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Aspergillus fumigatus is a globally present opportunistic fungal pathogen that plays a key role in degrading organic matter. A. fumigatus can cause a vast array of diseases, collectively known as aspergilloses. The most serious of these is invasive aspergillosis, that has a mortality rate of 30 to 95% with treatment. Recent studies have indicated that the global A. fumigatus population consists of multiple divergent genetic clusters that are broadly distributed geographically. However, most of the previously analyzed samples have come from continental Eurasia and the Americas where the effects of historical or contemporary gene flow is difficult to distinguish. My thesis project, therefore, focused on analyzing the genetic diversity of the Auckland, New Zealand A. fumigatus population, as it is geographic distant from all previously analyzed populations. Here, we obtained 104 A. fumigatus isolates from Auckland and compared the genotypes of these isolates to population data obtained from nine other countries from Europe, Africa, North America, and Asia. The goal was to analyze the potential effects of historical differentiation and gene flow within this population. We determined that the Auckland population had a low, non-significant level of differentiation compared to most previously surveyed global populations. However, the Auckland population also contained unique genetic elements not present within populations from other geographic regions. Though the hypothesis of random recombination was rejected, we found abundant evidence for phylogenetic incompatibility and recombination within the Auckland A. fumigatus population. Lastly, we identified two triazole resistant strains within the Auckland population, with one carrying the common TR34/L98H cyp51A mutation. Our results suggest that contemporary gene flow, likely due to anthropogenic factors, is a major force shaping the New Zealand A. fumigatus population. These results contribute to our understanding of the high levels of gene flow observed within and among many geographic populations of A. fumigatus. / Thesis / Master of Science (MSc) / Aspergillus fumigatus is a globally distributed fungal mold capable of causing serious diseases in individuals with weakened immune systems or lung damage. In the environment, A. fumigatus lives in the soil where it degrades organic matter and contributes to the cycling of nitrogen and carbon across the planet. Due to the airborne nature of its spores, people inhale this fungus daily, and those at risk may develop disease. These diseases, collectively known as aspergilloses, can result in long term chronic illnesses, and in the case of invasive aspergillosis, the death rate can be as high as 95%, even with treatment. Medical treatment of aspergilloses involves the use of antifungal drugs. However, some A. fumigatus strains have developed resistance. I am interested in the patterns of global genetic diversity of A. fumigatus populations. For my MSc thesis, I investigated the A. fumigatus population within Auckland, New Zealand, as it is both geographically isolated and distant from other previously surveyed populations. Our data illustrated that the New Zealand population contains pockets of unique diversity as well as high levels of similarity to the previously surveyed populations within Europe. My results suggest that human influences, likely due to travel and trade, have played a large role in shaping the genetic diversity of the A. fumigatus population from Auckland, New Zealand.

The planned widening of U.S. Highway 17 along the east boundary of Great Dismal Swamp National Wildlife Refuge (GDSNWR) and a lack of knowledge about the refuge's bear population created the need to identify potential sites for wildlife crossings and estimate the size of the refuge's bear population. I collected black bear hair in order to collect DNA samples to estimate population size, density, and sex ratio, and determine road crossing locations for black bears (Ursus americanus) in GDSNWR in southeastern Virginia and northeastern North Carolina. I also investigated bear/vehicle collisions to determine patterns of road crossing. Genetic analysis of 344 hair samples collected on 2 trapping grids identified 85 unique individuals which I used in a mark-recapture analysis. Estimated population size on the trapping grids was 105 bears (95% CI = 91-148) and average density was 0.56 bears/km². This density estimate projected over the entire Great Dismal Swamp ecosystem yielded a population estimate of 308 bears (550 km2 X 0.56 bears/km²). Similar population estimates generated by Hellgren (1988), Tredick (2005), and this study suggest a stable bear population in the Great Dismal Swamp ecosystem over a 20-year period. I erected a 2.3-kilometer long strand of barbed wire along U. S. Highway 