O trabalho objetivou avaliar o efeito de probióticos e antibióticos utilizados como promotores do crescimento sobre o desempenho de frangos de corte e a capacidade dos agentes de alterar o ecossistema intestinal de aves criadas em baterias e sobre piso. Adicionalmente foi estudado o efeito dos probióticos sobre a presença de oocistos na cama das aves. Os antibióticos tiveram sua eficácia, como promotores de crescimento, comprovada para aves criadas sobre piso, mas não em bateria. Foram observadas alterações na estrutura da comunidade de Bacteria no trato intestinal de frangos criados em baterias e sobre piso e suplementados com antibióticos. Não houve evidência de efeito favorável dos probióticos sobre o desempenho e incidência de oocisto na cama das aves. Os probióticos não tiveram a capacidade de colonizar o epitélio intestinal de frangos de corte. Foram observadas discretas modificações na estrutura da comunidade de Bacteria de frangos criados em bateria e sobre piso e suplementados com dietas contendo probióticos. A estrutura da comunidade de Bacteria do intestino delgado de frangos foi modificada em função do ambiente no qual frangos, suplementados com probióticos e antibióticos, foram criados. Frangos isentos de qualquer tipo de promotor de crescimento apresentaram 15 unidades taxonômicas operacionais distintas na microbiota intestinal aderida ao epitélio, predominantemente Lactobacillus e Pseudomonas. / This study was conducted to evaluated the effects of growth promoter probiotic and antibiotics on the perfomance and organ morphometry of broiler chickens raised in floor pens and in batteries and the ability of the additive to promote changes in the intestinal ecosystem. Additionally, the effect of probiotics on the presence of coccidia oocysts in the litter was evaluated. The efficacy of antibiotics as growth promoters was observed when the chickens were raised in floor pens but not in batteries. Antibiotic supplementation caused changes in the structure of Bacteria community of the intestinal tract of chicken raised in floor pens or in batteries. The probiotic additives tested did not result in improvement in performance in both environmental or in oocyst incidence in the litter. Also, the probiotic did not have the ability to colonize the intestinal epithelium of the birds. Discrete changes in the structure of Bacteria community were observed when probiotics were supplemented to chickens raised in floor pens or in batteries. Bacteria community structure in the small intestine of chicken was modified as a function of the environmental in which the birds were raised. Chicken fed diets devoid of growth promoters had 15 distinct phylogenetic groups in the microbiota adhered to the intestinal epithelium.

El Net Promoter Score (NPS) es uno de los indicadores más conocidos y utilizados para la medición de lealtad de los clientes en las organizaciones, y existen diferentes perspectivas en torno a su utilidad como una medida de la conducta de compra de los clientes. Esta investigación consiste en una revisión de la literatura relevante y contrasta posturas a favor, en contra y neutras. Las primeras sostienen que el NPS conlleva un método de rápida y sencilla aplicación, cuyo uso genera un impacto positivo en las organizaciones; de acuerdo con una segunda postura, su uso es perjudicial en las empresas, pues la segmentación de las puntuaciones en grupos de detractores, pasivos y promotores no produce un indicador suficiente para medir la lealtad de los clientes; finalmente, según la postura neutra, no existe evidencia suficiente que respalde la utilidad o ineficacia del uso del NPS. Esta es la controversia central que se estudiará en el presente trabajo; para ello, en primer lugar, se resumieron los diferentes métodos existentes para la medición del comportamiento del cliente. Los resultados de esta investigación determinan que no existe un consenso entre las posturas existentes con respecto a su utilidad en las organizaciones; sin embargo, coinciden en que se deberá aplicar más de un método de medición. / The Net Promoter Score (NPS) is one of the best known and most widely used indicators for measuring customer loyalty in organizations, and there are different perspectives on its usefulness as a measure of customer purchasing behavior. This research consists of a review of the relevant literature and contrasts positions for, against and neutrals. The former argue that the NPS involves a method of quick and easy application, the use of which generates a positive impact on organizations; According to a second position, its use is detrimental in companies, since the segmentation of the scores into groups of detractors, liabilities and promoters does not produce a sufficient indicator to measure customer loyalty; finally, according to the neutral position, there is insufficient evidence to support the usefulness or ineffectiveness of using the NPS. This is the central controversy that will be studied in the present work; to do this, in the first place, the different existing methods for measuring customer behavior were summarized. The results of this research determine that there is no consensus between the existing positions regarding its usefulness in organizations; however, they agree that more than one measurement method should be applied. / Trabajo de Suficiencia Profesional

