Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases, the genetic basis is unknown. Via whole-exome sequencing and linkage analysis, we determined the most likely causative mutations in four families. While the mutations are in three different genes – FBLIM1, PLCG2 and PIP; all three genes are involved in Fcγ signaling and osteoclast activation. In a large cohort of 61 individuals with CRMO, we performed gene and pathway based association analysis using the 1000 genomes participants of European ancestry as controls. One gene from the family-based analyses, ANO6, was significantly enriched for rare variants in our cohort of cases. ANO6 is involved in P2RX7- mediated inflammasome activation and in the regulation of bone mineralization. While no pathways were enriched for rare variants in the CRMO cohort after genome-wide correction, four pathways were significantly enriched for rare variants in the control samples, indicating a protective effect of the variants. The second most significant pathway, activation of chaperone genes by XBP1s, is relevant to CRMO pathogenesis as XBP1s is a transcription factor that attenuates ER stress, and regulates the expression of genes involved in RANKL signaling and bone remodeling. An association analysis using a larger set of cases followed by functional validation of candidate genes is necessary to confidently declare the mutations isolated in the work presented here to be pathogenic. Our preliminary findings suggest that mutations in genes involved in both the inflammatory response and bone remodeling underlie the pathogenesis of CRMO.

The focus of this research project is the complex of infraspecific taxa that make up the crisp-leaf buckwheat species Eriogonum corymbosum (Polygonaceae), which is distributed widely across southwestern North America. This complex provides an ideal taxonomic group for research into population relationships and speciation. To avoid unnecessary debates about taxonomic validity or contentious issues regarding appropriate species definitions, the historical evolution of the species concept is first reviewed in detail, demythologizing an often-assumed species problem. Following that review, the E. corymbosum complex is examined specifically. Although eight varieties of E. corymbosum are currently recognized based on morphological characters, this group of large, woody shrubs has a history of revisions that demonstrates the uncertainty inherent in circumscriptions based on morphology alone. The apparent rarity of some E. corymbosum varieties also presents conservation and management challenges, demonstrating the need for taxonomic verification. To bring greater resolution to this group, I genetically tested samples from populations of six of the eight varieties of E. corymbosum, as well as a number of related buckwheat species. With 103 AFLP loci and chloroplast sequence data from 397 samples, I found strong support for the designation of the recently named E. corymbosum var. nilesii. This predominantly yellow-flowered variety had previously been considered part of a more common variety, and thus its management had not been of particular concern. But as a separate variety, its known distribution is quite limited, and management for this rare plant is now advised. An examination of the biogeography of the E. corymbosum complex provides further support for the apparent rarity of var. nilesii, as well as var. aureum. Both taxa are found at the periphery of the complex, and both may represent insipient species. While all other varieties appear more closely related to each other than to varieties aureum and nilesii, with overlapping ranges confined mostly to the Colorado Plateau, both var. aureum and var. nilesii appear to have allopatric ranges largely off the Colorado Plateau. It appears these two peripheral varieties may each entail a separate center of origin for two new taxa.

The hepatitis delta virus (HDV) is the smallest human pathogenic RNA virus and relies heavily on host proteins for its replication. The objective of my research was to observe the effect of HDV replication on host gene expression, using a HEK-293-based cell system engineered to mimic HDV replication. A high-throughput sequencing was performed and allowed to establish a total of 3,561 genes differentially expressed by HDV RNA. Among those genes, 3,278 were upregulated by HDV RNA and 283 downregulated. A Gene Ontology (GO) enrichment analysis was performed on those dysregulated genes and revealed that upregulated genes were predominantly part of these four pathways: RNA processing, G-protein coupled receptor signaling pathway, protein transport, and organelle organization. On the other hand, downregulated genes were part of the nucleosome assembly pathway. The expression of several genes was confirmed by RT-qPCR. Moreover, protein complexes whose expression at the gene level was affected were identified. A total of 30 complexes were found to be significantly affected by HDV replication. Among them, we found many chromatin and histone related complexes. Lastly, a flow cytometry analysis revealed an increase in cell cycle arrest in G0/G1 and a reduction in the percentage of cell in S phase. Moreover, there was a difference in cell size for arrested cells in G0/G1 in HDV replicating cells. Overall, my results support the hypothesis that HDV replication induces cell cycle dysregulation.

The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation. / Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms. / The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation. / DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere. / Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands. / This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere.

