<p>In this thesis, different genetic tools are used to investigate pre- and postzygotic barriers to gene exchange and their role in speciation in the pied flycatcher (<i>Ficedula hypoleuca</i>) and the collared flycatcher (<i>F. albicollis</i>). This species complex consists of four genetically distinct clades that apparently diverged in allopatry (I). Sequencing of introns from autosomal and Z-linked genes from the two species reveals signs of selection on the Z-chromosome. Sexual selection acting on Z-linked genes might explain this pattern (II). By using large-scale genotyping of single nucleotide polymorphisms (SNPs), introgression is observed at autosomal- but not Z-linked loci, mostly from the pied- to the collared flycatcher. Male plumage characters and genes involved in hybrid fitness are largely mapped to the Z-chromosome (III). By studying mate choice of female hybrids I show that there is a link between female preferences and the Z chromosome (IV). The rate of introgression in island versus clinal hybrid zones is consistent with regional differences in hybrid fertility. Asymmetric gene flow from allopatry on the islands may oppose reinforcement, leading to introgression and a partial breakdown of postzygotic isolation. Adaptive introgression may explain the high rate of introgression observed at one of the genetic markers (V). For late breeding female collared flycatchers it appears to be adaptive to use pied flycatchers as social fathers but conspecific males as genetic fathers. Additionally, females in mixed species pairs may reduce hybridization costs by producing an excess of male hybrid offspring that are more fertile than females (VI).</p><p>In conclusion, the Z-chromosome appears to play a major role in flycatcher speciation. Sexual selection and reinforcement are important mechanisms in the divergence of these birds. However, gene flow from allopatry, introgression of adaptive genes and adaptive hetrospecific pairing by late breeding collared flycatcher females may work in the opposite direction.</p>

<p>Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. </p><p>In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis.</p><p>In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.</p>

Graduation date: 2005

Lymphomas are a heterogeneous group of neoplasias originating from B- or T-lymphocytes. In this thesis, we determined the genetic and immunophenotypic characterization of DLBCL and their prognostic impact. Moreover, genomic alterations associated with the transformation to DLBCL from Hodgkin lymphoma (HL) and follicular lymphoma (FL) were elucidated. In order to outline the impact of cytogenetic as well as immunophenotypic prognostic markers in DLBCL, we firstly studied a series of 54 DLBCL tumors using comparative genomic hybridization (CGH) and we identified several frequently occurring chromosomal imbalances. Loss of 22q was more often found in the diagnostic tumors with a more advanced clinical stage, while gain of 18q21 was more commonly identified in relapses. Secondly, we correlated the expression patterns of CD10, bcl-6, IRF-4 and bcl-2 with clinical parameters in a series of 173 de novo DLBCL patients. Patients with a germinal center (GC) phenotype displayed a better survival than the non-GC group. Expression of bcl-6 and CD10 was correlated with a better survival while bcl-2 expression was associated with a poor prognosis. In approaching the HL transformation, two novel B-cell lines (U-2932 and U-2940), derived from patients with DLBCL following HL, were characterized. Interestingly, a translocation with materials from 2q and 7q as well as loss of material on 6q was found in both cell lines. For FL transformation, we assessed chromosomal alterations in a panel of 28 DLBCL patients with a previous history of FL. The DLBCL tumors displayed more chromosomal imbalances compared to FL tumors. Loss of 6q16-21 and gain of 7pter-q22 were more commonly found in the DLBCL counterparts, suggesting the chromosomal location of putative genes that may be involved in the transformation process.

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.

