TAR1 is a protein coding gene situated antisense to the 25S rRNA in S. cerevisiae. Tar1p is localized to the mitochondrial inner membrane, and expression is enhanced under conditions of respiratory dysfunction. One common cause of respiratory dysfunction in S. cerevisiae are selfish mitochondrial mutants known as ρ- mitotypes. ρ- mitotypes exhibit drive within the cell following sexual reproduction ; outcompeting host cells and inducing respiratory dysfunction. Respiratory dysfunction activates the Retrograde Response, which involves the expression of genes to compensate for loss of anabolic activity that accompanies respiratory dysfunction. The Retrograde Response also leads to the formation of lifespan shortening Extrachromosomal rDNA circles. Amplification of rDNA circles has the effect of increasing TAR1 at the same time as lifting transcription repression. This observation led to the hypothesis that the formation of rDNA circles was a positive effect of the Retrograde Response, and that TAR1 may serve to ameoleriate the spread of respiratory incompetent mitochondria following sexual reproduction. In this thesis, experiments are conducted that show that TAR1 does suppress the drive of selfish mitochondrial mutants. Additional bioinformatic analyses show that the Retrograde Response may be a recent adaption to selfish mitochondrial mutants.

In S. cerevisiae, the accumulation of dysfunctional mitochondria activates a retrograde signal that is mediated through multiple cytosolic regulators. Central to activation is the cytosolic regulator Rtg2p which through its interaction with Mks1p, promotes the nuclear translocation of Rtg1p/3p. Nuclear localized Rtg1p/3p promotes transcription of target genes. Prior work has shown Rtg2p interaction with Mks1p is required for downstream signaling, however the Mks1p binding site within Rtg2p is unknown. To identify this motif, random mutations were generated in RTG2 and a red-white screening strategy was used to assess 14,001 clones. Sequence analysis identified four mutants with amino acid mutations in the carboxy-terminus of Rtg2p that gave rise to defects in CIT2 transcription and loss of Mks1p interaction. Relative to RTG2, all mutants had reduced Rtg2p protein half-lives. Together these results suggest that the carboxy-terminal domain of Rtg2p is essential for retrograde signaling as it may contain the Mks1p binding site.

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 73-81).

Thesis (M.Med.Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 73-81). Also available in print.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Zugl.: Saarbrücken, Univ., Diss., 2003.

Abstract Retrograde cerebral perfusion (RCP) techniques have been adopted in aortic arch surgery for clinical use. The clear benefits of RCP are that it reduces embolic injury and prolongs the permissible period of hypothermic circulatory arrest (HCA). At the same time, however, there is a great deal of evidence according to which RCP may be associated with an increased risk of fluid sequestration and cerebral edema. In the current study intermittent RCP was compared with continuous RCP and HCA alone to clarify if periodical RCP decreases fluid sequestration (I). HCA is an effective method of cerebral protection, but is associated with long cardiopulmonary bypass times, and coagulation disturbances. We tested the hypothesis that deep hypothermic RCP could improve cerebral outcome during moderate HCA (II and III). Glutamate excitotoxicity plays an important role in the development of ischemic brain injury. The purpose of the present study was to determine the efficacy of lamotrigine, a Na+ channel blocker, to mitigate cerebral injury after HCA (IV). A chronic porcine model was used in the present series of studies. Hemodynamic, electrophysiologic, and metabolic monitoring were performed until four hours after the instigation of rewarming. S-100β was measured up to 20 hours. Daily behavioral assessment performed until death or elective sacrifice on the seventh postoperative day. After continuous RCP the median fluid sequestration volume was 145 (0–250) ml compared with -50 (-100 - 0) ml after intermittent RCP (p = 0.04). In comparison of 15°C RCP to HCA alone during moderate 25°C hypothermia, 5/6 animals in the RCP group survived seven days compared with 2/6 in the HCA group (p = 0.04). The total histopathologic scores in the RCP(15°C) group were lower than those for the RCP(25°C) group during moderate 25°C hypothermia (p = 0.04). EEG bursts were recovered better in the RCP(15°C) group at 3 hours after the start of rewarming compared to HCA group (p = 0.05). The rate of EEG burst recovery was higher in lamotrigine treated animals compared to placebo treated animals after 4 hours during the rewarming (p = 0.02). Among the animals that survived for 7 days, the median behavioral score was higher in the lamotrigine group (8) compared with controls (7) (p = 0.02). The results indicate that intermittent RCP decreases the rate of fluid sequestration after continuous RCP. The cold RCP at moderate systemic hypothermia seems to provide a better neurological outcome than that with moderate temperature RCP, a finding suggesting that enhanced cranial hypothermia is the major beneficial factor of RCP. The Na+ channel blocker lamotrigine improves neurological outcome after a prolonged period of HCA. In conclusion, two refinements in the RCP concept are to administer it at low temperatures and if longer periods of perfusion are necessary, RCP should be applied intermittently.