Intraspecific relationships among the anchialine cave shrimp Typhlatya mitchelli were examined by sequencing a total of 1505 bp from portions of three mitochondrial DNA genes. Cytochrome b, cytochrome oxidase I, and 16S rRNA were partially sequenced and analyzed for specimens from six different cenotes (water-filled caves) across the Yucatan Peninsula, Mexico. The conspecific Typhlatya pearsei that is sympatric with T. mitchelli was also sequenced and used as the outgroup. Comparisons among specimens of T. mitchelli yielded low sequence divergence values (0-1.7%), with the majority being less than 0.4%. Phylogenetic tree topologies reconstructed with neighbor-joining, maximum likelihood, and maximum parsimony were in agreement in regards of the resolution of deep branches. Also, there was no obvious geographic differentiation among the majority of T. mitchelli samples, with the exception of specimens from Cenote San Antonio Chiich (Yokdzonot, Yucatan, Mexico) which all clustered into an extremely well supported monophyletic group. The level of differentiation of this group, together with the nearly total absence of differentiation among T. mitchelli from distant cave systems, suggests that this is an Evolutionary Significant Unit (ESU), which may correspond to a new species. This unidentified Typhlatya from Cenote San Antonio Chiich was helpful in establishing a period in which the epigean ancestor colonized the cenotes. Based on pairwise distance data and previously published shrimp molecular clocks (Baldwin et al., 1998), T. mitchelli and the putative new Typhlatya species last shared a common ancestor between 3-5 million years ago (mya), during the mid-Pliocene era, while T. mitchelli and T. pearsei was approximately 7-10 mya (middle to late Miocene). The ancestor to T. mitchelli and the unidentified Typhlatya species abandoned its shallow coastal water existence in the early Pliocene and eventually expanded its range across the peninsula. Approximately 4 mya, Cenote San Antonio Chiich became isolated from the remaining gene pool thereby halting gene flow. As the regional water table fluctuated in response to the rise and fall of Pleistocene sea levels, T. mitchelli actively colonized the peninsula. The discovery of a single, continuous subterranean freshwater system provides for a better understanding of anchialine biogeography within the Yucatan Peninsula.

A Matrix-assisted Laser Desorption/Ionization (MALDI) – Ion Mobility (IM) – Surface-induced Dissociation (SID) – Time-of-Flight (TOF) instrument with three different collision source configurations was developed in order to improve the SID performance in high throughput peptide sequencing. The first version of the instrument was equipped with an angle resolved SID source in order to maximize the collection efficiency of the SID scattering ions. An orthogonal TOF was also implemented as the second MS stage in this instrument to increase mass resolution. The second version of the instrument was developed towards simplifying the coupled configuration of the IM, SID and TOF components by using a combined SID/TOF source with a confinement ring electrode as the collision target. The fragmentation efficiency of SID in this configuration was increased up to 50% due to the surface normal impact angle used as compared with the results from a previous experiment using 45 degree impact angle. The third version of the instrument was equipped with a dual-source/dual-detector TOF to facilitate high throughput tandem analysis of peptides through simultaneous separation, fragmentation and mass analysis, while retaining precursor ion identity in the same experimental sequence. A series of small organic molecules, model peptides and tryptic peptides from a protein digest were analyzed to demonstrate the utility of these new designs for enhanced SID performance and peptide sequencing capability. Finally, a new mobility drift cell using a periodic focusing mechanism has been designed and fabricated to replace the previous uniform field drift cell. Improvement in ion transmission has been observed in the periodic focusing drift cell instrument without sacrificing the mobility resolution.

<p>The tubulin homolog FtsZ provides the cytoskeletal framework for bacterial cell division. FtsZ is an essential protein for bacterial cell division, and is the only protein necessary for Z-ring assembly and constriction force generation in liposomes in vitro. The work presented here utilizes structural and genomic analysis methods to investigate FtsZ function for cell division with three separate questions: (1) What is the function of the C-terminal linker peptide in FtsZ? (2) Are there interacting proteins other than those of the divisome that facilitate FtsZ function? (3) Do lateral contact sites exist between protofilaments in the Z ring, resulting in an organized Z-ring substructure?</p><p>The FtsZ protein has an ~50 aa linker between the protofilament-forming globular domain and the C-terminal (Ct) membrane-tethering peptide. This Ct linker is widely divergent across bacterial species, and has been thought to be an intrinsically disordered peptide (IDP). We have made chimeras where we have swapped the <italic>Escherichia coli</italic> IDP for Ct linkers from other bacteria, and even for an unrelated IDP from human &alpha-adducin. Most of these substitutions allowed for normal cell division, suggesting that sequence of the IDP did not matter -any IDP appears to work (with some exceptions). Length, however, was important: IDPs shorter than 39 or longer than 89 aa's had compromised function. We conclude that the Ct linker of FtsZ functions as a flexible tether between the globular domain of FtsZ in the protofilament, and its attachment to FtsA and ZipA at the membrane. As a worm-like-chain, the Ct linker will function as a stiff entropic spring linking the constricting protofilaments to the membrane. </p><p>Previous work from our laboratory found that mutant and foreign FtsZ that do not normally function for cell division can function upon acquisition of a second site suppressor mutation, somewhere in the <italic>E. coli</italic> genome. We expect that some mutant or foreign FtsZ are partially functional for division in <italic>E. coli</italic>. As such, these FtsZ require another mutation that further enables their function. These suppressing mutations may reveal proteins interacting with FtsZ and the divisome, that have previously been unknown. In the present study, we have identified, via whole genome re-sequencing, single nucleotide polymorphisms that allow 11 different foreign and mutant FtsZ proteins to function for cell division. While we see a trend toward mutations in genes related to general metabolism functions in the cell, we have also identified mutations in two genes, <italic>ispA</italic> and <italic>nlpI</italic>, that may be interacting more directly with the cell division mechanism.</p><p>Finally, we have devised a screen to identify mutations in FtsZ that may be involved in lateral bonding between protofilaments. There are presently two proposed models of FtsZ substructure: the scattered or the ribbon model. A major difference between these models is that the scattered model proposed no interaction between adjacent protofilaments in the Z ring, while the ribbon model suggests that adjacent protofilaments are bonded laterally to create an organized substructure of aligned protofilaments. Our screen was designed to identify complementary surface-exposed residues that may be involved in lateral bonding. We initially identified two lateral contact candidate residues: R174, and E250 and mutated them to abrogate FtsZ function. We also mutated L272, which is known to make contacts across the protofilament interface, to look for compensating mutations in these contact residues. Using the screen, we identified a number of secondary mutations in FtsZ that can complement these initial loss-of-function mutations. While this screen has not yielded strong candidates for lateral bonding partners, it has emerged as a high-throughput method for screening large libraries of mutant FtsZ proteins in order to identify compensating mutation pairs.</p> / Dissertation