17 to monitor road crossing patterns near the Northwest River drainage. Genetic analysis identified 6 bears (4 males, 1 female, 1 unknown) that apparently crossed the highway in a 10-month period. Five of 6 bears deposited hair in a 171-m section which included the Northwest River corridor. The 6 bears detected crossed the road at least 11 times. I investigated 10 reports of bear/vehicle collisions on the periphery of the refuge from June 2000 to May 2002. Six bears (4M:1F:1 unknown) were confirmed killed during this time period. Based on reported bear/vehicle collisions from Hellgren (1988), the Virginia Department of Game and Inland Fisheries database, and this study, a minimum of 4 to 5 bears are struck by vehicles each year on the periphery of the refuge. I identified 2 areas of multiple bear/vehicle collisions: highway 58 on the north side of the refuge near Hampton Airport and Highway 17 on the eastern side of the refuge in the vicinity of the Northwest River corridor. / Master of Science

Since the dawn of the genomics era, the genetics of numerous human disorders has been understood which has led to improvements in targeted therapeutics. However, the focus of most research has been primarily on protein coding genes, which account for only 2% of the entire genome, leaving much of the remaining genome relatively unstudied. In particular, repetitive sequences, called microsatellites (MST), which are tandem repeats of 1 to 6 bases, are known to be mutational hotspots and have been linked to diseases, such as Huntington disease and Fragile X syndrome. This work represents a significant effort towards closing this knowledge gap. Specifically, we developed a next generation sequencing based enrichment method along with the supporting computational pipeline for detecting novel MST sequences in the human genome. Using this global MST enrichment protocol, we have identified 790 novel sequences. Analysis of these novel sequences has identified previously unknown functional elements, demonstrating its potential for aiding in the completion of the euchromatic DNA. We also developed a disease risk diagnostic using a novel target specific enrichment method that produces high resolution MST sequencing data that has the potential to validate, for the first time, the link between MST genotype variation and cancer. Combined with publicly available exome datasets of non-small cell lung cancer and 1000 genomes project, the target specific MST enrichment method uncovered a signature set of 21 MST loci that can differentiate between lung cancer and non-cancer control samples with a sensitivity ratio of 0.93. Finally, to understand the molecular causes of MST instability, we analyzed genomic variants and gene expression data for an autosomal recessive disorder, Fanconi anemia (FA). This first of its kind study quantified the heterogeneity of FA cells and demonstrated the possibility of utilizing the DNA crosslink repair dysfunctional FA cells as a suitable system to further study the causes of MST instability. / Ph. D. / The field of genetics has enjoyed substantial growth since the conclusion of the human genome project, which was declared complete in the year 2003. The human genome project produced the first framework for the human DNA sequence, the human genome. With the availability of this framework, the understanding of the genetic basis for a number of diseases has significantly grown, which has resulted in better methods of clinical diagnosis and treatment. While the current focus on understanding the genomic regions that are responsible for making proteins has inarguably helped, it has also created a gap in knowledge. Protein coding regions of the human genome account only for 2% of the entire human genome and a large part (47%) of the genome is occupied by repetitive DNA. DNA sequences can be complex, with the nucleotides arranged in no particular order, e.g. ATCGTACGA, or DNA sequences can be repetitive, e.g. ATATATATAT. Repetitive sequences, which have repeating units of 1 to 6 bases, are called microsatellites (MST). MSTs have been shown to be unstable and they have been linked to diseases such as Huntington disease and Fragile X syndrome. This work helps to close this knowledge gap by developing molecular methods and computational tools focused on identifying MST variations. Research conducted with this aim has resulted in three major accomplishments. One, we developed novel molecular and computational methods which we used to detect 790 previously unknown sequences in the human genome. This work proved the ability of our method to uncover functional elements in the human genome that can potentially answer numerous biological questions. Two, we developed another novel method for the production of high resolution MST sequence data that not only can improve MST research in general but also shows the potential for the development of new genetic diagnostics and cancer therapeutics. We identified a signature set of 21 MST sequences that can differentiate between lung cancer patient genomes and non-cancer control genomes. These results represent the first potential validation for a proposed link between MST sequence length (genotype) variation and cancer. Three, we attempt to understand a possible molecular cause and consequences of MST instability in a disease called Fanconi anemia. The results from this work not only, for the first time, quantify the effects of this disease on the genome but also establishes Fanconi anemia as a suitable system for studying MST instability in detail.