Le S23906-1 est un dérivé diacétate de la benzo-[b]-acronycine. Sélectionné pour son potentiel cytotoxique in vitro et son activité anti-tumorale in vivo, ce composé très prometteur est entré en essai clinique en 2006-2007. Cet agent alkylant présente la particularité d’interagir avec l’azote en position 2 des guanines localisé dans le petit sillon de l’ADN et d’induire ensuite une ouverture locale de la double hélice. La conformation de ce composé est importante pour son activité puisque la présence du groupement réactif acétate en orientation S induit une plus forte déstabilisation de l’ADN par rapport à l’orientation R, ceci en corrélation avec une plus forte cytotoxicité et une meilleure activité anti-tumorale. Cependant le mécanisme moléculaire mis en place, pour conduire à la mort de la cellule tumorale reste peu connu. Afin de mieux comprendre le lien entre la déstabilisation de l’ADN et l’activité cytotoxique du S23906-1, nous avons cherché à déterminer la nature des protéines nucléaires qui reconnaissent spécifiquement cette lésion. Une approche protéomique par chromatographie d’affinité suivie d’une électrophorèse séparative et d’analyse en MALDI-TOF nous a permis d’identifier la GAPDH. Des expériences de retard en gel ont validé l’interaction de la GAPDH à l’adduit S23906-1/ADN simple ou double brin. La recherche d’une éventuelle séquence consensus de fixation à l’ADN de la GAPDH a été entreprise par méthode de CASTing. Si aucun consensus proprement-dit n’est clairement apparu, une prévalence de fixation pour les séquences riches en guanines a été observée. Des études de relation structure/fonction ont montré les adduits formés par les dérivés diacétylés ou benzo-[a]-diacétylés de l’acronycine déstabilisent également l’ADN et que la GAPDH se fixe également de manière efficace à ces adduits. Par comparaison à d’autres composés interagissant au niveau de l’azote 2 des guanines, nous n’avons observé aucune interaction de la GAPDH avec l’adduit ET-743/ADN (l’ET-743 stabilise l’ADN), alors que d’autres auteurs ont mis en évidence une interaction avec l’adduit QAD/ADN, où QAD est un dérivé de la Saframycine A, molécule structurellement proche de l’ET-743. Au niveau cellulaire, un traitement au composé QAD engendre une translocation de la GAPDH du cytoplasme vers le noyau. Nous n'avons pas retrouvé une telle translocation en utilisant le S23906-1. De plus, et contrairement à ce dérivé de la Saframycine A, nos cellules traitées avec un siRNA dirigé contre la GAPDH sont plus résistantes au S23906-1, suggérant un rôle anti-apoptotique de la GAPDH suite au traitement au S23906-1, contrairement à l’effet pro-apoptotique observé avec le dérivé QAD de la Saframycine A. En parallèle, nous nous sommes intéressés à de nouveaux agents cytotoxiques, dont des composés de série benzylamine bis-8-hydroxyquinoline. En utilisant des approches spectrométriques et biochimiques, nous avons montré que le composé principal JLK1486 ne cible pas l'ADN, mais réagit de manière covalente avec des groupements thiol comme celui du glutathion. Cependant, des études cellulaires de déplétion du glutathion endogène ont montré que le glutathion n’était pas la cible du composé JLK1486 mais participait à sa détoxification. Afin d’identifier la ou les cible(s) protéique(s) responsable(s) de l’effet cytotoxique du JLK1486, nous avons mis au point une approche dérivée de l’électrophorèse 2D-DIGE. / S23906-1 is a diacetate benzo-[b] acronycine derivative. Selected for its cytotoxic potential in vitro and its antitumoral activity in vivo, this very promising compound entered clinical trial in 2006-2007. This alkylating agent presents the peculiarity to interact with the nitrogen in position 2 (N2) of guanines, localized in the minor groove of the DNA, and to subsequently lead local opening of the double helix. The conformation of this compound is important for its activity. Indeed, the presence of the active acetate group in the S-orientation leads to a stronger destabilization of the DNA with regard to the R-orientation, in correlation with a stronger cytotoxicity and a better antitumoral activity of the S- rather than R-stereoisomer. However, the molecular mechanism leading to the death of treated tumoral cell lines remains poorly understood. To address the link between the destabilization of the DNA and the cytotoxic activity of the S23906-1, we tried to determine the nature of nuclear proteins that specifically recognize this lesion. A proteomic approach, using chromatographic affinity followed by SDS-PAGE electrophoresis and MALDI-TOF analysis, identified the GAPDH protein. EMSA experiments validated the interaction of GAPDH to the S23906-1/DNA adduct (as a single- or double-stranded DNA template). The search for a possible consensus DNA-binding sequence of GAPDH was performed using a CASTing method. Even though no consensus site was clearly identified, prevalence for G-rich and GT-rich sequences was evidenced. From structure/activity relationships studies, we showed that DNA alkylation using the diacetylated or benzo-[a]-diacetylated forms of acronycine also destabilize the DNA helix and that GAPDH also binds in an efficient manner to these adducts. By comparison to other compounds also interacting with the N2 group of guanines, no interaction of the GAPDH with the ET-743/ADN adduct was evidenced (ET-743 stabilizes the DNA), while other authors evidenced an interaction with DNA of QAD, a Saframycine A derivative and a molecule structurally related to ET-743. At the cellular level, cellular treatment with the QAD resulted in GAPDH translocation from the cytoplasm to the nucleus. We did not observe such a translocation using S23906-1. Furthermore cells treated with a siRNA directed to the GAPDH are more resistant to S23906-1, suggesting an anti-apoptotic role of GAPDH after S23906-1 treatment, by contrast with the pro-apoptotic effect observed in the literature with QAD . In parallel, we were interested in new cytotoxic agents, among which abis-8-hydroxyquinoline benzylamine series. Using spectrometric and biochemical approaches, we showed that the lead compound JLK1486 does not target DNA, but covalently reacts with thiol groups as that of the glutathione. However, cellular studies using depletion of the endogenous glutathione showed that the glutathione was not the target of JLK1486 but that it participated in its detoxification as it does for S23906-1. To identify the protein(s) target(s) responsible for the cytotoxic effect of JLK1486, we developed an approach derived from the 2D-DIGE electrophoresis which clearly evidenced proteins that are the cellular targets for JLK1486.

