This thesis deals with early vertebrate fossil remains from the Lower Palaeozoic strata of the Barrandian area (Bohemian Massif). It is presented as a compilation of four papers that were published or submitted in scientific peer-reviewed journals with impact factor. All included papers are focused on the most abundant and diverse vertebrate fauna from the Barrandian area - the placoderms. Placodermi are early vertebrates characterized by articulated head and trunk shields composed of dermal bony plates. In the Barrandian area, they occur exclusively in the Devonian of the Prague Basin. The main objectives of the thesis are modern taxonomic revisions of the placoderm taxa from the Prague Basin as well as discussions about trends in their abundance, diversity, and distribution in space and time, taphonomy, and the evolutionary and phylogenetic implications of their morphology. Two characteristic placoderm associations were distinguished. In the Lochkovian and Pragian, members of the group Acanthothoraci dominate the placoderm record. Acanthothoracid placoderms are among the most basal jawed vertebrates and thus a detailed study of their morphology has a great potential to illuminate the relationships and evolutionary patterns among the stem jawed vertebrates. They are most abundant in the...

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DNA methylation is a crucial, abundant mechanism of gene regulation in vertebrates. It is less prevalent in many other metazoan organisms and completely absent in some key model species, such as D. melanogaster and C. elegans. In this thesis we report on a comprehensive study of the pres- ence and absence of DNA methyltransferases (DNMTs) in 138 Ecdysozoa covering Arthropoda, Nematoda, Priapulida, Onychophora, and Tardigrada. We observe that loss of individual DNMTs independently occured multiple times across ecdysozoan phyla. In several cases, this resulted in a loss of DNA methylation. In vertebrates, however, there is no single species known which lost DNA methylation. Actually, DNA methylation was greatly expanded after the 1R/2R whole genome duplication (WGD) and became a genome-wide phe- nomena. In our study of vertebrates we are not looking for losses of DNA methyltransferases and DNA methylation but are rather interested in the gain of additional DNA methyltransferase genes. In vertebrates there were a number of WGD. Most vertebrates only underwent two WGD but in the teleost lineage a third round of WGD occured and in some groups, e.g. Salmoniformes and some Cypriniformes even a forth WGD occured. The Carp-specific WGD (4R) is one of the most recent vertebrate WGD and is estimated to have occured 12.4 mya. We performed the most comprehen- sive analysis of the evolution of DNA methyltransferases after vertebrate whole-genome duplications (WGD) so far. We were able to show that the conservation of duplicated DNMT3 genes in Salmoniformes is more diverse than previously believed. We were also able to identify DNA methyltrans- ferases in Cypriniformes which have, due to their recent WGD, quite com- plex genomes. Our results show that the patterns of retained and lost DNA methyltransferases after a forth round of WGD differ between Cypriniformes and Salmoniformes. We also proposed a new nomenclature for teleost DNMT genes which correctly represents the orthology of DNMT genes for all teleost species. Next to these purely computational projects we collaborated with the Aluru lab to investigate the effects of different disturbances on zebrafish DNA methylation. One disturbance is the inactivation of DNMT3aa and DNMT3ab as single knockouts as well as a double knockout. This was the first double knockout of DNMT genes in zebrafish which was ever generated. It allows us to study the subfunctionalization of the two DNMT3a genes their effect on genome-wide DNA methylation. Given our results we hypothesize that DNMT3aa and DNMT3ab can compensate for each other to a high de- gree. DNMT3a genes have likely been subfuntionalized but their loss can be compensated by DNMT3b genes. This compensation by DNMT3b genes works well enough that no notable phenotype can be observed in double knockout zebrafish but a difference is notable on the epigenome level. The second disturbance we studied is the exposure of zebrafish to the toxic chemi- cal PCB126. We detected a moderate level of DNA methylation changes and a much larger effect on gene expression. Similar to previous reports we find little correlation between DNA methylation and gene expression changes. Therefore, while PCB126 exposure has a negative effect on DNA methyla- tion it is likely that other gene regulatory mechanisms play a role as well, possibly even a greater one. How do genes evolve and how are genes regulated are two of the main questions of modern molecular biology. In this thesis we have tried to shed more light on both questions. we have broadly expanded the phylogenetic range of species with a manually curated set of DNA methyltransferases. We have done this for ecdysozoan species which have lost all DNA methylating enzymes as well as for teleost fish which acquired more than ten copies of the, originally, two genes. We were also able to generate new insight into the subfunctionalization of the DNA methylation machinery in zebrafish and how it reacts to environmental effects.:1 Introduction 1.1 Biological introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.2 Detecting DNA methylation . . . . . . . . . . . . . . . . . . . . . . . . 7 2 Evolution of DNA methylation across Ecdysozoa 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3 Evolution of DNA methyltransferases after vertebrate whole genome duplications 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4 The effect of DNMT3aa and DNMT3ab knockout on DNA methyla- tion in zebrafish 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 5 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish exposed to Pentachlorobiphenyl 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 5.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 6 Conclusions 6.1 Evolution of DNA methylation across Ecdysozoa . . . . . . . . . . . . . 95 6.2 Evolution of DNA methyltransferases after vertebrate whole genome duplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 6.3 Role of DNA methylation in altered testis gene expression patterns in adult zebrafish (Danio rerio) exposed to Pentachlorobiphenyl (PCB 126). . . 107 6.4 Knockout of DNMT3aa and DNMT3ab in zebrafish (Danio rerio) . . . . . . 108 Bibliography 119