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25352号 / 農博第2618号 / 新制||農||1108(附属図書館) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 日本 典秀, 教授 田中 千尋, 准教授 小野 肇 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Erreger des Leishmania donovani Komplexes (LDC) verursachen viszerale Leishmaniose (VL), eine der häufigsten durch Sandmücken übertragenen Infektionskrankheiten beim Menschen. Die vorliegende von der EU geförderte Dissertation untersucht die weltweite Populationsstruktur des LDC mit besonderem Schwerpunkt auf dem Indischen Subkontinent (ISC), wo das gehäufte Auftreten von Therapieversagen ein Problem für die geplante Eliminierung von VL darstellt. Zwei hoch diskriminierende molekulare Typisierungsverfahren wurden angewendet. 845 LDC-Isolate wurden mittels Multi-Lokus-Mikrosatelliten-Typisierung (MLMT) charakterisiert. Die Parasiten wurden in Gebieten mit endemischer VL aus unterschiedlichen Wirten isoliert und repräsentieren verschiedene klinische Formen der Leishmaniose. Eine 125 Parasiten umfassende Teilprobe wurde vollständig sequenziert und in einem next-generation Multi-Lokus-Sequenz-Ansatz (ng-MLSA) typisiert, welcher auf der Analyse von genomweiten Single-Nukleotid-Polymorphismen (SNP) beruht. Sowohl die MLMT- als auch die SNP-Daten wurden mit den gleichen populationsgenetischen Methoden ausgewertet. Der ng-MLSA Ansatz bestätigte weitgehend die Populationsstrukturen des mit dem MLMT analysierten größeren Datensatzes, die genetische Struktur korrelierte mit der geographischen Herkunft der Isolate. Die Unempfänglichkeit der Parasiten gegenüber Antimon- oder Miltefosin sowie die in vitro gemessene Resistenz der Isolate vom ISC konnten nicht auf einen spezifischen Genotyp zurückgeführt oder mit einem spezifischen genetischen Merkmal verknüpft werden. Die Gesamtgenomsequenzierung konnte bisher keine Mutationen im Parasitengenom nachweisen, die in Zusammenhang mit der Antimon- und Miltefosin-Unempfindlichkeit bzw. dem Therapieversagen gebracht werden könnten. Analysen basierend auf ausgewählten Sequenzen deuten auf eine variable chromosomale Ploidie und eine erhöhte Kopienzahl einiger Gene hin, die zur Entstehung von Arzneimittelresistenzen beitragen könnten. / Parasites of the Leishmania donovani species complex (LDC) cause most cases of visceral leishmaniasis (VL), one of the most fatal vector-borne parasitic human diseases. As part of an EU funded project, this dissertation has investigated the worldwide genetic population structure of parasites of the LDC, with special focus on the Indian subcontinent (ISC) where unresponsiveness to anti-leishmanial drugs has recently become an urgent problem for the containment of VL. Two types of highly discriminatory approaches have been used. Multi-locus microsatellite typing (MLMT) has been applied to 845 LDC isolates from numerous Old and New World foci of VL, from different clinical forms of the disease and from various hosts. A subset of 125 fully sequenced isolates, reflecting the worldwide distribution of the LDC, was analysed using a next-generation multi-locus sequence approach (ng-MLSA) including single nucleotide polymorphisms (SNP). Both microsatellite and SNP data sets were analysed using, in general, the same population genetic tools. The ng-MLSA approach has, in general, corroborated the population structures obtained with MLMT for the larger data set. With the exception of non MON-1 parasites, the genetic structure revealed was largely associated with the geographic origin of the isolates, but not with the clinical presentation, host specificity and the immune status of the host or year of parasite isolation. Unresponsiveness to antimony or miltefosine treatment as well as the respective resistances measured in vitro could not be linked to a specific genotype or genetic trait. Wg sequencing also failed, so far, to identify mutations, which could be related to the unresponsiveness of LDC isolates from the ISC to antimony and miltefosine therapy. Analyses of selected targets have revealed extensive variation in chromosomal ploidy in all wg sequenced isolates under study and copy number variations for some genes possibly involved in drug resistance.