Spermatogenesis is a complex process of male germ cell development in which the diploid spermatogonia undergo series of mitotic divisions and differentiation steps culminating into the preleptotene spermatocytes which then enter into the meiotic prophase following a single replication cycle. This phase is characterized by meiotic recombination and is followed by reduction division resulting in haploid round spermatids. These cells then undergo extensive morphological and nuclear changes to form a unique cell, spermatozoa. This entire germ cell differentiation process occurs in a unique environment present inside the seminiferous tubules which is created by the Sertoli cells, the somatic cells in the tubules by forming junctions with each other thus providing unique milieu to the developing germ cells. Within the tubule, the germ cells are also arranged in an orderly manner called stages of spermatogenesis indicating a complex mechanism of germ cell differentiation. This complex differentiation process is a consequence of developmentally and precisely regulated differential gene expression (Eddy, 2002). Unraveling the molecular mechanisms involved in the male germ cell development is an uphill task due to the complexity of the cyto-architecture existing in the tubules and further complicated by unavailability of established germ cell lines and lack of cell culture systems that facilitate the germ cell differentiation in vitro. Comparative gene expression analysis of spermatogenesis in nematodes, flies and rodents revealed highly conserved transcriptomes and have provided some insights into its regulation (Schlecht and Primig, 2003). However, these data fail to represent the genetic and biological complexity of human spermatogenesis. In the present study, an attempt has been made to identify the genes that are differentially expressed in human tetraploid and haploid germ cells and to investigate the mechanism of regulation of the genes expressed in the post-meiotic germ cells. To identify the cell type specific genes, expression profiling of the human tetraploid and haploid germ cells was carried out using cDNA microarray. These cells were purified by centrifugal elutriation (Meistrich et al., 1981; Shetty et al., 1996) from the human testicular tissues obtained from the patients undergoing orchidectomy as treatment for prostate cancer. Purity of the enriched population of the germ cells was ascertained by DNA flow cytometry and by RT-PCR analysis using the known cell-specific markers and ruling out contamination of the somatic cells such as the Sertoli cells and the Leydig cells. Microarray experiments were carried out with the RNA isolated from each cell type and labeling the cDNA with Cy3/Cy5-dUTP and hybridizing to the human 19K array chip (University Health Network, Toronto, Canada) containing 19,200 ESTs. Two independent hybridizations were carried out using the germ cells isolated from two individuals and the microarray data were analyzed using Avadis 3.1 software (Strand Life Sciences, India). Analysis of the microarray data following normalization revealed that 723 transcripts showed higher expression in the meiotic cells whereas 459 transcripts showed higher expression in the post-meiotic germ cells. Microarray data were validated further by RT-PCR analysis of some of the differentially regulated genes. The DAVID analysis (Database for Annotation, Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov/) of these genes revealed that many genes associated with diverse functions and pathways appeared to be differentially expressed in both cell types. It is known that many biological systems exhibit distinct temporal gene expression profiles during different processes related to cell cycle, stress response and differentiation. Similarly, there are sets of genes, which respond to specific stimuli, appear to be synchronized in their expression. Such ‘synexpressed’ genes have been shown to be regulated by common transcription regulatory processes and have similar upstream transcription factor binding sites (Niehrs and Pollet, 1999). And therefore, having identified genes that appeared to be differentially expressed in the haploid and the tetraploid germ cells, attempt was made to analyze transcription factor binding sites in the promoter of those genes. In silico promoter analysis of several genes showing higher post-meiotic expression was carried out in order to identify the common regulatory motifs. Analysis of the annotated promoters (available from Eukaryotic Promoter Database; http://www.epd.isb-sib.ch/) of about forty genes highly expressed in the post-meiotic germ cells using TFSEARCH program (http://www.cbrc.jp/ research/db/TFSEARCH.html) confirmed that many genes had common transcription factor binding sites. Interestingly, almost all of the analyzed genes harbored SRY (Sex determining Region in Y)/SOX (SRY-box containing) binding motifs. In addition, the promoters of genes such as Protamine 1 and 2, Transition protein 1 and 2, A kinase (PRKA) anchor protein 4 that are known to be expressed post-meiotically, also harbor SRY binding sites suggesting that SRY may be one of the key regulators of the post-meiotic gene expression. SRY is a HMG-box containing member of Sox-family of architectural transcription factors. SRY is encoded by the Y chromosome and was first discovered as the testis-determining factor in mammals (Koopman et al., 1991). SRY HMG-box is eighty amino acids conserved motif that binds to the minor groove of the DNA in a sequence-dependent manner resulting in its bending and thus regulating the gene expression. The RT-PCR analysis of the human haploid and tetraploid germ cells showed very high expression of SRY in the post-meiotic cells further suggesting key role of SRY in the post-meiotic gene regulation. Role of SRY in the post-meiotic gene expression was investigated by determining the effect of SRY on human Protamine 1 (PRM1) promoter, a gene known to be exclusively expressed in the round spermatids and as indicated above, harbors many SRY binding sites in its promoter. SRY cDNA was cloned into the mammalian expression vector, pcDNA3.1 and the PRM1 promoter was cloned into the promoter-less pGL3 Basic vector upstream of the Luciferase reporter gene. Co-transfection of both constructs led to up-regulation of PRM1 promoter activity in both HeLa cells and LNCaP cells in a dose-dependent manner clearly demonstrating the role of SRY in PRM1 gene expression. Sequential deletion of the SRY binding sites in the PRM1 promoter led to the identification of the critical SRY binding motif important for SRY-mediated upregulation of PRM1 gene expression. This was confirmed by demonstrating in vitro binding of SRY to its critical binding site in the PRM1 promoter by gel shift assay using the nuclear extract of the HeLa cells transfected with FLAG-tagged SRY. The human SRY is an atypical transcription factor that binds DNA through its HMG, but unlike the mouse Sry and other Sox proteins, lacks the trans-activation domain and therefore requires other factors for its actions. Recently, the glutamine-rich, zinc-finger containing transactivator, Specificity protein 1 (Sp1) has been identified as one such interacting partner (Wissmuller et al., 2006). RT-PCR analysis showed that human SP1 is highly expressed in the haploid germ cells and could up-regulate PRM1 expression which harbors two SP1 binding sites in its promoter. When co-transfected, SRY and SP1 up-regulated PRM1 promoter in co-operative manner suggesting that SP1 may act in coordination with SRY in regulating PRM1. All these data taken together clearly signifies a critical role of SRY in post-meiotic germ cell gene expression. Recent reports suggest that SRY is also expressed in the adult human brain and prostate. However, its role in these tissues is not clearly understood. The Y chromosome has been shown to be frequently lost in prostate cancer and has also been shown to suppress the tumorigenicity of the PC-3 prostate cancer cells suggesting that the Y chromosome encoded genes may be involved in tumor suppression. SRY can physically interact with the androgen receptor (AR) and thereby interfere in its downstream signaling (Yuan et al., 2001). Since the prostate tumors show initial androgen-dependency, it was interesting to look at the role of SRY in the prostate cancer. To decipher the effect of SRY on the androgen-responsive LNCaP cells, stable clones of LNCaP expressing human SRY were generated. These clones showed significant decrease in growth in response to 5α-dihydrotestosterone (DHT) compared to the vector transfected or the parental LNCaP cells. In the soft agar colony formation assay, the SRY expressing LNCaP formed smaller colonies as compared to the controls in presence of DHT. Preliminary experiments in male athymic nude mice demonstrated that one of the SRY expressing clones showed reduced tumor growth compared to control cells suggesting that SRY may play a role in prostate cancer progression by decreasing the sensitivity to DHT. To summarize, the present study has identified several genes differentially expressed in the human haploid and tetraploid germ cells and further showed that SRY may be one of the key regulators of the post-meiotic gene expression.

