Chromosomal rearrangements occur in all organisms and are important both in the evolution of species and in pathology. In this dissertation I show that in Saccharomyces cerevisiae, or budding yeast, one type of chromosomal rearrangement occurs when inverted repeats fuse, likely during DNA replication by a novel mechanism termed "faulty template switching". This fusion can lead to the formation of either a dicentric or acentric chromosome, depending on the direction of the replication fork. Dicentric chromosomes are inherently unstable due to their abnormal number of centromeres, and thus undergo additional chromosomal rearrangements and chromosome loss.

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism that delays the initiation of anaphase by inhibiting the Anaphase Promoting Complex (APC) until all kinetochores have achieved bipolar attachment on the mitotic spindle. Mad1-3, Bub1, and Bub3, components of the SAC, are conserved from yeast to humans. These proteins localize to unattached kinetochores, though it is unknown with which kinetochore proteins they interact and how these interactions transduce information about microtubule attachement. Here, purification of the checkpoint proteins from Saccharomyces cerevisiae suggests that Mad1 interacts with the outer kinetochore protein Ndc80 in a SAC, cell cycle, and DNA dependent manner. Ndc80 is thought to mediate attachment of kinetochores to microtubules so the interaction between Mad1 and Ndc80 suggests a mechanism by which cells sense kinetochore-microtubule attachment. The SAC is of special importance in some types of cancer where genetic damage and aneuploidy is correlated with mutated SAC genes. A better understanding of the SAC mechanism will aid in the development of targetted cancer therpeutics.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

The Saccharomyces cerevisiae iron-responsive transcription factor, Aft1p, has a well established role in regulating iron homeostasis through the transcriptional induction of iron-regulon genes. However, recent studies have implicated Aft1p in other cellular processes independent of iron-regulation such as chromosome stability. In addition, chromosome spreads and two-hybrid data suggest that Aft1p interacts with and co-localizes with kinetochore proteins, however the cellular implications of this have not been established. Here, we demonstrate that Aft1p associates with the kinetochore complex through Iml3p. Furthermore, we show that Aft1p, like Iml3p, is required for the increased association of cohesin with the pericentromere and that aft1Δ cells display sister chromatid cohesion defects in both mitosis and meiosis. Our work defines a new role for Aft1p in the sister chromatid cohesion pathway.

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism that delays the initiation of anaphase by inhibiting the Anaphase Promoting Complex (APC) until all kinetochores have achieved bipolar attachment on the mitotic spindle. Mad1-3, Bub1, and Bub3, components of the SAC, are conserved from yeast to humans. These proteins localize to unattached kinetochores, though it is unknown with which kinetochore proteins they interact and how these interactions transduce information about microtubule attachement. Here, purification of the checkpoint proteins from Saccharomyces cerevisiae suggests that Mad1 interacts with the outer kinetochore protein Ndc80 in a SAC, cell cycle, and DNA dependent manner. Ndc80 is thought to mediate attachment of kinetochores to microtubules so the interaction between Mad1 and Ndc80 suggests a mechanism by which cells sense kinetochore-microtubule attachment. The SAC is of special importance in some types of cancer where genetic damage and aneuploidy is correlated with mutated SAC genes. A better understanding of the SAC mechanism will aid in the development of targetted cancer therpeutics.

A large fraction of the proteome displays cell cycle-dependent expression, which is important for cells to accurately grow and divide. Cyclical protein expression requires protein degradation via the ubiquitin proteasome system (UPS), and several ubiquitin ligases (E3) have established roles in this regulation. Less is understood about the roles of deubiquitinating enzymes (DUB), which antagonize E3 activity. A few DUBs have been shown to interact with and deubiquitinate cell cycle-regulatory E3s and their protein substrates, suggesting DUBs play key roles in cell cycle control. However, in vitro studies and characterization of individual DUB deletion strains in yeast suggest that these enzymes are highly redundant, making it difficult to identify their in vivo substrates and therefore fully understand their functions in the cell. To determine if DUBs play a role in the cell cycle, I performed a screen to identify specific DUB targets in vivo and then explored how these interactions contribute to cell cycle control. I conducted an in vivo overexpression screen to identify specific substrates of DUBs from a sample of UPS-regulated proteins and I determined that DUBs regulate different subsets of targets, confirming they display specificity in vivo. Five DUBs regulated the largest number of substrates, with Ubp10 stabilizing 40% of the proteins tested. Deletion of Ubp10 delayed the G1-S transition and reduced expression of Dbf4, a regulatory subunit of Cdc7 kinase, demonstrating Ubp10 is important for progression into S-phase. We hypothesized that compound deletion strains of these five DUBs would be deficient in key cellular processes because they regulated the largest number of cell cycle proteins from our screen. I performed genetic analysis to determine if redundancies exist between these DUBs. Our results indicate that most individual and combination deletion strains do not have impaired proliferation, with the exception of cells lacking UBP10. However, I observed negative interactions in some combinations when cells were challenged by different stressors. This implies the DUB network may activate redundant pathways only upon certain environmental conditions. While deletion of UBP10 impaired proliferation under standard growth conditions, I discovered that deletion of the proteasome-regulatory DUBs Ubp6 or Ubp14 rescues the cell cycle defect inubp10∆ cells. This suggests in the absence of Ubp10 substrates such as Dbf4 are rapidly degraded by the proteasome, but deletion of proteasome-associated DUBs restores cell cycle progression. Our work demonstrates that in unperturbed cells DUBs display specificity for their substrates in vivo and that a coordination of DUB activities promotes cell cycle progression.