In this study the composition of the denitrifying bacterial community among constructed wetlands in agricultural areas was investigated. Thirty-two constructed wetlands located in Southern Sweden were surveyed, and biofilm samples from each were analyzed by applying denaturing gradient gel electrophoresis, to investigate the community composition of the three denitrifying bacterial enzyme genes nirK, nirS and nosZ. The DNA sequences of the enzyme genes were compared to known DNA sequences in GeneBank using BLAST. The results of the denitrifying bacterial enzyme genes indicated that these habitats may harbour a heterogeneous denitrifying bacterial community. Individual analysis of the enzyme genes revealed that nirS was more heterogeneous than both nirK and nosZ. Most sequences from the present study clustered with known sequences from species belonging to the group of α-Proteobacteria, and to a lesser extent with β- Proteobacteria and γ-Proteobacteria, and only nirS clustered with a member of gram-positive bacteria. / <p>Included in doctoral thesis: Milenkovski, Susann. Structure and Function of Microbial Communities in Constructed Wetlands - Influence of environmental parameters and pesticides on denitrifying bacteria. Lund University 2009.</p>

The purpose of this thesis is to develop a heuristic solution for the static problem of resequencing unblocked vehicles as a part of an ongoing research project at Daimler AG. The target client of this project is Mercedes-Benz Cars. An unblocked vehicle is defined as a vehicle that for some reason could not be processed in its given time slot but at a later point in time needs to be inserted into the production sequence. Work overload is defined as work that the worker is unable to finish prior to reaching the station border. The resequencing problem can be described as finding new positions for a set of unblocked vehicles in a sequence of previously not blocked vehicles, such that the new sequence containing the previously not blocked vehicles and the additional unblocked vehicles causes as little work overload as possible. A decision has to be made in real-time, forcing the solution method to return a solution within a cycle time. Today, Mercedes-Benz Cars uses the sequencing approach “car sequencing”. This approach relies on so called spacing constraints, which basically means, trying to distribute work intensive vehicles as evenly as possible over the planning horizon and thereby enabling a hopefully smooth production. The car sequencing approach needs limited information. The difficulty is to find spacing constraints that fits the high level of product customization characterizing a modern car manufacturer. To overcome these difficulties, a new approach is being considered, namely the mixed-model sequencing, which takes more detailed data into account than the car sequencing approach but on the other hand is more costly in terms of computation. To this end, a simple but promising tabu search scheme was developed, that for many instances was able to find the optimal solution in less than 30 seconds of computing time and that also clearly outperformed all benchmark heuristics.

A Matrix-assisted Laser Desorption/Ionization (MALDI) – Ion Mobility (IM) – Surface-induced Dissociation (SID) – Time-of-Flight (TOF) instrument with three different collision source configurations was developed in order to improve the SID performance in high throughput peptide sequencing. The first version of the instrument was equipped with an angle resolved SID source in order to maximize the collection efficiency of the SID scattering ions. An orthogonal TOF was also implemented as the second MS stage in this instrument to increase mass resolution. The second version of the instrument was developed towards simplifying the coupled configuration of the IM, SID and TOF components by using a combined SID/TOF source with a confinement ring electrode as the collision target. The fragmentation efficiency of SID in this configuration was increased up to 50% due to the surface normal impact angle used as compared with the results from a previous experiment using 45 degree impact angle. The third version of the instrument was equipped with a dual-source/dual-detector TOF to facilitate high throughput tandem analysis of peptides through simultaneous separation, fragmentation and mass analysis, while retaining precursor ion identity in the same experimental sequence. A series of small organic molecules, model peptides and tryptic peptides from a protein digest were analyzed to demonstrate the utility of these new designs for enhanced SID performance and peptide sequencing capability. Finally, a new mobility drift cell using a periodic focusing mechanism has been designed and fabricated to replace the previous uniform field drift cell. Improvement in ion transmission has been observed in the periodic focusing drift cell instrument without sacrificing the mobility resolution.