This diploma thesis focuses on the education of genetics to grammar schools and students' comprehension of genetics. This abstract scientific discipline is considered difficult to learn so I decided to prepare educational materials with the aim to improve students' understanding of the topic. The main aim of the thesis was to prepare and test a didactic game focused on Mendelian inheritance and compare the effectivity of this kind of interactive education compared with the classical explanatory style of education. Other aims included evaluating the influence of other variables; comparing the difficulty of individual terms in genetics and the connection within chosen triplet of terms; and identifying the most common misconceptions. The data was collected in four classes of upper graders at two grammar schools by newly prepared questionnaire focused on demographic and knowledge. The results showed that the didactic game was as efficient as the classical explanatory method at creating short term knowledge Students had a bigger problem with explaining the connection among the terms than defining the individual terms. The most complicated term reported by the students was chromosome. It was the only term in which there wasn't any improvement between pre-test and post-test. Part of this thesis is also...

Tight barriers are crucial for animals. Insect respiratory cells establish barriers through their extracellular matrices. These chitinous-matrices must be soft and flexible to provide ventilation, but also tight enough to allow oxygen flow and protection against dehydration, infections, and environmental stresses. However, genes that control soft, flexible chitin-matrices are poorly known. We investigated the genes of the chitinolytic glycosylhydrolase-family 18 in the tracheal system of Drosophila melanogaster. Our findings show that five chitinases and three chitinase-like genes organize the tracheal chitin-cuticles. Most of the chitinases degrade chitin from airway lumina to enable oxygen delivery. They further improve chitin-cuticles to enhance tube stability and integrity against stresses. Unexpectedly, some chitinases also support chitin assembly to expand the tube lumen properly. Moreover, Chitinase2 plays a decisive role in the chitin-cuticle formation that establishes taenidial folds to support tube stability. Chitinase2 is apically enriched on the surface of tracheal cells, where it controls the chitin-matrix architecture independently of other known cuticular proteins or chitinases. We suppose that the principle mechanisms of chitin-cuticle assembly and degradation require a set of critical glycosylhydrolases for flexible and not-flexible cuticles. The same glycosylhydrolases support thick laminar cuticle formation and are evolutionarily conserved among arthropods.

The aim of this project was to study the population genetics of Echinostoma trivolvis, a parasitic trematode that uses multiple hosts in its lifecycle and has a significant impact on amphibian populations. Microsatellite markers were to be identified and isolated because of their highly variable nature and reported ease of use with PCR. Parasite DNA was extracted from planorbid snails from several locations within California including: Point Reyes National Seashore, Lake Tahoe, and the San Francisco Bay Area. In addition, parasite samples were obtained from Manitoba, Canada. Several microsatellites were identified and 29 PCR primers sets were designed, six of which were capable of amplifying consistently. Sequencing other published molecular markers, COl, NDl, and ITS, unveiled intriguing phylogenetic relationships and potential cryptic species. The echinostome population in central California, as a result of this project, may be much more diverse than has long been reported in the literature.

A Thesis Submitted to the School of Graduate Studies in the Partial Fulfillment of the Requirements for the Degree Master of Science / Caenorhabditis elegans and one of its close relatives, Caenorhabditis briggsae, are animal models that are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although the two species appear nearly identical morphologically, comparative genomic analyses have revealed interesting differences between the genomes. Whether such differ- ences contribute to changes in developmental mechanisms and signalling pathways is an active area of research. One of the most well studied phenotypes associated with C. elegans signalling pathways are those that affect the specification of vulval tissue. Within the system of vuval development, mutants that exhibit the Mul- tivulva (Muv) phenotype are important as they show inappropriate divisions of vulva cells, which model tumour formation. Comparing gene function in different species genetic backgrounds can lead to an understanding of how genetic differ- ences contribute to different responses in cancer development. Genetic screens, conducted in our laboratory, yielded several genes whose loss of function results in a Muv phenotype and identified a novel regulator of C. briggsae vulval devel- opment, Cbr-ivp-3. Using the nematode C. briggssae as experimental system, we have characterized the tumour suppressor gene, Cbr-ivp-3, which impacts cell sig- nalling and cell division. I have carried out molecular genetic analyses of ivp-3 in both C. briggsae and C. elegans and have begun to characterize the functional role of Cbr-ivp-3. The findings in this thesis suggest that Cbr-ivp-3 is functioning to negatively regulate EGF/Cbr-lin-3. / Thesis / Master of Science (MSc) / The nematodes Caenorhabditis elegans and Caenorhabditis briggsae, are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although both species appear morphologically similar, comparative genomic analyses reveal differences between genomes. Comparing gene function in different genetic backgrounds can lead to an understanding of how genetic differences contribute to different responses in cancer development. Genetic screens have yielded several genes whose loss of function results in a Multivulva phenotype, showing inappropriate division of vulva cells, modeling tumor formation. We have carried out molecular genetic analyses of ivp-3, a novel regulator of C. briggsae vulval development, in both species and have found that Cbr-ivp-3 is regulating vulva development by negatively regulating EGF/Cbr-lin-3.