No description available.

Green computing, an emerging field of research that seeks to reduce excess power consumption in high performance computing (HPC), is gaining popularity among researchers. Research in this field often relies on simulation or only uses a small cluster, typically 8 or 16 nodes, because of the lack of hardware support. In contrast, System G at Virginia Tech is a 2592 processor supercomputer equipped with power aware components suitable for large scale green computing research. DIRECT is a deterministic global optimization algorithm, implemented in the mathematical software package VTDIRECT95. This thesis explores the potential energy savings for the parallel implementation of DIRECT, called pVTdirect, when used with a large scale computational biology application, parameter estimation for a budding yeast cell cycle model, on System G. Two power aware approaches for pVTdirect are developed and compared against the CPUSPEED power saving system tool. The results show that knowledge of the parallel workload of the underlying application is beneficial for power management. / Master of Science

Mathematical modeling has become increasingly popular as a tool to study regulatory interactions within gene-protein networks. From the modeler's perspective, two challenges arise in the process of building a mathematical model. First, the same regulatory network can be translated into different types of models at different levels of detail, and the modeler must choose an appropriate level to describe the network. Second, realistic regulatory networks are complicated due to the large number of biochemical species and interactions that govern any physiological process. Constructing and validating a realistic mathematical model of such a network can be a difficult and lengthy task. To confront the first challenge, we develop a new modeling approach that classifies components in the networks into three classes of variables, which are described by different rate laws. These three classes serve as "building blocks" that can be connected to build a complex regulatory network. We show that our approach combines the best features of different types of models, and we demonstrate its utility by applying it to the budding yeast cell cycle. To confront the second challenge, modelers have developed rule-based modeling as a framework to build complex mathematical models. In this approach, the modeler describes a set of rules that instructs the computer to automatically generate all possible chemical reactions in the network. Building a mathematical model using rule-based modeling is not only less time-consuming and error-prone, but also allows modelers to account comprehensively for many different mechanistic details of a molecular regulatory system. We demonstrate the potential of rule-based modeling by applying it to the generation of circadian rhythms in cyanobacteria. / Ph. D.

