Structural characteristics affecting functions of two actin regulating proteins

Structural biology is a branch of science that concentrates on the relationship between the structure and function of biological macromolecules. The prevalence of a large number of three dimensional structures offers effective tools for bio-scientists to understand the living world. Actin is the most abundant cellular protein and one of its main functions is to produce movement in living cells. Actin forms filaments that are dynamic and which are regulated by a number of different proteins. A class of these regulatory proteins contains actin depolymerizing factor homology (ADF-H) domains. These directly interact with actin through their ADF-H domains. Although ADF-H domains possess very similar three dimensional structures to one another, they vary in their functional properties. One example of this is the ability to bind to actin monomers or filaments. During the work for this thesis two structures of ADF-H domains were solved by nuclear magnetic resonance spectroscopy (NMR). The elucidated structures help us understand the binding specificities of the ADF-H family members.

Aspects and Applications of Pulse Sequence Design for Solution-State Nuclear Magnetic Resonance Spectroscopy

NMR spectroscopy enables the study of biomolecules from peptides and carbohydrates to proteins at atomic resolution. The technique uniquely allows for structure determination of molecules in solution-state. It also gives insights into dynamics and intermolecular interactions important for determining biological function. Detailed molecular information is entangled in the nuclear spin states. The information can be extracted by pulse sequences designed to measure the desired molecular parameters. Advancement of pulse sequence methodology therefore plays a key role in the development of biomolecular NMR spectroscopy. A range of novel pulse sequences for solution-state NMR spectroscopy are presented in this thesis. The pulse sequences are described in relation to the molecular information they provide. The pulse sequence experiments represent several advances in NMR spectroscopy with particular emphasis on applications for proteins. Some of the novel methods are focusing on methyl-containing amino acids which are pivotal for structure determination. Methyl-specific assignment schemes are introduced for increasing the size range of 13C,15N labeled proteins amenable to structure determination without resolving to more elaborate labeling schemes. Furthermore, cost-effective means are presented for monitoring amide and methyl correlations simultaneously. Residual dipolar couplings can be applied for structure refinement as well as for studying dynamics. Accurate methods for measuring residual dipolar couplings in small proteins are devised along with special techniques applicable when proteins require high pH or high temperature solvent conditions. Finally, a new technique is demonstrated to diminish strong-coupling induced artifacts in HMBC, a routine experiment for establishing long-range correlations in unlabeled molecules. The presented experiments facilitate structural studies of biomolecules by NMR spectroscopy.

Eurasian Neogene large herbivorous mammals and climate

The development and changes in the distribution of herbivorous mammal communities during the Neogene is complex. The Eurasian scale environmental patterns reflect the large scale geographical and climatic patterns. The reorganization of these affect the biome distribution throughout the continent. The distribution of mammal taxa was closely associated with the distribution of biomes. In Eurasia the Neogene development of environments was twofold. The early and middle Miocene that seemed to have been advantageous for mammals was followed by drying of environments during the late Neogene. The mid-latitude drying was the main trend, and it is the combined result of the retreat of Paratethys, the uplift of Tibetan Plateau and changes in the ocean currents and temperatures. The common mammals were "driving" the evolution of mammalian communities. During the late Miocene we see the drying affecting more and more regions, and we see changes in the composition of mammalian communities.

Methods to improve gene signal: Application to cDNA microarrays

Microarrays are high throughput biological assays that allow the screening of thousands of genes for their expression. The main idea behind microarrays is to compute for each gene a unique signal that is directly proportional to the quantity of mRNA that was hybridized on the chip. A large number of steps and errors associated with each step make the generated expression signal noisy. As a result, microarray data need to be carefully pre-processed before their analysis can be assumed to lead to reliable and biologically relevant conclusions. This thesis focuses on developing methods for improving gene signal and further utilizing this improved signal for higher level analysis. To achieve this, first, approaches for designing microarray experiments using various optimality criteria, considering both biological and technical replicates, are described. A carefully designed experiment leads to signal with low noise, as the effect of unwanted variations is minimized and the precision of the estimates of the parameters of interest are maximized. Second, a system for improving the gene signal by using three scans at varying scanner sensitivities is developed. A novel Bayesian latent intensity model is then applied on these three sets of expression values, corresponding to the three scans, to estimate the suitably calibrated true signal of genes. Third, a novel image segmentation approach that segregates the fluorescent signal from the undesired noise is developed using an additional dye, SYBR green RNA II. This technique helped in identifying signal only with respect to the hybridized DNA, and signal corresponding to dust, scratch, spilling of dye, and other noises, are avoided. Fourth, an integrated statistical model is developed, where signal correction, systematic array effects, dye effects, and differential expression, are modelled jointly as opposed to a sequential application of several methods of analysis. The methods described in here have been tested only for cDNA microarrays, but can also, with some modifications, be applied to other high-throughput technologies. Keywords: High-throughput technology, microarray, cDNA, multiple scans, Bayesian hierarchical models, image analysis, experimental design, MCMC, WinBUGS.