project is to uncover the molecular mechanisms of misfolding, namely, post trafficking of CPY & CPY*, whether these misfolded proteins renature and continue trafficking or whether they are degraded by cellular machinery. Alternatively, whether there is evidence of competition between these processes. This would also shed light on the kinetics of these processes and the likelihood of clearance of these misfolded proteins from the ER. To probe these processes, the maturation of pre-cursor forms of both CPY and CPY* have been studied, as they undergo cellular trafficking across the secretory pathway from the ER to the Golgi to the vacuole. The initial experiments have been used to test whether CPY/CPY* can be detected in a western blot through SDS-PAGE gels, and whether CPY/CPY* can be detected in an immunoprecipitate. The final experiment was used to assess whether the dose-dependent cell-cycle regulator 2 (DCR2) plays a role in ER-induced stress, by specifically affecting CPY* degradation. All such experiments employ classical molecular biology techniques. These findings could shed light on whether degradation, by means of disulphide bond breaking and thus slower migration on SDS-PAGE gels, or renaturation, by means of cellular mechanisms, is the dominant mechanism within the ER

Measurement of Magnetic Relaxation in the peak regime of V3Si

Magnetization relaxation measurements are carried out in the Peak effect regime of superconducting V3Si crystal, using Quantum Design SQUID magnetometer. Relaxation in the increasing field scan is logarithmic in time, consistent with the theory of flux creep. The relaxation on the decreasing field scan however exhibits athermal behavior which is predominantly governed by the flux avalanches triggered by the small external field perturbation experienced by the superconductor during measurement scan in an inhomogeneous field.Comment: PDF, 17 pages including 9 figure

Determinization of One-Counter Nets

One-Counter Nets (OCNs) are finite-state automata equipped with a counter that is not allowed to become negative, but does not have zero tests. Their simplicity and close connection to various other models (e.g., VASS, Counter Machines and Pushdown Automata) make them an attractive model for studying the border of decidability for the classical decision problems. The deterministic fragment of OCNs (DOCNs) typically admits more tractable decision problems, and while these problems and the expressive power of DOCNs have been studied, the determinization problem, namely deciding whether an OCN admits an equivalent DOCN, has not received attention. We introduce four notions of OCN determinizability, which arise naturally due to intricacies in the model, and specifically, the interpretation of the initial counter value. We show that in general, determinizability is undecidable under most notions, but over a singleton alphabet (i.e., 1 dimensional VASS) one definition becomes decidable, and the rest become trivial, in that there is always an equivalent DOCN

Irreversible magnetization in thin YBCO films rotated in external magnetic field

The magnetization M of a thin YBaCuO film is measured as a function of the angle $\theta$ between the applied field H and the c-axis. For fields above the first critical field, but below the Bean's field for first penetration H*, M is symmetric with respect to $\theta =\pi$ and the magnetization curves for forward and backward rotation coincide. For H>H* the curves are asymmetric and they do not coincide. These phenomena have a simple explanation in the framework of the Bean critical state model.Comment: 14 pages, 7 PostScript figure