<p>Genetic variation is a natural occurrence in the genome that contributes to the phenotypic differences observed between individuals as well as the phenotypic outcomes of various diseases, including infectious disease and cancer. Single nucleotide polymorphisms (SNPs) have been identified as genetic factors influencing host susceptibility to infectious disease while the study of copy number variation (CNV) in various cancers has led to the identification of causal genetic factors influencing tumor formation and severity. In this work, we evaluated the association between genomic variation and disease phenotypes to identify SNPs contributing to host susceptibility in Staphylococcus aureus (<italic>S. aureus</italic>) infection and to characterize a nervous system brain tumor, known as oligodendroglioma (OD), using the CNV observed in tumors with varying degree of malignancy.</p><p>Using SNP data, we utilized a computational approach, known as in silico haplotype mapping (ISHM), to identify genomic regions significantly associated with susceptibility to <italic>S. aureus</italic> infection in inbred mouse strains. We conducted ISHM on four phenotypes collected from <italic>S. aureus</italic> infected mice and identified genes contained in the significant regions, which were considered to be potential candidate genes. Gene expression studies were then conducted on inbred mice considered to be resistant or susceptible to <italic>S. aureus</italic> infection to identify genes differentially expressed between the two groups, which provided biological validation of the genes identified in significant ISHM regions. Genes identified by both analyses were considered our top priority genes and known biological information about the genes was used to determine their function roles in susceptibility to <italic>S. aureus</italic> infection.</p><p> We then evaluated CNV in subtypes of ODs to characterize the tumors by their genomic aberrations. We conducted array-based comparative genomic hybridization (CGH) on 74 ODs to generate genomic profiles that were classified by tumor grade, providing insight about the genomic changes that typically occur in patients with OD ranging from the less to more severe tumor types. Additionally, smaller genomic intervals with substantial copy number differences between normal and OD DNA samples, known as minimal critical regions (MCRs), were identified among the tumors. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques. </p><p> The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments. The genomic regions with copy number changes were interrogated for genes and assessed for their biological roles in the tumors' phenotype and formation. This information was used to assess the validity of using genomic variation in these tumors to further classify these tumors in addition to standard classification techniques.</p><p> The studies described in this project demonstrate the utility of using genetic variation to study disease phenotypes and applying computational and experimental techniques to identify the underlying genetic factors contributing to disease pathogenesis. Moreover, the continued development of similar approaches could aid in the development of new diagnostic procedures as well as novel therapeutics for the generation of more personalized treatments.</p> / Dissertation

Symbiosis is commonly present in marine invertebrates. Many corals and sponges have symbiotic algae or bacteria. In the previous studies of the sponge Cinachyra australiensis, 85% of the bacteria associated with the sponge have high similarity (88.65%) with the symbiotic chemoautotrophic sulfur-oxidizing bacteria of the deep-sea hydrothermal vent mussel, Solemya reidi. This study aims to investigate the localization of the chemoautotrophic sulfur-oxidizing bacteria associated with Cinachyra australiensis. The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) large-subunit genes for autotrophic organisms were amplified by polymerase chain reaction from the sponge samples. The phylogenetic relationship of the RubisCO large subunit genes was analyzed. A total of 26 clones were selected and sequenced. They could be divided into two groups. One (9 clones) belongs to form I type IB (cynobacteria and green algae). The other (17 clones) belongs to form II type IA (chemoautotrophic symbiotic bacteria). The location of the sulfur-oxidizing chemoautotrophic bacteria was shown to be intracellular symbiosis within the mesoglial cells by fluorescence in situ hybridization.

Yellow rust disease is one of the most important problems in wheat production. It causes substantial yield losses throughout the world. There are resistant and susceptible wheat varieties to various yellow rust pathotypes. In this thesis genes that are induced in wheat, in virulence and avirulence conditions upon yellow rust inoculations were investigated. Consequently, it was aimed to identify genes that may be playing critical roles in the disease resistance mechanism. The strategy was to construct subtracted cDNA libraries from resistant and susceptible plants and analyse the sequences obtained from these libraries. The subtraction approach in this study differs from the common subtraction designs implicated in plant-pathogen interactions / instead of comparing a compatible or an incompatible interaction with a control, one of the subtractions in this study is done taking a compatible interaction as the tester and an incompatible one as the driver, and the second subtraction, vice versa. Therefore, it was intended to compare the transcriptomes from compatible and incompatible plant-pathogen interactions directly. Suppression Subtractive Hybridization method was used to construct subtracted cDNA libraries. Two subtractions were performed / SSH1 (D-R), taking a compatible interaction as the tester sample and an incompatible one as the driver sample, and SSH2 (R-D), taking an incompatible interaction as the tester sample and a compatible one as the driver. In the end, two subtracted cDNA libraries, SSH1 (D-R) library (1536 clones) and SSH2 (R-D) library (1152 clones) were obtained and the libraries were sequenced. Sequence results were subjected to BlastN and BlastX analysis. We looked for a group of genes that were frequently emphasized in plant disease related studies when we searched within the Blast N homology results of the two libraries. We found out that 19 such genes are present in our libraries. We discussed supposed induction of these genes in the interactions investigated in our study. The fact that these genes were found to be present in our libraries enhances the reliability of our results suggesting that the gene sequences we found indeed belong to genes differentially expressed in the respective comparisons investigated in our study. As such, it also implies that other sequences that were found similar to genes of known functions may represent candidate genes as subjects of further studies investigating wheat-yellow rust interactions.