Šio magistro baigiamojo darbo tikslas yra atlikti išsamią ženklo pripažinimo plačiai žinomu kriterijų analizė, išskiriant ir nurodant svarbiausius šio statuso aspektus. Magistro baigiamojo darbo tyrimo objektas yra kriterijai, galintys būti reikšmingais sprendžiant dęl prekių ženklo pripažinimo plačiai žinomu. Magistro baigiamajame darbe naudojant lyginamąją, analizės, sisteminį – loginį, istorinį, lingvistinį metodus buvo analizuojami tarptautiniai, Europos Sąjungos ir nacionaliniais teisės aktais, užsienio bei Lietuvos teismų praktika, teisės specialistų moksliniai darbai. Ypatingas dėmesys skiriamas Paryžiaus sąjungos dėl pramoninės nuosavybės apsaugos asamblėjos ir Pasaulinės intelektinės nuosavybės organizacijos generalinės asamblėjos 1999 m. priimtai bendrai rekomendacijai dėl plačiai žinomų ženklų apsaugos (angl. Joint Recommendation Concerning Provisions on the Protection of Well – Known Marks). / The main goal of this master thesis is to make particular analysis of the criteria of the well-known trademark by showing and setting main aspects of this status. The object of this master thesis is criteria, which can be significant defining trade mark as well-known trademark. Using comparative, analytical, systematic – logical, historical, linguistic methods were analyzed international, European Union and national law acts/certificates, foreign and Lithuanian court practice, researches of law professionals. Particular attention and consideration was given to Joint Recommendation Concerning Provisions on the Protection of Well – Known Marks adopted in 1999.

