A Study Of Genetic Representation Schemes For Scheduling Soft Real-Time Systems

This research presents a hybrid algorithm that combines List Scheduling (LS) with a Genetic Algorithm (GA) for constructing non-preemptive schedules for soft real-time parallel applications represented as directed acyclic graphs (DAGs). The execution time requirements of the applications\u27 tasks are assumed to be stochastic and are represented as probability distribution functions. The performance in terms of schedule lengths for three different genetic representation schemes are evaluated and compared for a number of different DAGs. The approaches presented in this research produce shorter schedules than HLFET, a popular LS approach for all of the sample problems. Of the three genetic representation schemes investigated, PosCT, the technique that allows the GA to learn which tasks to delay in order to allow other tasks to complete produced the shortest schedules for a majority of the sample DAGs

Sexual pheromone modulates the frequency of cytosolic Ca2+ bursts in Saccharomyces cerevisiae

Transient and highly regulated elevations of cytosolic Ca2+ control a variety of cellular processes. Bulk measurements using radioactive Ca2+ and the luminescent sensor aequorin have shown that in response to pheromone, budding yeast cells experience a rise of cytosolic Ca2+ that is mediated by two import systems composed by the Mid1-Cch1-Ecm7 protein complex, and the Fig 1 protein. Although this response has been largely studied, there is no report on Ca2+ dynamics at the single cell level. Here, using protein calcium indicators we show that both vegetative and pheromone-treated yeast cells exhibit discrete and asynchronous Ca2+ bursts. Most bursts reach maximal amplitude in 1-10 secs, span between 7 and 30 secs and decay fitting a single exponential model. In vegetative cells bursts are scarce but preferentially occur when cells are transitioning G1 and S phase. Upon pheromone presence Ca2+ burst occurrence increases dramatically, persisting during cell growth polarization. Pheromone concentration modulates burst frequency in a mechanism that depends on Mid1, Fig 1 and a third, still unidentified, import system. We also show that the calcineurin-responsive transcription factor Crz1 experiences nuclear localization bursts during the pheromone response.Fil: Carbo, Natalia. Instituto Pasteur de Montevideo; UruguayFil: Tarkowski, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Perez Ipiña, Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Ponce Dawson, Silvina Martha. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; ArgentinaFil: Aguilar, Pablo Sebastián. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentin

Abnormal cognition, sleep, EEG and brain metabolism in a novel knock-in Alzheimer mouse, PLB1

Peer reviewedPublisher PD

Exploring new applications for photophysically encoded microcarriers

The first goal of this thesis was to investigate the potential of two cationic polysaccharides (PQ-4 and PQ-10) for DNA delivery. We have shown that, compared to PEI based polyplexes, they were less efficient in transfecting cells. However, as they had very low toxicity, further tailoring of the nature and extent of cationic side chains on cationic hydroxyethylcellulose may be a promising avenue to further enhance their DNA delivery properties. As a second goal we investigated the applications of digitally encoded microcarriers for cell based assays. We succeeded to show that encoded microcarriers were suitable to grow cells on. Neither the coating at the surface of the beads (which facilitates the growth of the cells), nor the cells themselves hampered the decoding of the beads, even when the cells covering the microcarriers exhibited green or red fluorescence due to the expression of GFP and RFP respectively. We were able (a) to immobilize DNA, siRNA or adenoviral particles on the surfaces of the encoded microcarriers by the use of polyelectrolytes and, subsequently, (b) to grow cells on top of the nucleic acids/adenoviral particles. The DNA and siRNA immobilized on the surface of the microcarrier were not able to transfect cells. However, we showed that the cells growing on the polyelectrolyte layer could indeed become transduced with adenoviral particles hosted by the polyelectrolyte layer. In conclusion, a proof of principal to use photophysically encoded microcarriers as transfected microarray has been shown. As a third goal we investigated the use of digitally encoded microcarriers as tool to combat counterfeiting of tablets. We showed that the codes in the Memobeads in tables produced by granulation did not deform during tabletting and that the code in the beads remained readable. We also found evidence that, after oral intake, the encoded microparticles are highly unlikely toxic to humans