The interaction between nanoscience and biomedicine is one of the important developing areas of modern science. The usage of functional nanoparticles with biological molecules provides sensitive and selective detection, labeling and sensing of biomolecules. Until today, several novel types of tagging materials have been used in bioassays, such as plasmon-resonant particles, quantum dots (QDs), and metal nanoshells. However, nowadays, Surface enhanced raman scattering (SERS) tags have been attracting considerable attention as a tagging system. SERS-tags provide high signal enhancement, and they enable multiplex detection of biomolecules due to high specificity. This thesis is focused on the designing proper SERS nanotags for DNA studies. SERS nano-tags are nanostructures consisting of core nanoparticle generally silver, Raman reporter molecule for labeling, and shell to make surface modifications and to prevent deterioration arising from environmental impact. Based on this information, silver core synthesized by thermal decomposition and chemical reduction methods. Thermal decomposition method provides synthesis of silver nanoparticles in hydrophobic medium, resulting in proper silica coating by reverse microemulsion method. On the other hand, silver nanoparticles sythesized by chemical reduction method exhibit hydrophilic property. Due to capping reagents, negatively charged silver nanoparticles could easily attach with positively charged Raman dye which is brilliant cresyl blue (BCB). After addition of Raman active molecule, silica coating process was done by using modified St&ouml / ber method. The resulting particles were characterized by Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX) ,UV-vis Spectrometry (UV-vis) and Surface-Enhanced Raman Spectroscopy (SERS). In recent years, DNA detection has gained importance for cancer and disease diagnosis and the detection of harmful microorganisms in food and drink. In this study, gene sequences were detected via SERS. For this, probe sequences were labelled with Raman reporter molecule, BCB,and SERS nano-tags and were called as SERGen probes. Then, after hybridization of DNA targets to complementary probe sequences onto gold substrate, SERS peak was followed.

Novel techniques are needed to investigate the genetic variation revealed in the first draft of the human genome sequence. Padlock probes are recently developed reagents, suitable for detecting single-nucleotide variations of DNA and RNA in situ or in solution. The probes are oligonucleotides of about 70-140 nucleotides that can be circularized by ligation in the presence of a correct target sequence. Standard chemical synthesis of padlock probes is difficult due to the requirement for intact 5' and 3' ends of these long oligonucleotides. A novel PCR-based method is presented in this thesis, whereby longer, densely labeled padlock probes can be made as compared to conventional chemical synthesis. PCR-generated padlock probes produced a stronger signal and a more resolved staining pattern, compared to chemically synthesized probes in fluorescence in situ analysis of an alpha-satellite sequence variant present in human chromosomes 13 and 21. Padlock probes used for in situ analysis of metaphase chromosomes had an optimal length of 140 nucleotides. They were used to identify individual chromosomes 7 and 15, and to follow the transmission of chromosome homologues for two consecutive generations. The specificity of the padlock probes to detect single copy genes in genomic DNA samples was demonstrated by detecting a single-nucleotide mutation in the ATP7B gene. It has not previously been known if T4 DNA ligase can be used for RNA sequence analysis. In this thesis, it is demonstrated that T4 DNA ligase can be used for distinguishing single-nucleotide RNA sequence variants. Reaction conditions were defined where most mismatches could be discriminated by a factor of 80 and all mismatches by a factor of at least 20. Under these conditions padlock probes could detect and distinguish RNA sequence variants with ligation efficiency almost as high as on the corresponding DNA sequence. A detailed study of the parameters influencing RNA-templated DNA ligation revealed that DNA ligation on RNA templates proceeds at a much slower rate compared to the same reaction on DNA, and that a molar excess of enzyme is required. Furthermore, the ligation reaction is inhibited by high concentrations of the cofactor ATP and NaCl. The work presented in this thesis demonstrates that PCR-generated padlock probes can detect and distinguish single-nucleotide variation in both RNA and DNA.

Uncovering and understanding the processes that have led to the biological diversity we observe today are of fundamental interest in biology. Since direct observation of speciation is usually impossible, knowledge about the processes behind species formation can be gathered by studying mutations, natural/sexual selection, and genetic drift. In this thesis I aim to identify evolutionary processes that cause species divergence and, ultimately, speciation using Swedish cyprinid fish as a model system. Assuming that the demographic history of a population is mirrored in the genome, I studied the effects of a bottleneck on genetic variability in populations of roach. As expected, I found that a decrease in population size caused a decrease in genetic variability, a pattern that was obtained from both microsatellite and mitochondrial data. The importance of hybridization for speciation is debated, however, by analyzing morphology and microsatellites I could show that common bream and white bream and their interspecific hybrids are phenotypically and genetically differentiated and that ongoing geneflow is mainly unidirectional. Ongoing geneflow antagonizes the effect of genetic drift, but by studying isolated populations (= no gene flow) the impact of genetic drift can be assessed. Long-term isolated populations of roach and perch surprisingly showed stable levels of genetic diversity over time despite decreasing effective population size. However, each population genetically diverged during the period of investigation, a finding that is consistent with the effect of drift. An analysis of the systematic relationship of the 18 species of Swedish cyprinids revealed low congruence of phylogenies based on two different genetic markers. The position of the tench remains unresolved and the relationship of common bream and white bream as sister species cannot be confirmed. Within cyprinid fishes, diversification rates reveal a slowdown with time, a pattern that I found also in other fish clades and that is consistent with density-dependent cladogenesis. Overall, based on the findings presented in this thesis I emphasize that the maintenance of genetic variation in populations is essential since genetic variation is the key element for processes of divergence to act upon.