DNA arrays have become a major bioanalytical method as they enable high-throughput screening and they can be manufactured on different surfaces depending on the nature of diagnostic purpose. However, current technologies to produce and detect arrays of DNA probes are expensive due to the requirement of specialized instrumentation. In this study we have established an array platform in sandwich hybridization format for the detection of label-free nucleic acid targets. Unlike direct hybridization, which is the main microarray hybridization principle, sandwich assay enables unlabeled target detection, lowering the cost and assay time. To this end, sequence specific signal development was achieved by a sandwich complex which is composed of a surface immobilized capture DNA probe (Probe1) and a fluorescein-tagged signal DNA probe (Probe 2), which are partially complementary to the sequence to be analyzed (target oligonucleotide). As the solid support of the array platform both 3-aminopropyl-3-methoxysilane (APTMS) activated and commercially purchased poly-L lysine coated glass slides were used and due to the less background noise property the latter one was preferred. Similarly, for the immobilization of the capture Probe (P1) onto the solid support two different methods were tried / heat immobilization and immobilization via a heterobifunctional cross-linker (HBCL). In regard to the experiments, it is observed that using a cross-linker instead of heat immobilization reduces the ratio of false negative control results in a significant manner. Following the solid support and immobilization method choice comparative optimization studies which include cross-linker type, probe concentration, sensitivity of the platform and hybridization conditions (sequence, temperature and duration) were conducted. Optimum hybridization signal was obtained with a 32.5

Advances in DNA micro and macroarray technologies made these high-throughput systems good candidates for the development of cheaper, faster and easier qualitative and quantitative detection methods. In this study, a simple and cost effective sandwich hybridization-based method has been developed for the rapid and sensitive detection of various unmodified recombinant elements in transgenic plants. Attention was first focused on the optimization of conditions such as time, concentration and temperature using commercial ssDNA, which in turn could be used for real sample detection. In this sandwich-type DNA chip platform, capture probes complementary to the first half of recombinant element (target adapter) were immobilized onto poly-L-lysine covered conventional microscope slides. PCR-amplified un-purified target adapter and biotin labeled detection probe, which is complementary to the second half of target adapter, were hybridized in solution-phase to complementary capture probes to create a sandwiched tripartite complex. Later, hybridization signal was visualized by the attachment of streptavidin conjugated Quantum Dot to the sandwiched complex under UV illumination. Sandwich based array system that has been developed in this study allows multiplex screening of GMO events on a single DNA chip platform. 35S promoter, NOS terminator, CRY1Ab and BAR target sequences were successfully detected on the same DNA chip platform. The platform was able to detect unlabeled PCR amplified DNA fragments of CaMV 35S promoter sequence and NOS terminator and BAR transgene sequences from transgenic potato plants and NK603 Certified GMO Reference material, respectively. The DNA-chip platform developed in this study will allow multiple detection of label-free PCR-amplified transgenic elements from real GMO samples on a single slide via a cost effective, fast, reliable and sensitive sandwich hybridization assay.