No / Yellow leaf syndrome (YLS) of sugarcane is associated with sugarcane yellow leaf virus (SCYLV), a member of the family Luteoviridae. A fragment of the coat protein and readthrough domain of SCYLV was expressed in a bacterial expression system. The resulting protein was purified and used to immunize mice for monoclonal antibody (MAb) production. Two MAbs, 3A2E3 and 2F7H5, were selected following the screening of hybridoma cells using both plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) and tissue blot immunoassay (TBIA). These MAbs can be incorporated into the TBIA assay currently used for the routine detection of SCYLV but could not be used in triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The two antibodies selected have slightly different specificities. Antibody 3A2E3 gave equivalent results to a polyclonal antiserum (raised to purified virus) in comparative testing using TBIA. The MAbs produced should provide a widely available, uniform reagent for SCYLV diagnosis with the potential to help manage YLS.

No / In the November issue (2001) of Neuroscience Letters, Holmin et al. (Neurosci. Lett. 314 (2001) 151) reported that the synthesis of the intermediate filament protein nestin was upregulated by potassium-induced depolarization in the rat cortex. In this letter, we provide supplementary evidence that repeated cortical spreading depression elicited by potassium induces a delayed upregulation of nestin. However, we argue against the authors' conclusion, Nestin expression was N-methyl-D-aspartate (NMDA)-receptor dependent since dizocilpine (MK-801) treatment abolished the response because spreading depression itself is very sensitive to NMDA-receptor block, and the drug treatment was initiated prior to potassium application to the cortex in Holmin et al.'s study.

No / Red deer stags produce an androgen-dependent mane of long hairs only in the breeding season; in the non-breeding season, when circulating androgen levels are low, the neck hair resembles the rest of the coat. This study was designed to determine whether androgen receptors are present in deer follicles throughout the year or only in the mane (neck) follicles when circulating testosterone levels are high in the breeding season. Although androgens regulate much human hair growth the mechanisms are not well understood; they are believed to act on the hair follicle epithelium via the mesenchyme-derived dermal papilla. The location of androgen receptors in the follicle was investigated by immunohistochemistry and androgen binding was measured biochemically in cultured dermal papilla cells derived from mane and flank follicles during the breeding season and from neck follicles during the non-breeding season. Immunohistochemistry of frozen skin sections using a polyclonal antibody to the androgen receptor localised nuclear staining only in the dermal papilla cells of mane follicles. Saturation analysis assays of 14 primary dermal papilla cell lines using [(3)H]-mibolerone demonstrated high-affinity, low-capacity androgen receptors were present only in mane (breeding season neck) cells; competition studies with other steroids confirmed the specificity of the receptors. Androgen receptors were not detectable in cells from either the breeding season flank nor the non-breeding season neck follicles. The unusual biological model offered by red deer of androgen-dependent hair being produced on the neck in the breeding, but not the non-breeding season, has allowed confirmation that androgen receptors are required in follicle dermal papilla cells for an androgen response; this concurs with previous human studies. In addition, the absence of receptors in the non-breeding season follicles demonstrates that receptors are not expressed unless the follicle is responding to androgens. Androgen receptors may be induced in mane follicles by seasonal changes in circulating hormone(s).