Baigiamajame magistriniame darbe susietas Lietuvos moreninių molinių gruntų pavadinimas (pagal LST EN ISO 14688-2:2006) su statinio zondavimo (CPTu) duomenimis. Darbą sudaro teorinė ir praktinė dalys. Teorinėje dalyje aptartos sudarytos gruntų klasifikacijos pagal CPT ir CPTu duomenis, aprašytos tyrime naudojamų gruntų vietovės inžinerinės-geologinės sąlygos bei sudaryta tyrimo metodologija, kurioje pateiktos formulės CPT ir CPTu rodiklių santykio apskaičiavimui, aprašomas grunto pavadinimo nustatymas pagal granuliometrinę sudėtį bei taikomas metodas molinių gruntų granuliometrinės sudėties nustatymui. Trumpai aprašomi tyrime taikyti principai. Praktinėje dalyje išsamiai aprašyti ir išanalizuoti skaičiavimuose naudojami duomenys, surastos priklausomybės, sudaryta gruntų pavadinimo nustatymo schema bei įvertinta gruntų klasifikacija pagal aptartus 4 metodus. Pateiktos išvados ir siūlymai tolimesniam darbui bei gautų rezultatų patikslinimui. Darbą sudaro 8 pagrindinės dalys: įvadas, atliktų darbų apžvalga, vietovės inžierinės-geologinės sąlygos, tyrimo metodologija, tyrimo rezultatai, išvados, literatūros sąrašas ir priedai. Darbo apimtis – 50 p. teksto be priedų, 59 p. priedų, 20 literatūros šaltiniai, 30 pav. ir 5 lentelės. / The present Master Thesis forms a link between the names of the Lithuanian moraine argillaceous soils (according to LST EN ISO 14688-2:2006) and the data of the cone penetration test (CPTu). The paper consists of a theoretical part and a research. The theoretical part is devoted to discussing formulated soil classifications according to the data of CPT and CPTu, describing the engineering/ geological conditions of the soil locations investigated in the research and presenting the research methods. The research methods include the formulas for calculating the CPT and CPTu indicator ratio, description of determining the soil name according to the particle size distribution and application of the method for determining the particle size distribution of argillaceous soils. The principles employed in the research are also briefly described. The research includes a detailed description and analysis of the data used in the calculations and the discovered dependencies, the formulated soil name determination plan and the soil classification assessed according to the discussed four methods. At the end of the paper, conclusions and recommendations are given for further work and specification of the received results. The study consists of the following eight parts: introduction, review of the earlier studies, engineering/ geological conditions of the location, research methods, research results, conclusions, references and annexes. The paper consists of 50 pages without annexes, 59 pages... [to full text]

Les téléostes, le clade possédant le plus d’espèces parmi les vertébrés, emploient une saisissante diversité de mécanismes de détermination du sexe, incluant des mécanismes génétiques et environnementaux. Des études récentes ont identifié de nombreux nouveaux régulateurs génétiquesdu développement sexual des poissons et ont introduit la notion de “terrain de jeux évolutif”. Le but de ce projet de thèse est de donner une vue complète des dynamiques évolutives des déterminants du sexe dans un ordre de téléostes, les Esociformes. Dans notre espèce focale, Esox lucius, nous avons identifié une duplication d’un membre de la famille des TgfB – une famille qui a émergé en tant que fondamentale dans la régulation du sexe chez les téléostes – comme MSD (gène controlant la détermination du sexe). Nous avons obtenu des preuves fonctionnelles du rôle de ce gène en tant que MSD, de son interaction avec des facteurs environnementaux, ainsi que des nouvelles informations sur les processus évolutifsCe gène est perdu dans une population de la même espèce en Amérique du Nord, mais il est conservé parmi les autres Esociformes. En parallèle, d’autres systèmes de détermination du sexe ont été identifiés dans des espèces proches. De plus, dans des clades plus distants comme Umbra et Dallia, le MSD d’Esox n’est pas présent, et d’autres mécanismes implicants de nouveaux MSD ont été identifiés, complefixiant l’histoire évolutive des MSD dans ce groupe, qui reflète la plasticité génétique observée dans les téléostes en général. / Teleost fishes, the most species-rich clade among vertebrates, employs an astonishing diversity of sex-determining (SD) mechanisms, including environmental and genetic systems. Recent studies identified many new genetic regulators in fish sexual development that lead to the notion of an 'evolutionary playground'.This thesis project aims to provide a complete picture of the evolutionary dynamic of SD systems within a small order of teleost, Esociformes. In our focal species Esox lucius, we idenWhile the gene is lost in another population of the same species rapidly possibly during post-glacial recolonization process, it is well conserved among different species. Meanwhile, additional transition of SD system have also been identified in a sister Esox species. Moreover, in the most distant genera Dallia and Umbra, the Esox master SD gene is not present and we found different SD mechanisms with novel SD genes that adds additional layers of complexity to this group, which mirrors the observed high genetic plasticity in teleost SD