The Tumor Suppressor Function of LGI1

Several evidences supporting the tumor suppressor role of LGI1 have been presented here. The discovery of chromosomal rearrangements leading to loss of LGI1 expression in glioblastoma cells, mutations in LGI1 gene specifically associated with gliobastoma and the downregulation of LGI1 expression in several tumors all point out a role of LGI1 in tumor suppression. Furthermore the findings that re-expression of LGI1 in glioblastoma cells impaired cell growth and migration ability through inhibition of the ERK1/2 pathway, with consequent downregulation of MMPs expression, support a role in the suppression of metastasis formation and tumor vascularization. This is in line with the downregulation of LGI1 expression observed in the malignant progression of gliomas. The findings that increased expression of LGI1 impaired growth and survival of neuroblastoma cells further strengthen the tumor suppressor role of LGI1. The involvement of LGI1 in the negative regulation of the PI3K/AKT pathway supporting cell proliferation and survival explains the mechanism of spontaneous cell death triggered by the elevation of LGI1 levels in neuroblatoma cells. The activation of intrinsic apoptosis triggered by LGI1 is consistent with a blockage of AKT activity, which regulates Bcl-2 family members involved in the control of mitochondrial membrane permeability. Furthermore, the interaction of LGI1 protein with voltage gated potassium channels (Kv1.1) shown to prevent channel inactivation by Kv1b subunit, provides an additional link with apoptosis since these channels are important regulators of cell survival. Because suppression of apoptosis in cancer cells is one of the main strategies to achieve the survival advantage required for malignant progression, it is feasible that alterations of LGI1 gene or downregulation of expression often observed in cancer cells might be required to suppress apoptosis through the inhibition of survival pathways linked to growth factor receptors and of Kv channels activity

Vascular cambium regeneration and vessel formation in wounded inflorescence stems of Arabidopsis

Synchronized tissue polarization during regeneration or de novo vascular tissue formation is a plant-specific example of intercellular communication and coordinated development. According to the canalization hypothesis, the plant hormone auxin serves as polarizing signal that mediates directional channel formation underlying the spatio-temporal vasculature patterning. A necessary part of canalization is a positive feedback between auxin signaling and polarity of the intercellular auxin flow. The cellular and molecular mechanisms of this process are still poorly understood, not the least, because of a lack of a suitable model system. We show that the main genetic model plant, Arabidopsis (Arabidopsis thaliana) can be used to study the canalization during vascular cambium regeneration and new vasculature formation. We monitored localized auxin responses, directional auxin-transport channels formation, and establishment of new vascular cambium polarity during regenerative processes after stem wounding. The increased auxin response above and around the wound preceded the formation of PIN1 auxin transporter-marked channels from the primarily homogenous tissue and the transient, gradual changes in PIN1 localization preceded the polarity of newly formed vascular tissue. Thus, Arabidopsis is a useful model for studies of coordinated tissue polarization and vasculature formation after wounding allowing for genetic and mechanistic dissection of the canalization hypothesis

Synthese, Assemblierung und intrazellulärer Transport von Mitgliedern der cys-loop- und P2X-Familie liganden-gesteuerter Ionenkanäle