La cytokinèse est un processus fondamental prenant place à la fin de la mitose et permettant la séparation des deux cellules filles. Un défaut de cytokinèse peut mener à une ségrégation anormale des chromosomes et engendrer des phénomènes de cancer. Dans beaucoup d'organismes eucaryotes, la cytokinèse nécessite l'assemblage et la contraction d'un anneau d'actomyosine permettant la formation d'un sillon et la réorganisation de la membrane cellulaire au site de clivage. Dans la plupart de ces organismes, des protéines du cytosquelette appelées septines participent à la cytokinèse. Chez la levure bourgeonnante, Saccharomyces cerevisiae, cinq septines sont exprimées durant la mitose (Cdc3, Cdc10, Cdc11, Cdc12 et Shs1). Ces protéines ont la capacité de s'assembler en un anneau au niveau du site de bourgeonnement, lieu de séparation entre la cellule mère et la cellule fille. Cet anneau de septines permet la fixation et le recrutement de nombreuses protéines intervenant dans la cytokinèse. La dynamique des septines change durant le cycle cellulaire, ce qui a une importance dans la régulation de la cytokinèse. La stabilisation de cet anneau est accompagnée d'un changement du niveau de phosphorylation des septines, mais les kinases responsables de ces modifications restent inconnues. Les travaux de l'équipe de Simonetta Piatti ont mis en évidence un nouveau rôle de la GTPase Rho1 et de sa cible, la protéine kinase C (Pkc1), dans la régulation de la dynamique des septines. Le but de ce travail de thèse était de déterminer les voies moléculaires par lesquelles la protéine Pkc1 intervient dans le recrutement et la stabilisation de l'anneau de septines. Pour se faire nous avons purifié le complexe de septines chez la levure bourgeonnante en présence ou en absence de la protéine Pkc1 et nous l'avons analysé par spectrométrie de masse. Cette analyse nous a permis d'observer que le niveau de phosphorylation d'un cluster (îlot) de 5 sérines était diminué sur Shs1. L'alignement de séquence nous a permis de constater que ce domaine était conservé dans la septine Cdc11. Par ailleurs ces deux protéines sont connues pour jouer un rôle dans l'assemblage des filaments et la formation de l'anneau de septines. Il a déjà été observé qu'un mutant phosphomimétique du cluster de sérine de la septine Shs1 empêche la formation des filaments in-vitro. Nous avons voulu caractériser le rôle de ce cluster dans la protéine Cdc11 en créant un mutant non-phosphorylable (CDC11-9A) et un mutant phosphomimétique (CDC11-9D). De manière très évidente, le mutant phosphomimétique provoque des problèmes de cytokinèse dans les cellules dont le gène codant la protéine Shs1 a été supprimé. A l'inverse le mutant non-phosphorylable améliore le phénotype des cellules ne comportant pas Shs1. Ces résultats sont en parfait accord avec l'observation selon laquelle les protéines Shs1 et Cdc11 pourraient avoir des fonctions très similaires, et mettent en avant le rôle important du cluster de sérines phosphorylées de Cdc11 lors de la cytokinèse. Nous avons constaté que Pkc1 ne phosphoryle pas directement les septines, mais agit par l'intermédiaire de kinases et de phosphatases impliquées dans la régulation des septines. Nous avons pu montrer que Pkc1 régule l'interaction de Gin4 avec les septines, cette kinase étant connue pour sa capacité à phosphoryler Shs1. De plus, nous avons observé que Pkc1 impacte sur le niveau de phosphorylation des deux autres kinases de la même famille, Hsl1 et Kcc4. Par ailleurs, la délétion de PKC1 diminue drastiquement la quantité de protéines Kcc4 dans la cellule.L'absence de Pkc1 augmente également l'interaction entre les septines et Bni4, une sous-unité régulatrice de la phosphatase PP1. Nous avons également observé que Bni4-PP1 peut déphosphoryler Cdc11, expliquant la diminution de son niveau de phosphorylation en cas d'absence de la protéine Pkc1.Ces travaux mettent en évidence que Pkc1 est un nouveau régulateur majeur des septines dans la levure. / Cytokinesis is the last step of mitosis and is the fundamental process leading to the physical separation of two daughter cells. Defects in cytokinesis generate polyploid cells that are prone to chromosome missegregation and cancer development. In animal cells and fungi, cytokinesis requires the formation and contraction of an actomyosin ring that drives ingression of the cleavage furrow. Additional cytoskeletal proteins called septins contribute to cytokinesis. In the budding yeast Saccharomyces cerevisiae, five different septins are expressed during the mitotic cell cycle (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1). All septins, except for Shs1, are essential for cell viability. Yeast septins form filaments that in turn organize into a ring at the bud neck, which is the constriction between the mother and the future daughter cell where cytokinesis takes place. The septin ring then expands into a rigid septin collar that acts as scaffold for cytokinesis by recruiting most cytokinetic proteins to the bud neck. Cell cycle-regulated changes in septin ring dynamics are thought to be important for its cytokinetic functions and formation of the rigid septin collar is accompanied by septin phosphorylation. However, the kinases responsible for these modifications have not been fully characterized. Unpublished data from our laboratory indicate that the Rho1 GTPase, which is essential for actomyosin ring formation and contraction, and its target protein kinase C (Pkc1) contribute to deposition and stabilization of the septin ring. Here, we have addressed how Pkc1 regulates septin ring deposition and/or stability. To this end, septin complexes were purified from yeast and analyzed by mass spectrometry, comparing wild type and pkc1Δ mutant cells. This mass spectrometry analysis clearly showed that phosphorylation of a cluster of residues in Shs1 decreased in the absence of Pkc1. Interestingly, we found that this cluster is conserved in the septin Cdc11, which together with Shs1 is known to play an important role in the assembly of high-order structures like filaments and rings. Phosphomimetic mutations of the phosphorylatable cluster in Shs1 have been previously shown to disrupt filament formation in-vitro. We therefore proceeded to mutagenise the same cluster in Cdc11, generating a phosphomimetic (CDC11-9D) and in a non-phosphorylatable mutant (CDC11-9A). Strikingly, the phosphomimetic CDC11-9D caused cytokinesis defects in cells lacking Shs1, whereas the non-phosphorylatable CDC11-9A allele partially rescued the sickness of shs1∆ mutant cells. These observations are in agreement with the notion that Cdc11 and Shs1 share overlapping functions and highlight an important role of the phosphorylatable cluster of Cdc11 for cytokinesis. We also found that Pkc1 does not phosphorylate septins directly, but rather regulates the activity of septin kinases and phosphatases. Consistently, we show that Pkc1 affects the interaction between septins and the bud neck kinase Gin4, which is known to interact with septins and to phosphorylate them. In addition, Pkc1 impacts on the phosphorylation of two additional bud neck kinases, Hsl1 and Kcc4, which are part of the same family of Nim1-related kinases as Gin4. In addition, PKC1 deletion leads to a dramatic decrease in the levels of Kcc4 , so that it is barely detected at the bud neck.Deletion of PKC1 affects also the interaction between septins and the Bni4 protein, which is a regulatory subunit for the PP1 phosphatase at the bud neck. In turn, we found that Bni4-PP1 modulates Cdc11 phosphorylation, thereby explaining how the latter is decreased in the absence of Pkc1. Altogether, our data strongly suggest that Pkc1 is a novel major regulator of septins in yeast.