Mitochondria are some of the most complex organelles of eukaryotic cell. They have their own genome and transcriptional apparatus and maintain several key cellular functions. A substantial part of cellular energetic metabolism happens in the mitochondria, as well as formation of iron-sulfur complexes, synthesis of several key molecules and they are also the essential organelles for the apoptotic pathway. In order to maintain the quality of proteins in their oxidative environment, mitochondria have developed a complex system of proteases that reaches all the mitochondrial compartments that degrade damaged proteins and thus promote mitochondrial turnover. The aim of this work was to characterise function of ClpP subunit of ClpXP matrix protease, which role was not yet extensively investigated in human cells. Therefore, we used RNA-interference to silence expression of ClpP in HEK 293 cells and then we performed rescue experiment during which we reintroduced ClpP in cells. Our results show that the ClpP subunit does not actively participate in apoptotic pathway, nevertheless it is essential for correct assembly of all the respiratory complexes as well as the quality of mitochondria itself. We have also shown that the system of mitochondrial proteases is highly functional and that a lack of ClpP...

A biofilter removes biodegradable contaminants from air by passing it through a biologically-active packed bed. The microorganism community is of fundamental interest but has been the focus of few studies. This work is an investigation of the bacterial community based on the potential functional diversity of the community. <br /><br /> A number of experiments were performed in laboratory-scale biofilters using ethanol as a model contaminant. All biofilters were able to remove the ethanol with elimination capacities in the range 80 to 200gVOCm^-3h^-1; these values are comparable with published literature. Natural organic media (peat or compost) was used as packing. <br /><br /> The potential functional diversity of the community was assessed by Community-Level Physiological Profiling (CLPP) using sole-Carbon Source Utilisation Profile (CSUP). Community samples were used to inoculate Biolog EcoPlates^TM: microplates containing a selection of 31 different carbon-substrates and an indicator dye responding to bacterial growth. This technique was found to be sensitive to changes in the community structure over time and location. <br /><br /> Results showed that the community in samples taken close together (over a scale of a few centimetres) are similar and that relatively small media samples (0. 5 to 1 g) provide reproducible information. A study of a single biofilter indicated stratification of the community occurring with the community near the inlet diverging from that near the middle and outlet of the unit; this is attributed to the ethanol being degraded in the upper part of the column and the lower part of the column not being subjected to ethanol loading. In a study of two units at a higher loading rate, stratification was not observed over a period of weeks; it is suggested that the stratification may develop over this timescale as a result of the presence or absence of the Volatile Organic Compound (VOC) and not due to differences in concentration. <br /><br /> An acclimation period of 7 to 10 days was observed before near-complete removal of ethanol was attained. Monitoring of the community suggested a subsequent shift in diversity. It is suggested that the initial acclimation period is due to biofilm formation and the subsequent shift in community diversity is due to re-organisation of the community as species specialise. In a portion of the biofilter with minimal ethanol exposure, a sudden shift in community is observed after a period of some weeks. This may reflect changes as a result of starvation and indicates that periods of shut-down (when the biofilter is not loaded) may affect the community. <br /><br /> Two studies of biofilters operating in parallel were carried out. The first provided evidence of a divergence in the communities over a period of two weeks. In the second, communities in the two units underwent changes over time but observations from both units at any one time were similar. This demonstrates that biofilters set-up and operated in a similar manner may maintain similar communities but that this is not necessarily the case. This has implications for the reproducibility of laboratory experiments and for the variation of community structure with horizontal position in industrial units.

A biofilter removes biodegradable contaminants from air by passing it through a biologically-active packed bed. The microorganism community is of fundamental interest but has been the focus of few studies. This work is an investigation of the bacterial community based on the potential functional diversity of the community. <br /><br /> A number of experiments were performed in laboratory-scale biofilters using ethanol as a model contaminant. All biofilters were able to remove the ethanol with elimination capacities in the range 80 to 200gVOCm^-3h^-1; these values are comparable with published literature. Natural organic media (peat or compost) was used as packing. <br /><br /> The potential functional diversity of the community was assessed by Community-Level Physiological Profiling (CLPP) using sole-Carbon Source Utilisation Profile (CSUP). Community samples were used to inoculate Biolog EcoPlates^TM: microplates containing a selection of 31 different carbon-substrates and an indicator dye responding to bacterial growth. This technique was found to be sensitive to changes in the community structure over time and location. <br /><br /> Results showed that the community in samples taken close together (over a scale of a few centimetres) are similar and that relatively small media samples (0. 5 to 1 g) provide reproducible information. A study of a single biofilter indicated stratification of the community occurring with the community near the inlet diverging from that near the middle and outlet of the unit; this is attributed to the ethanol being degraded in the upper part of the column and the lower part of the column not being subjected to ethanol loading. In a study of two units at a higher loading rate, stratification was not observed over a period of weeks; it is suggested that the stratification may develop over this timescale as a result of the presence or absence of the Volatile Organic Compound (VOC) and not due to differences in concentration. <br /><br /> An acclimation period of 7 to 10 days was observed before near-complete removal of ethanol was attained. Monitoring of the community suggested a subsequent shift in diversity. It is suggested that the initial acclimation period is due to biofilm formation and the subsequent shift in community diversity is due to re-organisation of the community as species specialise. In a portion of the biofilter with minimal ethanol exposure, a sudden shift in community is observed after a period of some weeks. This may reflect changes as a result of starvation and indicates that periods of shut-down (when the biofilter is not loaded) may affect the community. <br /><br /> Two studies of biofilters operating in parallel were carried out. The first provided evidence of a divergence in the communities over a period of two weeks. In the second, communities in the two units underwent changes over time but observations from both units at any one time were similar. This demonstrates that biofilters set-up and operated in a similar manner may maintain similar communities but that this is not necessarily the case. This has implications for the reproducibility of laboratory experiments and for the variation of community structure with horizontal position in industrial units.