Changes in gene expression in response to mitochondrial dysfunction are mediated by components of the retrograde signaling pathway. The mitochondrial signal is recognized and transferred to the nucleus by dynamic interactions between regulatory proteins Rtg2p, Mks1p and Bmh1p. Retrograde signaling genes have been well characterized in the budding yeast Saccharomyces cerevisiae but very little is known about the retrograde response of other fungi. To identify retrograde signaling proteins in other fungi, the protein sequence encoded by the S. cerevisiae RTG2 gene was used to search for fungal homologs using NCBI BlastP and the T-Coffee Multiple Sequence Alignment program. We selected four species having uncharacterized ORFs with more than 66% amino acid identity to Rtg2p for further analysis: Ashbya gossypii, Candida glabrata, Vanderwaltozyma polyspora and Kluyveromyces lactis. In S. cerevisiae, cells deleted for RTG2 are glutamate auxotrophs, and have reduced expression of Aco1p and Cit2p proteins. To determine whether the putative RTG2 genes we identified encode bonefide regulators of the retrograde response pathway, we used standard yeast genetic approaches and molecular biology tools to investigate their ability to complement the defects associated with the rtg2Ä mutant using our S. cerevisiae RTG2 shuffle strain. We investigated functional roles of Rtg2p homologs by comparing Cit2p and Aco1p protein levels, glutamate auxotrophy, as well as analyzing the interaction between Rtg2p homologs and Mks1p. We also analyzed sensitivity of mutant strains under various stress conditions to address possible signaling cross talk between the retrograde signaling pathway and the TOR pathway. Our data show that the fungal Rtg2p homologs from C. glabrata, V. polyspora and K. lactis are functional in mediating the mitochondrial signal through known components of the retrograde signaling cascade. Our immunoprecipitation data suggest that TOR and retrograde signaling may exhibit cross pathway activation under rapamycin treatment. We show that Mks1p, the negative regulator of retrograde signaling pathway is required for Cit2p expression under rapamycin treatment. Given that all Rtg2p homologs showed low affinity for Mks1p which was in turn paralleled by a higher affinity of Mks1p for Bmh1p suggests that Rtg2p may have an additional functional role in influencing the association of Mks1p with Bmh1p.

No description available.

<p>We are interested in backward-in-time solution techniques for evolutionary PDE problems arising in fluid mechanics. In addition to their intrinsic interest, such techniques have applications in recently proposed retrograde data assimilation. As our model system we consider the terminal value problem for the Kuramoto-Sivashinsky equation in a l D periodic domain. The Kuramoto-Sivashinsky equation, proposed as a model for interfacial and combustion phenomena, is often also adopted as a toy model for hydrodynamic turbulence because of its multiscale and chaotic dynamics. Such backward problems are typical examples of ill-posed problems, where any disturbances are amplified exponentially during the backward march. Hence, regularization is required to solve such problems efficiently in practice. We consider regularization approaches in which the original ill-posed problem is approximated with a less ill-posed problem, which is achieved by adding a regularization term to the original equation. While such techniques are relatively well-understood for linear problems, it is still unclear what effect these techniques may have in the nonlinear setting. In addition to considering regularization terms with fixed magnitudes, we also explore a novel approach in which these magnitudes are adapted dynamically using simple concepts from the Control Theory.</p> / Thesis / Master of Science (MSc)