Antibiotics that target the bacterial ribosome make up about half of all clinically used antibiotics. We have studied two ribosome targeting drugs: Fusidic acid and Viomycin. Fusidic acid inhibits bacterial protein synthesis by binding to elongation factor G (EF-G) on the ribosome, thereby inhibiting translocation of the bacterial ribosome. Viomycin binds directly to the ribosome and inhibits both the fidelity of mRNA decoding and translocation. We found that the mechanisms of inhibition of these two antibiotics were unexpectedly complex. Fusidic acid can bind to EF-G on the ribosome during three separate stages of translocation. Binding of the drug to the first and most sensitive state does not lead to stalling of the ribosome. Rather the ribosome continues unhindered to a downstream state where it stalls for around 8 seconds. Dissociation of fusidic acid from this state allows the ribosome to continue translocating but it soon reaches yet another fusidic acid sensitive state where it can be stalled again, this time for 6 seconds. Viomycin inhibits translocation by binding to the pre-translocation ribosome in competition with EF-G. If viomycin binds before EF-G it stalls the ribosome for 44 seconds, much longer than a normal elongation cycle. Both viomycin and fusidic acid probably cause long queues of ribosomes to build up on the mRNA when they bind. Viomycin inhibits translational fidelity by binding to the ribosome during initial selection. We found that the concentration of viomycin required to bind to the ribosome with a given probability during decoding is proportional to the accuracy of the codon∙anticodon pair being decoded. This demonstrated that long standing models about ribosomal accuracy cannot be correct. Finally, we demonstrated that a common viomycin resistance mutation increases the drug binding rate and decreases its dissociation rate. Our results demonstrate that ribosome targeting drugs have unexpectedly complex mechanisms of action. Both fusidic acid and viomycin preferentially bind to conformations of the ribosome other than those that they stabilize. This suggests that determining the structures of stable drug-bound states may not give sufficient information for drug design.

Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsK(C), which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex. We show that a 62-amino-acid fragment of FtsK(C) interacts directly with the XerD C-terminus in order to stimulate the cleavage by XerD of BSN, a dif-DNA suicide substrate containing a nick in the 'bottom' strand. The resulting recombinase-DNA covalent complex can undergo strand exchange with intact duplex dif in the absence of ATP. FtsK(C)-mediated stimulation of BSN cleavage by XerD requires synaptic complex formation. Mutational impairment of the XerD-FtsK(C) interaction leads to reduction in the in vitro stimulation of BSN cleavage by XerD and a concomitant deficiency in the resolution of chromosomal dimers at dif in vivo, although other XerD functions are not affected.

Thesis advisor: Marc-Jan Gubbels / Apicomplexan parasites like Toxoplasma gondii have a complex life cycle comprising of transitions between different hosts, different organ systems and between the extracellular and intracellular milieu. The parasite must thus adjust itself and its cellular processes in accordance with its environment. In this dissertation, I have focused on such stage specific behaviors of three distinct intermediate filament-like proteins as well as a glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH1). These proteins relocate from the cytosol to the unique cortical membrane skeleton of non-dividing parasites. The intermediate filament-like proteins IMC7, 12 and 14, localize exclusively to the mature cytoskeleton. One model of function was that these proteins differentially stabilized mother and budding daughter cytoskeletons in the division process, but we ruled out this role for the individual proteins, as they are not essential for the lytic cycle of the parasite. However, we determined that IMC7 and IMC14 are contributing to the maintenance of rigidity of the cytoskeleton under osmotic stress conditions in extracellular parasites. In addition, IMC14 is critical in cell cycle progression as its depletion results in the formation of multiple daughters per division round. When the parasite egresses from the host cell, glycolytic enzyme GAPDH1 translocates to the cortex. The functional role of GAPDH1 in the parasite and the mechanism of its cortical translocation are deciphered based on the 2.25Å resolution crystal structure of the GAPDH1 holoenzyme in a quaternary complex. These studies identified that GAPDH1’s enzymatic function is essential for intracellular replication but we confirmed the previous reports that glycolysis is not strictly essential in presence of excess L-glutamine. We identify, for the first time, S-loop phosphorylation as a novel, critical regulator of enzymatic activity that is consistent with the notion that the S-loop is critical for cofactor binding, allosteric activation and oligomerization. We show that neither enzymatic activity nor phosphorylation state correlate with the ability to translocate to the cortex. However, we demonstrate that association of GAPDH1 with the cortex is mediated by Cysteine 3 in the N-terminus, likely by palmitoylation. Overall, glycolysis and cortical translocation are functionally decoupled by post-translational modifications. Collectively, the discoveries made in this dissertation reveal unprecedented detail in mechanism and function of cortical protein translocation and thereby identifying new drug targets. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

The Twin arginine translocation pathway translocates fully folded proteins across cellular membranes and is only utilised by proteins that fold before translocation. It is a unique process that is found in many bacteria, archaea and also in plant chloroplasts. Investigation of the bacterial and thylakoidal systems has revealed much of the substrates and the components involved in their translocation. Unfortunately, there are still many unanswered questions such as how substrates are directed to the membrane and the actual mechanism of translocation. This thesis specifically investigates the Tat pathway of halophilic and thermophilic archaea. To date, there has been a lack of research into the archaeal Tat pathway and it is possible that there are unique adaptations because of the extreme environments that these organisms inhabit. Chapter 3 specifically investigates the thermophiles Sulfolobus solfataricus and Sulfolobus tokodaii and attempts to purify their Tat complexes. By doing so it was hoped to learn more about the Tat components and their interactions. Further experiments were also performed to determine if the two S. solfataricus Tat operons provide specificity to the Tat substrates that translocate. Four separate areas of the Tat pathway of halophilic archaea (haloarchaea) were investigated in Chapters 4-7. Firstly, site-directed mutagenesis was used to analyse the signal peptides of haloarchaeal Tat substrates in more detail. Consequently, the resulting data led to the use of bioinformatics to analyse the Haloarchaeal signal peptide. The bioenergetics of the Tat system was then determined by analysing the effect of a variety of ionophores on translocation of the Tat substrates AmyH and SptA. Finally, a series of folding and stability assays were used to increase our understanding of AmyH, which could provide further information on why this protein, like many other haloarchaeal proteins, requires the Tat pathway for translocation.