Potato production in tropical and subtropical countries suffers from damage caused by the potato tuber moth (PTM), Phthorimiaea operculella. Development of a germline transformation system and the identification of genes that are differentially expressed within the PTM midgut are the main goals of this research. We tested three components that are critical to genetic transformation systems for insects; promoter activity, marker gene expression, and transposable element function. We compared the transcriptional activities of five different promoters, hsp70, hsp82, actin5C, polyubiquitin and ie1, within PTM embryos. The ie1 promoter flanked with the enhancer element, hr5, showed a very high level of transcriptional activity compared with the other promoters. The expression of the enhanced green fluorescent protein (EGFP) was detected under UV-illumination within the embryonic soma demonstrating that it can be used as an effective marker gene for PTM. The transpositional activities of the Hermes, mariner and piggyBac transposable elements were tested in interplasmid transposition assays. The piggyBac element was shown mobile within the embryonic soma with a transposition frequency of 4.2 X 10-5 transposition/donor plasmid. The piggyBac mobility has been enhanced by incorporating a transactivator plasmid expressing the IE1 protein from the bacoluvirus Autographa californica nuclear polyhedrosis virus. Seven transformation experiments were performed. The experiments failed to produce a transgenic PTM. The insect midgut is a rich region of molecular targets involved in food processing that could be potentially used to design a new control strategy. The suppression subtractive hybridization (SSH) method was used to identify differentially expressed genes from the PTM midgut. From this subtracted library, 2984 clones were collected and screened. Of these clones, 637 clones are candidate differentially expressed genes within the PTM midgut. Sixty-nine cDNA clones were randomly selected for DNA sequencing. Tweleve clones were selected for further analysis using RT-PCR and Northern blot techniques. Eleven of the clones resulted in positive results for midgut expression. Five clones, showing homology with insect immune peptides, were used in the challenge experiment which revealed that these cDNAs are constitutively expressed in the midgut, as well as being up-regulated due to bacterial or viral challenge.

This study reports the localization and partial characterization of thioredoxin from the parasitic trematode Fasciola hepatica. Snails (Pseudosuccinia columella) were raised in culture and infected with F. hepatica so that Western blotting and immunohistochemical techniques could be utilized to determine the presence of thioredoxin in different stages of the parasite’s development. The results of these experiments showed that thioredoxin was present in the tegument, gut epithelium, excretory canal epithelium and sperm, of the adult parasite as well as in the tegument and gut of the redia and cercaria intermediate stages. In situ hybridization was used to determine the localization and possible differential mRNA expression of two different F. hepatica thioredoxin isotypes (Fh2020.A and Fh2020.SL) in the adult parasite. The in situ hybridization results showed that both isotypes are expressed in the tegument and gut epithelium. Fh2020.A stains with a greater intensity possibly demonstrating a difference in the amount of expression between the two isotypes. Recombinant F. hepatica thioredoxin expressed in bacteria using the pMAL™ Protein Fusion and Expression System was used to test its affects on the production of super oxide anion by murine peritoneal macrophages, bovine monocyte-derived macrophages and bovine whole blood neutrophils, and nitric oxide production by mouse peritoneal macrophages and bovine monocyte-derived macrophages. The results of the cellular assays were not definitive due to the fact that the maltose binding protein (MBP) moiety of the recombinant thioredoxin, when tested alone, increased production of nitric oxide by bovine monocyte-derived macrophages. Consequently, since the MBP could not be effectively separated from the thioredoxin portion of the recombinant, allowing the thioredoxin affects to be tested independently, no true conclusions regarding its affects on the host immune cells tested could be drawn. This is the first report of the localization of thioredoxin in both the adult F. hepatica as well as in specific intermediate stages of the parasite. These studies demonstrate the possible affects that a protein tag can have on experimental results and demonstrate how such data may be interpreted when a non-cleaved recombinant protein is used in cellular or other assays when compared to native or cleaved recombinant proteins.

A DNA sequence-based phylogenetic tree (Dillon et al., 2004) places the species of the genus Sorghum into two sister lineages, one with x = 5 and the other with x = 10 as a basic chromosome number. It has not been resolved whether or not these lineages are monophyletic or polyphyletic. A repetitive sequence, CEN38, found only in Sorghum and sugarcane, was used to assess evolutionary relationships among Sorghum species. The objectives of this research were to determine the taxonomic distribution of CEN38, its chromosomal position(s), and its organization in DNA. CEN38 was detected by filter hybridization to be present in the DNA of 16 of 21 Sorghum species analyzed, ranging from 15 to ~21,000 copies. It was detected by fluorescence in situ hybridization (FISH) only in chromosomes of species of the section Eu-sorghum, where it had a pericentromeric distribution. The low copy number and/or chromosomal distribution of CEN38 in other Sorghum species apparently does not allow for its detection by FISH. Analysis of restriction enzyme digested DNA with homology to CEN38 and of fragments amplified by PCR using primers selected to amplify S. bicolor CEN38 sequences showed that S. laxiflorum and S. macrospermum have tandemly arranged CEN38 sequences as is found in S. bicolor. This supports the close evolutionary affinity of the species in the x = 10 lineage. In the x = 5 lineage, DNA of 11 of 16 species analyzed hybridized with CEN38 by filter hybridization. In S. versicolor, large DNA fragments (4.36 kb to 23 kb) generated by digestion with restriction enzymes hybridized to CEN38. Since a ladder of smaller fragments was not detected, CEN38 may have been inserted into a transposable element in this species and dispersed throughout the genome. Among species of the x = 5 lineage, PCR using primers for S. bicolor CEN38 amplified only DNA fragments from S. timorense and these formed a ladder based on a ~125 bp repeat. Since hybridization of the CEN38 sequence to DNA of S. timorense was not detected by filter hybridizations, these sequences apparently are not similar to CEN38. Cloning and sequencing of DNA from species of the x = 5 lineage that hybridizes to CEN38 are needed to determine whether or not they are in the CEN38 family. A monophyletic or polyphyletic origin of the x = 5 and x = 10 lineages was not resolved.