Caenorhabditis elegans and its congener, C. briggsae are excellent animal models for the comparative study of developmental mechanisms and gene function. Gupta lab is using the vulval tissue in these nematodes as a system to investigate conservation and divergence in signal transduction pathways. Genetic screens conducted earlier in our laboratory recovered several mutants that cause multivulva (Muv) phenotype. The Muv genes act as tumor suppressors and negatively regulate the proliferation of vulval precursors. Genetic and molecular work on these genes has revealed that C. briggsae vulva developmental utilizes novel genes representing a new phenotypic class termed ‘Inappropriate Vulva cell Proliferation (IVP)’ (Sharanya et al., 2015). This indicates that the signaling mechanism in C. briggsae specifies vulval cell fates differently from C. elegans. Interestingly, it has been found that Cbr-ivp mutants show higher levels of Cbr-lin-3 (EGF) transcript, indicating that these genes act genetically upstream of Cbr-lin-3, similar to SynMuv family members in C. elegans. Moreover, RNAi knockdown of the Cbr-lin-3 transcript resulted in the suppression of the multivulva phenotype in mutant animals. Similar suppression was also observed when a MAP kinase inhibitor was used in the previous study. In addition, the role of two other novel negative regulators of cell proliferation, Cbr-lin(bh1) and Cbr-lin(bh3) was also investigated. Preliminary findings on these regulators suggested that both Cbr-lin(bh1) and Cbr-lin(bh3) exhibiting a heritable Muv phenotype and are found to be located on Chromosome I and III respectively. Identification of novel genes and further characterization will help us understand the molecular function of genes and their involvement in the regulation of vulval cell differentiation. The findings of my research work will provide a background for future studies to understand the role of novel genes in reproductive system development. Overall, these results provide evidence that although the morphology of vulva is similar in the two nematode species, underlying mechanisms of development appear to have diverged. / Thesis / Master of Science (MSc)

Background: The central nervous system is frequently affected in children with inherited metabolic disorders (IMD). The causes of the brain insult are incompletely understood, and novel methods are required for disease diagnosis and monitoring response to novel therapies. Aims & Methods: The study aimed to improve understanding of the pathogenesis of IMD-related neurodegeneration, and to identify potential disease biomarkers in specific IMD, by directly investigating alterations in brain tissue metabolite profiles using non-invasive in vivo magnetic resonance spectroscopy (MRS) in conjunction with conventional MRI brain scans. Results: MRI/MRS studies were performed on over 300 children. Normal brain metabolite profiles were established from a standard comparator cohort. A detailed quality analysis enabled combination of data from different scanner systems. Non-standard brain metabolites were detected in 2.3% of children. Metabolite-based methods of disease progression monitoring were evaluated in Hunter Syndrome. Mechanisms leading to strokes in patients with propionic acidaemia and to learning difficulties and epilepsy in argininosuccinic aciduria were explored using brain tissue metabolite profiling. Conclusions: Non-invasive in vivo brain tissue metabolite profiling is achievable using quantitative magnetic resonance spectroscopy in the routine clinical paediatric setting, and has utility in disease diagnostics, in monitoring disease progression and in investigating disease pathogenesis.

Fundamental to the complexity of the nervous system is the precise regulation in space and time of the production, maturation, and migration of neurons in the developing embryo. This is eloquently seen in the forming cranial sensory ganglia (CSG) of the peripheral nervous system. Placodes, which are transient pseudostratified neuroepithelia in the surface ectoderm of the embryo, are responsible for generating most of the neurons of the CSG. Placodal progenitors commit to the neuronal fate and delaminate from the epithelium as immature, multipolar neuroblasts. These neuroblasts reside in a staging area immediately outside the placode. Differentiation of the neuroblasts is intimately coupled to their adoption of a bipolar morphology and migration away from the staging area to the future site of the CSG. Thus the forming CSG is a highly tractable model to anatomically separate the three phases of a neuroblast’s lifetime: from neuroepithelial progenitor (in the placode), to immature neuroblast (in the staging area), to mature neuron (in the migratory stream). In this thesis, I used the forming CSG as a model to investigate the role of Regulator of G protein Signalling 3 (RGS3) in neuroblast commitment and differentiation. Promoters within introns of the RGS3 locus generate isoforms in which N-terminal sequences are sequentially truncated, but C-terminal sequences are preserved. Intriguingly, I found that expression of these isoforms in the forming CSG is temporally co-linear with their genomic orientation: longer isoforms are exclusively expressed in the progenitor placode; a medium isoform is expressed exclusively in the neuroblast staging area; and the shortest isoforms are expressed in the neuronal migratory stream. Furthermore, through loss- and gain-of-function experiments, I demonstrated that each of these isoforms plays a specific role in the differentiation state in which it is expressed: placode-expressed isoforms negatively regulate neurogenesis; the neuroblast-expressed isoform negatively regulates differentiation; and the neuron-expressed isoforms negatively regulate neuronal migration. The negative regulatory role which all isoforms play in different cell-biological contexts is intriguing in light of the fact that they all share a C-terminal RGS domain, which canonically negatively regulates G protein signalling. Through domain mutation and deletion, I showed that the RGS and N-terminal domains are important for the function of each isoform. Thus temporally co-linear expression within the RGS3 locus generates later-expressed isoforms which lack the regulatory N-terminal domains of the earlier-expressed isoforms, giving them new license to perform different biochemical functions. Lastly, I investigated the conservation and evolution of RGS3 and its isoforms. RGS3 was found to be present in all extant metazoans, and results from this thesis implicate it as the founding member of the R4 subfamily of RGS proteins. Furthermore, in the early vertebrate lineage, a critical domain was lost. This is intriguing in light of the fact that placodes in their stereotypic forms also emerged early in the vertebrate lineage. Ectopic overexpression of the full-length invertebrate RGS3 protein prevented pseudostratification of the vertebrate placode, suggesting that the domain loss in the early vertebrate lineage was important for the evolution of pseudostratified placodes and the expansion of the vertebrate nervous system. In summary, the work in this thesis has uncovered a previously unseen model of transcriptional regulation of a single locus: intragenic temporal co-linearity. Furthermore, the demonstrated functions of this regulation have profound implications on the generation and differentiation of vertebrate neurons, as well as the evolution of the vertebrate nervous system.