Medicago is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In the current study, a strategy was developed for over-expressing an “Osmotin-Chitinase” gene chimera in transgenic Medicago that could potentially confer resistance to different biotic and abiotic stresses. Seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula, and M. Rugosa) was tested to determine the cultivars with good germination rates. Among these, M. sativa ssp. sativa showed an average of 80% germination over a period of one week and was subsequently selected for regeneration and transformation experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for regeneration. Among these, hypocotyl explants showed highest (46.17 %) percent regeneration. Escherichia coli harboring Osmotin-Chitinase (OSM-CHI) gene chimera cloned into binary vector pBTEX with nptII as a selection marker was mobilized in Agrobacterium tumefaciens strain EHA105 which was employed in the transformation of hypocotyl explants of Medicago. Transformed calli were grown on callus inducing medium containing kanamycin for screening. Further screening of the positive transgenics was performed using PCR. Southern hybridization was carried out for further confirmation of successful transformation. Transformed shoots will be grown on the root inducing medium for developing into plantlets which would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.

To identify known genes or expressed sequence tags (ESTs) which are expressed specifically or preferentially in the chicken hypothalamus and pituitary gland related to highly reproductive performance, two reciprocal cDNA libraries were constructed using a subtractive hybridization strategy. Two different strains, L2 (dam line; n=12) and B (sire line; n=12) of Taiwan Country Chickens (TCCs), which were originated from one single strain and further subjected to 40-wk egg production and comb size, body weight, respectively since 1982, were used in our study. A total of 324 and 370 clones were identified from L2-subtract-B and B-subtract-L2 hypothalamus/pituitary cDNA libraries. 311 and 360 single inserted sequences from each cDNA library, 53 and 23 non-redundant candidate genes were identified. Quantitative reverse-transcription (RT)-PCR were used to validate the association of mRNA expression profiles of the identified candidate genes and high-egg yield trait in another 118 hypothalamuses and pituitary glands that were dissected from seven different chicken stocks, including B-, L2-, Black-, Red-feather TCCs, commercial Single-Comb White Leghorn (WL) layer at National Chung-Hsing University (NCHU) and Red-feather TCCs grouped into high eggs (Red-high) & low eggs (Red-low) to 40 wks of age at National Chiayi University (NCYU). Among identified genes including known genes and novel genes, involving 33 screened genes, Inhibitor-1 of protein phosphatase type 2A (ANP32A), 3-hydroxybutyrate dehydrogenase (BDH), Contactin (CNTN1), Deiodinase iodothyronine type II (DIO2), Inhibitor of growth family, member 3 (ING3), Lysosomal-associated transmembrane protein 4 beta (LAPTM4B), Neural cell adhesion molecule 1 (NCAM1), DJ-1 protein (PARK7), Prostaglandin D2 synthase (PGDS), Prolactin (PRL), Protocadherin 1 (PCDH1), Pleiomorphic adenoma gene 1 (PLAG1), GTP-binding protein SAR1a (SAR1A), Secretogranin II (SCG2), Stathmin 2 (STMN2), T-box protein 2 (TBX2) were up-regulated in B-subtract-L2 cDNA library. Among above-mentioned 16 identified genes, there were 9 genes related to high-egg yield in chickens., including BDH, NCAM1, PCDH1, PGDS, PLAG1, PRL, SAR1A, SCG2, STMN2.