Caseinolytic protease (ClpP) is a cylindrical protease forming the core of protein degradation machinery in eubacteria. ClpP is tightly regulated and is non-functional without a member of the Clp-ATPases. A new class of antibiotics, termed ADEPs, bind to ClpP and allow for activation without the Clp-ATPases; leading to cell death. A more efficient synthetic route to the ADEPs utilizing solid-phase peptide synthesis was investigated. A linear peptide was synthesized, however attempts to close the depsipeptidic macrocycle via macrolactonization failed. Further attempts of assembling a branched depsipeptide for ring closure via a macrolactamization resulted in products that were not stable to cleavage conditions. A group of molecules termed Activators of Self-Compartmentalizing Proteases (ACP) were identified through a screen for activity towards ClpP. Compound ACP1 was synthesized along with twelve analogs and their activity towards ClpP evaluated. The project resulted in a compound with a higher activity than its natural product counterpart.

No description available.

As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. / Dissertation / Doctor of Philosophy (PhD) / Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target.

No description available.

<p>Caseinolytic protease P (ClpP) is a compartmentalized bacterial protease tightly regulated by AAA+ proteins such as ClpA and ClpX within <em>Escherichia coli</em>. It is known that the amino terminus is required to gate the axial entry pores of ClpP however the conformation adopted during activation by ClpA and ClpX has not been properly characterized. Recently it has been determined that binding of a group of antimicrobials termed acyldepsipeptides induces the open conformation of the axial channel independent of ClpA or ClpX to mediate the translocation of unfolded proteins. To determine the structural determinates required to stabilize the open conformation of the axial channel during acyldepsipeptide binding we generated amino terminal variants by site directed mutagenesis. It was found that the formation and anchoring of a β-hairpin element at the amino terminus was crucial for the effective translocation of protein substrates. These results describe the structural requirements that mediate substrate translocation during acyldepsipeptide induced activation and provide a model for the structural requirements of the ClpP amino terminus during formation of the ClpAP and ClpXP holocomplexes.</p> / Master of Science (MSc)

Microorganisms are known to be agents involved in the decomposition of organic matter. However, little is known about the participation of the microbial communities during the decomposition of mammalian skeletal muscle tissue. This study investigates the capacity of the soil microbial community to adapt to the decomposition of skeletal muscle tissue in differing soils. This has implications for the study of mass graves and sites of repeated burial. A controlled laboratory experiment was designed to assess the adaptability of microbial communities present in three distinct soil types (sand, loamy sand and sandy clay loam) found near Perth, Western Australia. This experiment was split into two main stages. The initial decomposition stage involved the addition of porcine skeletal muscle tissue (SMT) (Sus scrofa) to each of the three soil types which were then left to decompose for a period of time. Controls were run in parallel, which had no porcine SMT present. The second decomposition stage involved a second addition of SMT to the soils obtained from the initial decomposition stage. Therefore, for each soil, SMT was either decomposed in the soil that had been pre-exposed to SMT or not. The rate of decomposition, microbial activity (CO2 respiration) and microbial biomass (substrate-induced respiration) were monitored during the second decomposition stage. The functional diversity of the microbial populations in the soil were assessed using Community-Level Physiological Profiling (CLPP). Across the three soil types, the re-introduction of SMT to the soil has led to its enhanced decomposition (measured by tissue mass loss and microbial activity) by the microbial communities. This microbial adaptation may have been facilitated by a functional change in the soil microbial communities.

Evolutionary biology is dependent on accurate phylogenies. In this thesis two branch support methods, Bayesian posterior probablities and bootstrap frequencies, were evaluated with simulated data and empirical data from the chloroplast genome. Bayesian inference was found to be more powerful and less conservative than maximum likelihood bootstrapping, but considerably more sensitive to choice of parameters. Bayesian inference increased in power when data were underparameterized, but the associated increase in type I error was comparatively larger. The chloroplast DNA phylogeny of the tribe Sileneae (Caryophyllaceae) was inferred by analysis of 33,149 aligned nucleotide bases representing 24 taxa. The position of the SW Anatolian taxa Silene cryptoneura and S. sordida strongly disagreed with previous studies on nuclear DNA sequence data, and indicate a possible case of homoploid hybrid origin. Silene atocioides and S. aegyptiaca formed a sister group to Lychnis and remaining Silene, thus suggesting that Silene may be paraphyletic, despite recent revisions based on molecular data. Several nodes in the phylogeny remained poorly supported, despite large amounts of data. Additional sequence sampling is not expected to solve this problem. The main reason for poor resolution is probably a combination of rapid radiation and substitution rate hererogeneity. Apparent incongruent patterns between different regions of the chloroplast genome are evaluated with ancient interspecific chloroplast recombination as explanatory model. Extremely elevated substitution rates in the exons of the plastid clpP gene was documented in Oenothera and three separate lineages of Sileneae. Introns have been lost in some of the lineages, but where present, intron sequences have a markedly slower substitution rate, similar to the rates found in other introns of their genomes. Three branches in the phylogeny show significant whole gene positive selection. In two of the lineages multiple partial copies of the gene were found.