The results presented here strongly indicate that ubiquitination of the recombinant human alpha1 GlyR at the plasma membrane of Xenopus oocytes is involved in receptor internalisation and degradation. Ubiquitination of the human alpha1 GlyR has been demonstrated by radio-iodination of plasma membrane-boundalpha1 GlyRs, whose subunits differed in molecular weight by additional 7, 14 or 21 kDa, corresponding to the molecular weights of one, two and three conjugated ubiquitin molecules, respectively, and by co-isolation of the non-tagged human alpha1 GlyR through hexahistidyl-tagged ubiquitin. Ubiquitin conjugated GlyRs where prominent at the plasma membrane, but could be hardly detected in total cell homogenates, indicating that ubiquitination takes place exclusively at the plasma membrane. Ubiquitination of the alpha1 GlyR at the plasma membrane was no longer detectable when the ten lysine residues of the cytoplasmic loop between transmembrane segments M3 and M4 were replaced by arginines. Despite this proteolytic cleavage continued to take place at the same extent as with the wild type alpha1 GlyR, suggesting that removal of GlyRs from the plasma membrane and routing to lysosomes for degradation were not dependent on ubiquitination. Also replacing a tyrosine in position 339, which was speculated to be part of an additional endocytosis motif, did not lead to a significant reduction of cleavage of the GlyR alpha1 subunits. However, a mutant lacking both, ubiquitination sites and 339Y, was significantly less processed. These results may suggest that the GlyR alpha1 subunit harbors at least two endocytosis motifs, which may act independently to regulate the density of alpha1 GlyR. Apparently, each of the two signals may be capable of compensating entirely the loss of the other. Part two of this Dissertation demonstrates that the correct topology of the glycine receptor alpha1 subunit depends critically on six positively charged residues within a basic cluster, RFRRKRR, located in the large cytoplasmic loop following the C-terminal end of M3. Neutralization of one or more charges of this cluster, but not of other charged residues in the M3-M4 loop, led to an aberrant translocation into the endoplasmic reticulum lumen of the M3-M4 loop. However, when two of the three basic charges located in the ectodomain linking M2 and M3 were neutralized, in addition to two charges of the basic cluster, endoplasmic reticulum disposition of the M3-M4 loop was prevented. We conclude that a high density of basic residues C-terminal to M3 is required to compensate for the presence of positively charged residues in the M2-M3 ectodomain, which otherwise impair correct membrane integration of the M3 segment. Part three of this Dissertation describes my contribution (blue native PAGE analysis of metabolically labeled alpha7 and 5HT3A receptors and the examination of the glycosylation state of metabolically labeled alpha7 subunits) to a work on the limited assembly capacity of Xenopus oocytes for nicotinic alpha7 subunits. While 5HT3A subunits combined efficiently to pentamers, alpha7 subunits existed in various assembly states including trimers, tetramers, pentamers, and aggregates. Only alpha7 subunits that completed the assembly process to homopentamers acquired complex-type carbohydrates and appeared at the cell surface. We conclude that Xenopus oocytes have a limited capacity to guide the assembly of alpha7 subunits, but not 5HT3A subunits to homopentamers. Accordingly, ER retention of imperfectly assembled alpha7 subunits rather than inefficient routing of fully assembled alpha7 receptors to the cell surface limits surface expression levels of alpha7 nicotinic acetylcholine receptors. Part four of this Dissertation describes my contribution (the biochemical analysis of the human P2X2 and P2X6 subtypes) to studies on the quaternary structure of P2X receptors. Armaz Aschrafi, the main author of the paper showed that subsequent to isolation under non-denaturing conditions from Xenopus oocytes the His-rP2X2 protein migrated on blue native PAGE predominantly in an aggregated form. The only discrete protein band detectable could be assigned to homotrimers of the His-rP2X2 subunit. Because of the exceptional assembly-behaviour of the rP2X2 protein compared to the rP2X1, rP2X3, rP2X4 and rP2X5 proteins, its human orthologue was investigated in the same manner. In contrast to rP2X2 subunits, hP2X2 subunits migrated under virtually identical conditions in a single defined assembly state, which could be clearly assigned to a trimer. P2X6 subunits represent the sole P2X subtype that is unable to form functional homomeric receptors in Xenopus oocytes. The blue native PAGE analysis of metabolically labeled hP2X6 receptors and the examination of the glycosylation state revealed that hP2X6 subunits form tetramers and aggregates that are not exported to the plasma membrane of Xenopus oocytes