Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Coarse Global Visual Localization of a Mobile Robot

Localization, which is the ability of a mobile robot to estimate its position within its environment, is a key capability for autonomous operation of any mobile robot. This thesis presents a system for indoor coarse and global localization of a mobile robot based on visual information. The system is based on image matching and uses SIFT features as natural landmarks. Features extracted from training images arestored in a database for use in localization later. During localization an image of the scene is captured using the on-board camera of the robot, features are extracted from the image and the best match is searched from the database. Feature matching is done using the k-d tree algorithm. Experimental results showed that localization accuracy increases with the number of training features used in the training database, while, on the other hand, increasing number of features tended to have a negative impact on the computational time. For some parts of the environment the error rate was relatively high due to a strong correlation of features taken from those places across the environment.

Mobile robot localization using sonar ranging and wlan intensity maps

 Main goal of this thesis was to implement a localization system which uses sonars and WLAN intensity maps to localize an indoor mobile robot. A probabilistic localization method, Monte Carlo Localization is used in localization. Also the theory behind probabilistic localization is explained. Two main problems in mobile robotics, path tracking and global localization, are solved in this thesis. Implemented system can achieve acceptable performance in path tracking. Global localization using WLAN received signal strength information is shown to provide good results, which can be used to localize the robot accurately, but also some bad results, which are no use when trying to localize the robot to the correct place. Main goal of solving ambiguity in office like environment is achieved in many test cases.

Multi-sensor Simultaneous Localization and Mapping

Simultaneous localization and mapping(SLAM) is a very important problem in mobile robotics. Many solutions have been proposed by different scientists during the last two decades, nevertheless few studies have considered the use of multiple sensors simultane¬ously. The solution is on combining several data sources with the aid of an Extended Kalman Filter (EKF). Two approaches are proposed. The first one is to use the ordinary EKF SLAM algorithm for each data source separately in parallel and then at the end of each step, fuse the results into one solution. Another proposed approach is the use of multiple data sources simultaneously in a single filter. The comparison of the computational com¬plexity of the two methods is also presented. The first method is almost four times faster than the second one.

Role and Function of c-Jun Protein Complex in Cancer Cell Behavior

Transcription factors play a crucial role in the regulation of cell behavior by modulating gene expression profiles. Previous studies have described a dual role for the AP-1 family transcription factor c-Jun in the regulation of cellular fate. In various cell types weak and transient activations of c-Jun N-terminal kinase (JNK) and c-Jun appear to contribute to proliferation and survival, whereas strong and prolonged activation of JNK and c-Jun result in apoptosis. These opposite roles played by c-Jun are cell type specific and the molecular mechanisms defining these antonymous c-Jun-mediated responses remain incompletely understood. c-Jun activity in transformed cells is regulated by signalling cascades downstream of oncoproteins such as Ras and Raf. In addition, the pro-proliferative role and the survival promoting function for c-Jun has been described in various cancer models. Furthermore, c-Jun was described to be overexpressed in different cancer types. However, the molecular mechanisms by which c-Jun exerts these oncogenic functions are not all clearly established. Therefore it is of primary interest to further identify molecular mechanisms and functions for c-Jun in cancer. Regulation of gene expression is tightly dependent on accurate protein-protein interactions. Therefore, co-factors for c-Jun may define the functions for c-Jun in cancer. Identification of protein-protein interactions promoting cancer may provide novel possibilities for cancer treatment. In this study, we show that DNA topoisomerase I (TopoI) is a transcriptional co-factor for c-Jun. Moreover, c-Jun and TopoI together promote expression of epidermal growth factor receptor (EGFR) in cancer cells. We also show that the clinically used TopoI inhibitor topotecan reduces EGFR expression. Importantly, the effect of TopoI on EGFR transcription was shown to depend on c-Jun as Jun-/- cells or cells treated with JNK inhibitor SP600125 are resistant to topotecan treatment both in regulation of EGFR expression and cell proliferation. Moreover, c-Jun regulates the nucleolar localization and the function of the ribonucleic acid (RNA) helicase DDX21, a previously identified member of c-Jun protein complex. In addition, c-Jun stimulates rRNA processing by supporting DDX21 rRNA binding. Finally, this study characterizes a DDX21 dependent expression of cyclin dependent kinase (Cdk) 6, a correlation of DDX21 expression with prostate cancer progression and a substrate binding dependency of DDX21 nucleolar localization in prostate cancer cells. Taken together, the results of this study validate the c-Jun-TopoI interaction and precise the c-Jun-DDX21 interaction. Moreover, these results show the importance for protein-protein interaction in the regulation of their cellular functions in cancer cell behavior. Finally, the results presented here disclose new exciting therapeutic opportunities for cancer treatment.