No / The excavation at Cnip, Isle of Lewis, Scotland of the largest, and only known family cemetery from the early Norse period in the Hehrides, provided a unique opportunity to use Sr isotope analysis to examine the origins of people who may have been Norwegian Vikings. Sr isotope analysis permits direct investigation of a person's place of origin rather than indirectly through acquired cultural and artefactual affiliations. Sr isotope data suggest that the Norse group at Cnip was of mixed origins. The majority were consistent with indigenous origins but two individuals, of middle-age and different sex. were immigrants. They were, however, not from Norway but were raised separately, most probably on Tertiary volcanic rocks (e.g. the Inner Hebrides or NE Ireland) or, for the female, on marine carbonate rocks.

No / Traditional methods of dietary reconstruction do not allow the investigation of dietary variability within the lifetimes of individual hominins. However, laser ablation stable isotope analysis reveals that the ¿13C values of Paranthropus robustus individuals often changed seasonally and interannually. These data suggest that Paranthropus was not a dietary specialist and that by about 1.8 million years ago, savanna-based foods such as grasses or sedges or animals eating these foods made up an important but highly variable part of its diet.

Spermatogenesis is characterized by the succession in time and space of specific germ cell associations (stages). There can be a single stage (e.g., rodents and some macaques) or more than one stage (e.g., chimpanzee and human) per tubular cross section. We analyzed the organization of the seminiferous epithelium and quantified testicular germ cell production and apoptosis in a New World primate, the common marmoset (Callithrix jacchus). Tubule cross sections contained more than one stage, and the human six-stage system could be applied to marmoset spermatogenesis. Stereological (optical disector) analysis (n = 5) revealed high spermatogenic efficiency during meiosis and no loss of spermatids during spermiogenesis. The conversion of type A to type B spermatogonia was several-fold higher than that reported for other primates. Highest apoptotic rates were found for S-phase cells (20%) and 4C cells (15%) by flow cytometric analysis (n = 6 animals); histological analysis confirmed spermatogonial apoptosis. Haploid germ cell apoptosis was <2%. Marmoset spermatogenesis is very efficient and involves substantial spermatogonial proliferation. The prime determinants of germ cell production in primates appear to be proliferation and survival of spermatogonia rather than the efficiency of meiotic divisions. Based on the organizational similarities, common marmosets could provide a new animal model for experimental studies of human spermatogenesis.

Lake Tanganyika is a complex, tropical ecosystem in East Africa, harboring an estimated 2,100 species. Extensive watershed deforestation threatens the biodiversity and ecological integrity of the lake. In this dissertation, ecological and paleoecological methods were employed to study the distribution of invertebrate biodiversity through space and time, with particular emphasis on linkages between biodiversity and land –use patterns. Ecological surveys of fish, mollusc, and ostracod crustacean diversity at sites in northern Lake Tanganyika representing different levels of watershed disturbance revealed a negative correlation between biodiversity and intensity of watershed disturbance. To elucidate the long -term relationship between disturbance and biodiversity, paleoecological records of invertebrates offshore from watersheds experiencing different degrees of anthropogenic disturbance were examined. Life, death, and fossil assemblages of ostracod valves were compared to assess the reliability and natural variability inherent to the paleoecological record. These comparisons indicated that paleoecological (i.e. death and fossil) assemblages reliably preserve information on species richness, abundance, and occurrence frequency at comparable -to- annual resolution. Unlike life assemblages, species composition of paleoecological assemblages reflects input of species from multiple habitat types. Ostracod paleoecological assemblages are characterized by spatiotemporal averaging that renders them representative of larger areas and longer time spans than life assemblages. Thus, paleoecological assemblages provide an efficient means of characterizing longer -term, site -average conditions. Natural variability in ostracod fossil assemblages from a sediment core representing the Late Glacial to the present indicates that abundance of individual ostracod species is highly variable. Ostracod assemblages were preserved in only the most recent 2,500 years of sediment. Species composition of ostracod assemblages reflects lake water depth. Core geochemical data indicate that the coring site may have been below the oxycline for ~2,000 years, inhibiting ostracod survival and preservation. Paleoecological, sedimentological, and stable isotope data revealed differences in biodiversity and watershed disturbance through time offshore from a pair of sites. The protected site is offshore from Gombe Stream National Park (Tanzania), the other offshore from a deforested watershed outside the park. Offshore from the deforested watershed, sedimentation rates increased, and turnover in ostracod species composition occurred during the past 50 years. Comparable changes were not observed offshore from the park.