Improving the identification ability of surfaced-immobilized nucleic acid probes for small size DNA or RNA targets, utilizing optical or electrochemical methods, has been the goal for the gene chip technology. This study focuses on new probe design for introducing hairpin structural features and locked nucleic acid modification. We use three kinds of probes (DNA-LN, DNA-HP and LNA-HP) to prepare recognition layers via self-assembly processes on a gold substrate, and utilize AFM-based nanolithography technique to produce nanofeatures to observe the stiffness changes of oligonucleotide chains resulting from the formation of rigid double stranded duplexes when target sequence hybridizes to the probe. We also monitor the topographic changes upon exposure to the single mismatched and non-complementary targets as a function of time. The results reveal LNA-HP probes exhibit the highest response to discriminating single-point mutation in the base sequence. In addition, we study the effects of salt concentration, reaction temperature and the small size on the hybridization efficiency.

This is the first research by using suppression subtractive hybridization (SSH) to analysis the gene expression in marine macroalga Ulva fasciata Delile against excess copper toxicity, and it gives us a comprehensive understanding of the tolerant mechanism while macroalgae face to the excess copper. Suppression subtractive hybridization was used to identify genes differentially expressed following exposure to 50 £gM CuSO4 for 6- 12h in a marine macroalga Ulva fasciata Delile. In this work, 69 genes were identified, of which 55 were up-regulated and 14 were down-regulated. According to the database of Gene Ontology (GO), these genes were classified into 10 categories as follows: 1. Transcription; 2. Translation, ribosomal structure and biogenesis; 3. Posttranslational modification, protein turnover, chaperones; 4. Photosynthesis; 5. Cell redox homeostasis; 6. Stress; 7. Metabolism; 8. Energy production and conversion; 9. Transport; 10. Function unknown. According to the results, we suggest that the responses of U. fasciata against excess copper toxicity are mainly through increase of the energy production for providing sufficient energy to many metabolic pathways, and control of the Fe homeostasis and redox form of thiol groups for maintaining the cellular redox homeostasis, moreover, expression of photosynthetic genes for letting the photosynthesis work. In addition, to scavenge the ROS is by expression of stress-related genes, meanwhile, the proteins, DNA and lipids damaged by ROS (reactive oxygen species) and copper are repaired by expression of the other categorical genes. Over and above, the genes expressing in the metabolism category might maintain the amino acids homeostasis and increase the purine content, and subsequently increase the tolerant capacity of U. fasciata against excess copper toxicity. In addition, the concentrations of antioxidants and the activities and gene expression of antioxidant enzymes were determined in Ulva fasciata Delile by a 4-day exposure to 0, 5, 10, 20 and 50 £gM CuSO4. These results demonstrate that the maintenance of antioxidant homeostasis and the induction of activities of antioxidant enzymes via enhanced gene expression are used by U. fasciata to cope with the Cu-induced oxidative stress, but the defense capacity cannot sufficiently alleviate oxidative damage occurring under the condition of higher Cu concentrations. Moreover, according to the results from the expression of genes involved in the control of redox homeostasis and antioxidant defense was studied in macroalga Ulva fasciata Delile in response to CuSO4 (5 and 50 £gM) and ROS (H2O2 and O2£»-), we suggest that ROS involved in up-regulation of antioxidant defense-related genes and the expression of genes of antioxidant defense enzymes and UfMsrA (methionine sulfoxide reductase A) are associated with long-term adaptation of U. fasciata to Cu excess and transcription of redox- related genes and UfGr (glutathione reductase) is up-regulated for short-term acclimation. Promoters play a key role in regulating gene expression. Based on the analysis of cis-acting elements on UfMsr promoters, we suggested that the signal transduction pathway of copper stress in U. fasciata is related to that of other stresses and of defense-related plant hormones, however, Ca2+ and calmodulin might participate in it. To sum up, U. fasciata could resist oxidative damage caused by excessive copper through the regulation on the molecular level.