The Significance of Hydrogen Bonding Interactions in the Cleavage of RNA

RNA is essential for all living organisms. It has important roles in protein synthesis, controlling gene expression as well as catalyzing biological reactions. Chemically RNA is a very stable molecule, although in biological systems many agents catalyze the cleavage of RNA, such as naturally occurring enzymes and ribozymes. Much effort has been put in the last decades in developing highly active artificial ribonucleases since such molecules could have potential in the therapeutic field and provide tools for molecular biology. Several potential catalysts have emerged, but usually detailed cleavage mechanism remains unresolved. This thesis is aimed at clarifying mechanistic details of the cleavage and isomerization of RNA by using simpler nucleoside models of RNA. The topics in the experimental part cover three different studies, one concerning the mechanism of catalysis by large ribozymes, one dealing with the reactivity of modified and unmodified RNA oligonucleotides and one showing an efficient catalysis of the cleavage and isomerization of an RNA phosphodiester bond by a dinuclear metal ion complex. A review of the literature concerning stabilization of the phosphorane intermediate of the hydrolysis and isomerization of RNA phosphodiester bond is first presented. The results obtained in the experimental work followed by mechanistic interpretations are introduced in the second part of the thesis. Especially the significance of hydrogen bonding interactions is discussed.

Novel CO2 Regulated Proteins in Synechocystis PCC 6803

The large biodiversity of cyanobacteria together with the increasing genomics and proteomics metadata provide novel information for finding new commercially valuable metabolites. With the advent of global warming, there is growing interest in the processes that results in efficient CO2 capture through the use of photosynthetic microorganisms such as cyanobacteria. This requires a detailed knowledge of how cyanobacteria respond to the ambient CO2. My study was aimed at understanding the changes in the protein profile of the model organism, Synechocystis PCC 6803 towards the varying CO₂ level. In order to achieve this goal I have employed modern proteomics tools such as iTRAQ and DIGE, recombinant DNA techniques to construct different mutants in cyanobacteria and biophysical methods to study the photosynthetic properties. The proteomics study revealed several novel proteins, apart from the well characterized proteins involved in carbon concentrating mechanisms (CCMs), that were upregulated upon shift of the cells from high CO₂ concentration (3%) to that in air level (0.039%). The unknown proteins, Slr0006 and flavodiiron proteins (FDPs) Sll0217-Flv4 and Sll0219-Flv2, were selected for further characterization. Although slr0006 was substantially upregulated under C_i limiting conditions, inactivation of the gene did not result in any visual phenotype under various environmental conditions indicating that this protein is not essential for cell survival. However, quantitative proteomics showed the induction of novel plasmid and chromosome encoded proteins in deltaslr0006 under air level CO₂ conditions. The expression of the slr0006 gene was found to be strictly dependent on active photosynthetic electron transfer. Slr0006 contains conserved dsRNA binding domain that belongs to the Sua5/YrdC/YciO protein family. Structural modelling of Slr0006 showed an alpha/beta twisted open-sheet structure and a positively charged cavity, indicating a possible binding site for RNA. The 3D model and the co-localization of Slr0006 with ribosomal subunits suggest that it might play a role in translation or ribosome biogenesis. On the other hand, deletions in the sll0217-sll218- sll0219 operon resulted in enhanced photodamage of PSII and distorted energy transfer from phycobilisome (PBS) to PSII, suggesting a dynamic photoprotection role of the operon. Constructed homology models also suggest efficient electron transfer in heterodimeric Flv2/Flv4, apparently involved in PSII photoprotection. Both Slr0006 and FDPs exhibited several common features, including negative regulation by NdhR and ambiguous cellular localization when subjected to different concentrations of divalent ions. This strong association with the membranes remained undisturbed even in the presence of detergent or high salt. My finding brings ample information on three novel proteins and their functions towards carbon limitation. Nevertheless, many pathways and related proteins remain unexplored. The comprehensive understanding of the acclimation processes in cyanobacteria towards varying environmental CO₂ levels will help to uncover adaptive mechanisms in other organisms, including higher plants.

