<p>Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from <i>Saccharomyces cerevisiae</i>, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner.</p><p>Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants.</p><p>We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.</p>

Intracellular membrane trafficking is regulated by a large number of protein complexes and lipids. Blocking of trafficking disrupts normal membrane dynamics and causes membrane stress. Two similar proteins from Saccharomyces cerevisiae, Myr1 and Smy2, each containing a polyproline-binding GYF domain, were discovered in separate screens for dosage suppressors of trafficking mutations. The functions of GYF domain proteins are poorly described despite its determined structure and a number of known polyproline peptide ligands. We predicted, using computational analysis, associations between mRNA decay factors and both Myr1 and Smy2, and further demonstrated that they localize to sites of mRNA degradation upon stress, in a GYF domain dependent manner. Ypt6 is a small GTPase that regulates vesicle docking at the late Golgi in budding yeast. Myr1 was found as a novel suppressor during the screening of a genomic library in a null ypt6 mutant. Myr1 additionally was capable of rescuing the temperature sensitive growth of a Ric1 deficient strain. Importantly, Ric1 is an activator of Ypt6 and is synthetic lethal with Myr1. Biochemical characterization of the Myr1 protein revealed a limited solubility and an ability to bind cellular membranes, likely relevant to the rescue of trafficking mutants. We further assayed the affinity of Myr1 domains to liposomes of distinct composition. Preference for negatively charged lipids suggested possible electrostatic interactions with polybasic clusters within C-terminal regions of Myr1. In contrast, the N-terminus with the GYF domain was found to be capable of self-association. Membrane stress caused by a lipid-bilayer perturbing drug resulted in induced formation of mRNA processing bodies. Cumulatively, these studies suggest that Myr1 functions in the regulation of mRNA stability via its GYF domain, and can sense membrane stress by binding to the lipid bilayer.

This diploma thesis deals with problem of design and calculation of membrane structure of stadium roof. This is a complex engineering problem, which includes many partial problems: finding of initial form of membrane, statically and architecturally suitable arrangement of catenaries, economical solution of boundary conditions (foundations). All components affect each other and cannot be dealt without mutual coordination. It always greatly depends on the experience and intuition of engineer who design such structure. Task which cannot be resolved according to the theory of the first order. Equilibrium forces on the deformed structure, which in many projected structures gives satisfactory results, did not correspond to reality. It is therefore necessary to consider equilibrium of forces on the deformed structure according to the theory of large deformations. Diploma thesis was entered with regard to the intention of the companies Ing. Software Dlubal s.r.o. and FEM consulting s.r.o., working together to develop software RFEM. These companies plan to complement this program system with a module MEMBRANE for searching of initial shapes of membrane structures. This work is a contribution to the creation of this module.

Conditions which promote reversible growth arrest, such as hypoxia and high cell density, lead to activation of a diverse network of proteins known as growth arrest specific (GAS) genes. Fatty acid binding protein 4 (FABP4), a lipid chaperone involved in the regulation of metabolic and inflammatory responses, has been shown to be part of the GAS program. While the induction of FABP4 in oxygen-deprived environments is well characterized, its functionality and regulation in such conditions remains unclear. In this study, we describe how mis-expression of FABP4 affects cell viability and survival within low oxygen conditions. Loss of FABP4 using shRNA was shown to be associated with a significant increase in oxidative stress and lipid peroxidation, a reduction in lipid droplet formation and a greater incidence of apoptosis. Hypoxia-mediated expression of FABP4 was also found to be positively correlated with cellular levels of C/EBP-beta, an essential activator of p20K in quiescence. FABP4 and p20K are both lipocalins that have been shown to share similar induction patterns and ability to assist in the maintenance of lipid trafficking in cellular stress circumstances. Unexpectedly, the depletion of FABP4 or p20K results in loss of the other in limited oxygen concentrations. This occurs independently of disruption to the broad GAS gene program, suggesting the two proteins may be co-regulated in a shared hypoxic-signalling pathway. C/EBP-beta appears to be the transcriptional activator shared by FABP4 and p20K in quiescence, and the three may be part of an intricate system to sense and respond to reactive oxygen species and lipid radicals. However, the forced expression of either FABP4 or p20K when the other is repressed only moderately restores cell survival through alleviating oxidative stress, indicating the two are both necessary for optimal response to hypoxia. In all, these studies suggest that analogous to the p20K lipocalin, FABP4 plays a critical role in lipid homeostasis and cell survival in conditions of limited oxygen concentrations, and its stimulation is dependent on C/EBP-beta activity. / Thesis / Master of Science (MSc) / A study investigating the role of FABP4 and p20K in conditions of reversible growth arrest with an emphasis on cell survival, lipid homeostasis and mitigating the effects of oxidative stress, and regulation of the two lipocalins by C/EBP-beta.