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas. / Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

The diploma thesis explores with the evaluation of the success of selected indices for determining the number of clusters used in cluster analysis. The aim of this thesis is on the basis of various combinations of clustering methods and distances verify whether, alternatively using which clustering methods and distances is it possible to rely on the results of indices for determining the number of clusters. The results of success rate presented in the third chapter suggest that not all of indices for determining the number of clusters can be used universally. The most successful index is Dunn index, which was able to determine the correct number of clusters in 37 % of cases, respectively Davies-Bouldin index with the share of 70 % when including deviation of one cluster. The success rate is affected by both used method and selected distance.

The dissertation deals with different approaches to determining the value of synergies of companies implementing mergers in the mechanical engineering industry in the Czech Republic. This topic was chosen because of the high number of mergers, as well as the failure to achieve the effects planned. As a result, the consequence of such transactions is not the expected value growth for the owners. In the theoretical part of the thesis, mergers are defined from a legal point of view, and the historical development in the studied area is described. Based on the results of previous research, the systematization of the motives of mergers was carried out. The most common reason is the planned economic benefits in the form of cost savings or revenue growth. This part also describes the merger efficiency evaluation procedures used in previous research. Two approaches were identified, the first based on financial analysis indicators, and the second on discounted cash flow. The main objective of the dissertation is to propose adjustments to the procedure of valuation of companies implementing mergers in order to make it possible to identify the formation of synergy. Therefore, the data of companies operating in the mechanical engineering industry from 2004 to 2011 was used. As part of factor research, statistical tests of significance and dependence were used; the methods of classification and regression trees, as well as the linear regression model, were used to predict synergy. First, using the revenue indicators and operating profit, the transactions were divided into successful mergers and unsuccessful mergers. Based on this division, statistically significant differences in the financial analysis indicators were identified between companies implementing successful and unsuccessful transactions. Statistically significant differences were identified in the indicators of labour cost to revenues, depreciation to revenues and asset turnover ratio. All of the companies examined (target, bidder and newly created) were valued using the discounted capital cash flow method. Based on this valuation, factors that affect the value of the synergy achieved before implementation of the merger were identified. The indicators of return on assets, short-term financial assets to assets, cash flow to assets, and cash flow to interests were identified as statistically significant. These indicators were subsequently used to create prediction models. The first model predicts whether synergies will be achieved, and the second model predicts what the value of the synergies will be. The second model was subsequently incorporated into the pre-merger company valuation model

The thesis deals with the determination of ash fusion temperatures. The first part of the thesis consist of research about the properties of ash, ash fusion temperatures and test methods, including a description of the problems associated with ash and their solution. In the second part, the ash fusion temperatures of the selected fuels are determined first experimentally and then by elemental analysis of ash. The results of both methods are compared at the end of the thesis.

This work deals with mergers of commertial companys in largers economic units. Chapters are systematicly organized and provide the reader with overview of the development, relevant issues and execution of mergers of commercial companies. The work shows styles of merger execution, company´s motives for merger and finally also legal regulations connected with this proccess. Other sections of this work show analyses of companies, which have undergone a merger. You can see characteristics of these organisations and compare their merger execution plans and detailed schedule for different stages of the merge project.

The aim is to determine the price of a building selected for different purposes, using itemized budget and methods for valuation of property under applicable laws. Explain basic concepts in property valuation, explain their rationale, show the procedure of formation value by different pricing methods and selected construction object to demonstrate their use.