As ecosystems become increasingly fragmented, there has been a proliferation of research into fields such as resource use, movement ecology and habitat connectivity. To understand how species may adapt to threats associated with habitat fragmentation it is necessary to study these processes in dispersing individuals. However, this is seldom done. Dispersal is one of the most important life-history traits involved in species persistence and evolution, but the consequences of dispersal are determined primarily by those that survive to reproduce. Although dispersal is most effectively studied as a three-stage process (departure, transience and settlement), empirical studies rarely do so and an investigation into the entire process has probably never been carried out on any one species. Here I investigate the survival, resource use, movement ecology and connectivity of African lions (Panthera leo) in all three dispersal phases in addition to adulthood. I make use of a longterm dataset incorporating radio-telemetry and observational data from lions in Hwange National Park, Zimbabwe. Dispersal is inherently risky and my results show that male lions that disperse while young suffer high mortality, young dispersal being brought about by high off-take of territorial males. Dispersing males may be aware of risks associated with territorial adults as they position themselves far from them and utilise habitats and resources differently. However, dispersers, compared to adult males and females, are far less averse of risky, anthropogenic landscapes, suggesting they are the demographic most prone to human-lion conflict. The ontogenetic movement behaviour of lions reflects a transition from directional movement during transience, suggestive of sequential search strategies, to random or periodic use of a fixed territory after settlement. In terms of habitat connectivity, I show that radically different conclusions emerge depending on which demographic is used to parameterise connectivity models. Understanding the shifting mechanisms that species adopt throughout ontogeny is critical to their conservation in an increasingly fragmented world.

Molecular biology and genetics are currently very fast developing disciplines. One of the possibilities how to increase the pupils' interest in studying of molecular biology and genetics, and to support their own curiosity is to use research-based teaching strategies. Although Czech Education tries to correspond with this progress, teaching of molecular biology and genetics is limited not only by financial and material limits but also by a lack of vocational education. The diploma thesis is divided into theoretical and practical part. The theoretical part deals with the position of molecular biology in teaching and model systems in the study and teaching of molecular biology and genetics. The practical part is divided into three chapters. The goal of the first chapter was to present available teaching materials suitable for a teaching at grammar schools, especially with regard to the availability of manuals for practical seminars. The second chapter summarized the results of a questionnaire survey among grammar school teachers, which concerns the position of molecular biology and genetics in teaching. Both of the above-mentioned goals have shown that more needs to be done about the molecular biology of genetics. According to the teachers, students who would like to continue to pursue this...

We review fold usage on completed genomes in order to explore protein structure evolution and assess the evolutionary relevance of current structural classification systems (SCOP and CATH). We assign folds on a set of 150 completed genomes using fold recognition methods (PSI-BLAST, SUPERFAMILY and Gene3D). The patterns of presence or absence of folds on genomes gives us insights into the relationships between folds and how we have arrived at the set of folds we see today. In particular, we develop a technique to estimate the relative ages of a protein fold based on genomic occurrence patterns in a phylogeny. We find that SCOP's `alpha/beta' class has relatively fewer distinct folds on large genomes, and that folds of this class tend to be older; folds of SCOP's `small protein' class follow opposite trends. Usage patterns show that folds with many copies on a genome are generally old, but that old folds do not necessarily have many copies. In addition, longer domains tend to be older and hydrophobic amino acids have high propensities for older folds whereas, polar - but non-charged - amino acids are associated with younger folds. Generally domains with stabilising features tend to be older. We also show that the reliability of fold recognition methods may be assessed using occurrence patterns. We develop a method, that detects false positives by identifying isolated occurrences in a phylogeny of species, and is able to improve genome wide fold recognition assignment sets. We use a structural fragment library to investigate evolutionary links between protein folds. We show that 'older' folds have relatively more such links than 'younger' folds. This correlation becomes stronger for longer fragment lengths suggesting that such links may reflect evolutionary relatedness.