Localization, Frequency, and Functions of Filled Pauses: Five American Politicians’ Use of er and erm in the Talk Show Larry King Live

Tämän pro gradu– tutkielman tavoitteena oli testata täytettyjen taukojen (er ja erm) esiintymistiheyttä, sijaintia kieliopillisessa rakenteessa sekä funktioita Kjellmerin (2003) korpus-tutkimuksessa. Materiaalina käytin viiden yhdysvaltalaisen poliitikon puhetta keskusteluohjelmasta Larry King Live. Tutkimuksessani sovelsin Kjellmerin tutkimusmenetelmiä, joita muokkasin huomattavasti suppeampaan materiaaliini sopiviksi. Lähestymistapani oli täten induktiivinen toisin kuin testatussa tutkimuksessa. Materiaalini oli tarkoituksellisesti rajattu, sillä halusin selvittää, kuvaavatko Kjellmerin laajaan materiaaliin perustuvat tutkimustulokset myös täytettyjen taukojen käyttöä suppeammassa materiaalissa. Materiaalini (kokonaisuudessaan 101 minuuttia) transkriboin ortografisesti. Analyysissäni arvioin täytettyjen taukojen esiintymistiheyden puhujakohtaisesti ja koko ryhmälle suhteuttamalla täytettyjen taukojen lukumäärän kokonaissanamäärään. Tämän jälkeen tein perinteisen kielioppianalyysin rakenteista, joita edeltää tai joissa esiintyy täytetty tauko, ja täytettyjen taukojen sijainnin perusteella luokittelin ne sana-, lauseke-, ja lausetasolle. Lopuksi analysoin täytettyjen taukojen käyttöä soveltaen Kjellmerin ehdottamia funktioita (hesitaatio, vuorottelujäsennyksen merkitseminen, huomion herättäminen ja kontaktin luominen, korostus ja korjaus) ja niiden piirteitä omaan materiaaliini. Tutkimukseni perusteella täytetyt tauot esiintyvät tutkitun viiden poliitikon puheessa suhteellisen usein. Puhujakohtaiset eroavaisuudet olivat kuitenkin huomattavat. Kieliopillisen luokitteluni mukaan sana-, lauseke- ja lausetasot eivät täysin kuvaa täytettyjen taukojen sijoittumista, sillä täytetyt tauot edelsivät mm. määre-lauseita, jotka eivät vastaa lausetasoa englannin kielessä. Materiaalini funktioanalyysi osoitti, että täytetyt tauot yleensä vastaavat yhtä tai useampaa Kjellmerin ehdottamaa funktioita. Lisäksi tutkimukseni mukaan täytetyillä tauoilla on ainakin yksi rakenteellinen funktio. Analyysini perusteella Kjellmerin tutkimustulokset ovat siis pääosin sovellettavissa suppeampaan materiaaliin. Puutteiksi hänen tutkimuksessaan osoittautuivat funktioanalyysille tärkeän kontekstuaalisen informaation puute sekä keskittyminen täytettyihin taukoihin, jotka esiintyvät vain tietyissä kielioppirakenteissa. Yleisesti voin tutkimukseni pohjalta todeta, että täytetyt tauot ovat vielä vajaasti tunnettuja ja että kieliopillisen sijoituksen ja funktioiden lisätutkimus on tarpeellista.

Two-dimensional atom localization and Raman cooling of tripod-type atoms

Both atom localization and Raman cooling, considered in the thesis, reflect recent progress in the area of all-optical methods. We focus on twodimensional (2D) case, using a four-level tripod-type atomic scheme for atom localization within the optical half-wavelength as well as for efficient subrecoil Raman cooling. In the first part, we discuss the principles of 1D atom localization, accompanying by an example of the measurement of a spontaneously-emitted photon. Modifying this example, one archives sub-wavelength localization of a three-level -type atom, measuring the population in its upper state. We go further and obtain 2D sub-wavelength localization for a four-level tripod-type atom. The upper-state population is classified according to the spatial distribution, which in turn forms such structures as spikes, craters and waves. The second part of the thesis is devoted to Raman cooling. The cooling process is controlled by a sequence of velocity-selective transfers from one to another ground state. So far, 1D deep subrecoil cooling has been carried out with the sequence of square or Blackman pulses, applied to -type atoms. In turn, we discuss the transfer of atoms by stimulated Raman adiabatic passage (STIRAP), which provides robustness against the pulse duration if the cooling time is not in any critical role. A tripod-type atomic scheme is used for the purpose of 2D Raman cooling, allowing one to increase the efficiency and simplify the realization of the cooling.

Indoor Localization Solutions for a Marine Industry Augmented Reality Tool

In this report are described means for indoor localization in special, challenging circum-stances in marine industry. The work has been carried out in MARIN project, where a tool based on mobile augmented reality technologies for marine industry is developed. The tool can be used for various inspection and documentation tasks and it is aimed for improving the efficiency in design and construction work by offering the possibility to visualize the newest 3D-CAD model in real environment. Indoor localization is needed to support the system in initialization of the accurate camera pose calculation and auto-matically finding the right location in the 3D-CAD model. The suitability of each indoor localization method to the specific environment and circumstances is evaluated.