Shen, Jinbo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 110-119). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Barley is an economically important cereal crop throughout most of the world. The study and elucidation of the genetic complex of this plant will add to its extensive usefulness to man, and will also provide valuable information for similar research projects on other crop plants.

No description available.

Utah prairie dogs (Cynomys parvidens) have been extirpated in 90% of their historical range. Because most of the population occurs on private land, this threatened species is continually in conflict with landowners. The Utah Division of Wildlife Resources has been relocating prairie dogs from private to public land since the 1970s, but relocations have been largely unsuccessful due to high mortality. Prairie dogs are highly social animals, but they are usually relocated without regard to their family group (coterie). I hypothesized that relocating Utah prairie dogs with their social structure intact may positively affect their survival rates and behavior. Utah prairie dogs were relocated from the golf course in Cedar City, Utah to two prepared sites near Bryce Canyon National Park, Utah in 2010 and 2011. Trapped animals were individually marked, and released at the new sites. Prairie dogs were relocated as coteries, or in a control group as randomly trapped individuals. To compare the two sites, vegetation transects were established at each site to document differences in composition and structure. Two months after relocation, traps were set to recapture released animals. Activity budgets were collected prior to, and following, relocation. Activity data were also collected on wild prairie dog populations for comparison. The best predictor of survival and recapture rate was the animal’s weight at initial capture. Larger animals had higher survival, but lower recapture rates. More research is needed to determine if this is due to better body condition, older animals having more experience, or both. Analysis showed no evidence of an advantage to relocating Utah prairie dogs by coteries. There was no benefit to survival, and no difference in behavior between coterie and control relocation strategies. Relocated animals behaved differently from non-relocated prairie dogs. While still significantly different, relocated individuals behaved more like wild prairie dogs than the animals at the urban source population. The vegetation at the two sites was significantly different. One site had significantly less grass cover, more invasive plant cover, and rockier soils. The sites also had different soil structures, which affect burrowing, and long-term retention rates. More research is needed to determine how site selection influences long-term success of a relocation site.

Barley (Hordrium sp.) is the most important spring sown cereal crop in Utah, both in acreage and in yield of total digestible nutrients per unit area. It also rates high as a feed crop in the United States. For this reason it is important that varieties are available which are high in yield, high in quality, and disease resistant, along with other desirable characteristics. In order to work toward this end most efficiently, it is first necessary that the genetics of the barley plant be worked out. Barley offers the plant breeders and geneticists a valuable material to use in the study of genetics. The fact that it has many easily distinguishable characters, that it is easy to grow large F2 populations, and that it has a low chromosome number make it desirable for this type of study. That all interspecific crosses are fertile make it all the more desirable for the plant breeder. This study is a by-product of the cereal breeding and improvement program being carried on at the Utah Experiment Station. Recently the study of translocations induced by X0rays and other means has gained wide interests among plant breeders, geneticists, and cytologists alike. Translocations or interchanges consist of the exchange of segments of non-homologous chromosomes. As a result semisterility occurs in plants which are heterozygous for the interchange. the objective of this study is to calculate any linkage which may be found in different crosses involving translocated stocks in barley in order to determine which linkage groups are involved in the translocation; and to note any characters now linked as a result of a translocation which were not linked in normal barley stocks. The ratios and interactions which occur in any of the characters found in this study will be calculated.