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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Iron-dependent regulation of gene expression in Corynebacterium pseudotuberculosis

Walker, Caray A. January 2009 (has links)
This study set out to analyse C. pseudotuberculosis within an environment relevant to that which would be encountered within its natural host. The impact of the availability of iron within the growth environment of numerous bacteria has been widely reported, and an equivalent investigation was conducted to determine whether the same was true of C. pseudotuberculosis. To this end, a novel chemically-defined medium was designed, which supported the growth of C. pseudotuberculosis, but in which the concentration of specific growth factors could be manipulated. Subsequently, iron was shown to be essential for C. pseudotuberculosis growth, and analysis of secreted protein profiles revealed differential expression between low- and high-iron growth conditions. Furthermore, growth experiments conducted in the defined medium revealed that C. pseudotuberculosis is capable of obtaining iron from the host iron-binding proteins, transferrin and lactoferrin. The results presented in this thesis confirm the importance of iron to C. pseudotuberculosis, and reveal the existence of an iron-dependent regulator which is involved in regulating the expression of multiple target genes.
2

Role of Chk1 and Chk2 in mitotic checkpoint control in vertebrate cells

Oehler, Verena January 2008 (has links)
Two conserved protein kinases, Chk1 and Chk2, are activated in response to genotoxic stress and mediate multiple cell cycle checkpoint mechanisms that ensure genomic integrity. The establishment of mitotic checkpoint delay in response to DNA damage or incompletely replicated DNA is conventionally thought to be accomplished through inhibition of the cyclin-dependent kinase, Cdc2. Both Chk1 and Chk2 have the potential to operate in this pathway. Therefore, the initial aim of this study was to investigate the relative requirement of Chk1 and Chk2 for mitotic delay mechanisms triggered by DNA damage and DNA replication arrest in avian and human cells. These studies demonstrated that Chk1 is the principal regulator of the G2/M checkpoint with a direct role in the establishment and maintenance of the mitotic delay in response to DNA damage. Chk1 was also found to be required for the S/M checkpoint in response to DNA replication arrest; however, detailed analysis indicated that its role is to maintain rather than initiate this checkpoint, as cells lacking functional Chk1 can initially delay mitosis for many hours before they enter a premature mitosis with unreplicated DNA. In avian cells, mitotic phosphorylation of cyclinB2 was found to be mediated by cyclin dependent kinases and suppressed by checkpoint signalling. However, accumulation of potentially active phospho-cyclinB2/Cdc2 complexes was observed during the initial mitotic delay in the absence of functional Chk1, suggesting that other factors apart from the conventionally known mechanisms can restrain mitotic Cdc2 activity. In addition, avian cells were able to delay mitosis effectively during replication arrest in the presence of the ATM/ATR inhibitor caffeine, further emphasizing the possibility of mitotic delay mechanisms that operate independently of ATM/ATR and Chk1. Furthermore, this study revealed that endogenous Cdc6 accumulates in a Chk1-dependent manner during replication arrest. To test whether Cdc6 might function upstream or downstream of Chk1 in the replication checkpoint pathway, Cdc6 was ectopically expressed in both checkpoint-proficient and checkpoint-deficient Chk1-depleted cells. The results from these intervention experiments give preliminary evidence that places Cdc6 downstream of Chk1 in the S/M checkpoint response. The ability of cells to delay the onset of mitosis while DNA replication is stalled independently of ATM/ATR/Chk1 is consistent with the general idea of an inherent relationship between the process of DNA replication and mitosis. The replication machinery might be able to signal either normal DNA replication in progress or the presence of stalled replication structures and thereby intrinsically link the successful completion of DNA synthesis to progression into mitosis.
3

Linkage mapping and genetic analysis of Trypanosoma brucei

Cooper, Anneli Clare January 2010 (has links)
Trypanosoma brucei is a protozoan parasite of major public health and economic importance in sub-Saharan Africa, where it is the causative agent of sleeping sickness in man and Nagana in cattle. The complete genome sequence of T.brucei is now available and the diploid genetic system has recently been demonstrated to be Mendelian. This opens up the possibility of using a classical genetic approach to identify genetic loci that determine important phenotypic traits in this parasite, such as host specificity, drug resistance, and pathogenicity. A genetic map of the non human-infective subspecies, T.b.brucei, has already been assembled and successfully used in quantitative trait analysis of a number of traits specific to this pathogen. This thesis describes the construction of a separate genetic map for the sub-species responsible for > 90% of human African trypanosomiasis infections, T.b.gambiense, which differs significantly from T.b.brucei in many key phenotypes. The genetic linkage map was constructed from the analysis of 119 polymorphic microsatellite markers in a population of 38 F1 progeny, obtained from the genetic cross of a T.b.gambiense group 2 strain, STIB 386, with a T.b.brucei strain, STIB 247. Eleven major linkage groups were resolved, one for each of the megabase chromosomes, resulting in a total genetic map length of 733 cM, and an average map unit size of 24 Kb/cM. The map provides a 90% probability of a marker being within 268 Kb of any genetic locus. A comparative analysis of the T.b.gambiense and T.b.brucei genetic maps revealed synteny and marker order to be conserved between the two sub-species. However, variation was observed in the location of regions of high and low recombination frequency (hot and cold spots) in the two maps. The genetic linkage map presented here is the first available for T.b.gambiense and can now be utilised to find the location within the genome of genes responsible for phenotypic traits in this clinically important sub-species. These traits include human infectivity, tsetse transmissibility and virulence, in addition to sensitivity to the trypanocidal drug, pentamidine, for which phenotypic variation between the parents was characterised both in vitro and in vivo in this thesis. The ability of the T.brucei genetic maps to pinpoint loci underlying phenotypic variation is limited by the number of recombination events, and therefore progeny, available for analysis. To increase the utility of this approach for future studies, an improved method for progeny isolation from uncloned genetic cross populations was also developed. This in vitro bloodstream cloning procedure is scalable and efficient, and replaces a time consuming and technically demanding in vivo method. Twelve new progeny clones were isolated by this approach during the trial and incorporated into the analysis, representing a step toward a higher resolution second-generation genetic map. Finally, whilst undertaking genotyping analysis with microsatellite markers the development of spontaneous chromosome 10 abnormalities was observed. A detailed investigation identified seven laboratory-adapted T.brucei lines in which loss of heterozygosity appeared to have occurred. These alterations to the karyotype significantly exceeded the well-characterised genomic rearrangements of subtelomeric regions that are frequently associated with antigenic variation in African trypanosomes. Microsatellite analysis, pulsed field gel electrophoresis and Illumina next generation sequencing demonstrated these changes to be the product of mitotic recombination events in the chromosome core, resulting in an extensive loss of heterozygosity of up to 75% of the chromosome and correlated with an improved growth phenotype. Further work is now required to determine the extent and frequency with which these abnormalities might occur, however these findings do highlight the potential instability of the molecular karyotype of T.brucei in prolonged in vitro culture.

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