The thesis focuses on customer satisfaction analysis of the company TEWECO GROUP s.r.o., which provides Internet connectivity in the district of Hodonin, and proposals to increase it. The theoretical part describes the problematics of marketing research. The practical part analyzes the results of marketing research of customers of TEWECO GROUP s.r.o. Based on this information, measures are proposed, which should increase customer satisfaction and bring higher revenue to the company.

Inactivation of tumor supressor gene BRCA1 causes a life-long risk of breast carcinoma development. Genetic screenings of indicated individuals from high-risk families help to identify large number of sequence variants in known predisposing genes. Majority of discovered variants doesn't have clinical significance yet which causes a big problem for diagnostics. Some of these variants are found within regulatory non-coding regions of gene. A part of the clinical classification of variants is their functional characterization. The goal of this thesis was to create a model system for functional characterization of variants in non-coding regions and to verify its function. Model system was based on targeted gene manipulation by co-transfecting CRISPR-Cas9 construct and donor construct that contained a portion of BRCA1 gene sequence with analyzed modifications, into U2 OS cells. The cells have stably integrated DR-GFP system which allows the activity of homologous recombination (HR) to be determined. Monoallelic modifications were induced into U2 OS cells. These modifications were in a Kozak sequence region of BRCA1 gene. Expression level of BRCA1 mRNA was determined by qRT-PCR, which showed the same levels of mRNA in all cells with analyzed alterations. Next, expression level of BRCA1 protein was...

La santé cardiovasculaire, la fonction immunitaire, l'hémostase et la réponse à d'autres maladies dépendent de l'abondance et des caractéristiques spécifiques des cellules sanguines. Au fil des années, un effort considérable a été fait pour trouver les variants génétiques, les gènes et les mécanismes de régulation impliqués dans la création de ces cellules. L'inactivation d'un allèle, appelée "perte de fonction" (LoF), est un type de variant codant que nous aimerions associer aux phénotypes sanguins. Comme ces mutations ne peuvent pas être artificiellement induites chez l'humain, pour des raisons éthiques évidentes, nous observons les occurences naturelles de ces pertes de fonction et espérons que la taille des cohortes sera suffisante pour trouver des associations statistiquement significatives. L'inactivation des deux allèles, appelée "knockout" (KO), peut avoir des conséquences plus fortes qu'une simple perte de fonction. Nous espérons également trouver des KO d'origine naturelle grâce à la taille des cohortes. La combinaison de deux variants LoF différents sur les deux allèles est appelée knockout hétérozygote composé. Nous nous intéressons également aux variants non codants qui affectent l'expression des gènes impliqués dans l'hématopoïèse. Certains de ces variants créent ou perturbent des sites de liaison des facteurs de transcription (TF), ces protéines qui se lient à des séquences d'ADN spécifiques et régulent l'expression des gènes. Les sites de liaison (TFBS) des facteurs de transcription se trouvent dans les promoteurs des gènes et dans les amplificateurs spécifiques au type cellulaire. Alors que certaines de ces mutations peuvent être bénignes ou même bénéfiques, la présence d'un LoF ou d'un KO peut être trop nuisible à la survie de l'individu. Les résultats de cette étude sont limités par le biais de survie. Comparée à une étude d'association pangénomique, cette étude se concentre sur un plus petit nombre de variants génétiques pour augmenter la puissance statistique et offrir une interprétation pour les résultats statistiquement significatifs. Le programme Trans-Omics for Precision Medicine (TOPMed) recueille et garantit la qualité des 45 000 séquences du génome entier que nous avons utilisées dans cette étude, ainsi que les bilans sanguins correspondants. Grâce à ces données, nous avons pu trouver plusieurs associations connues et nouvelles entre des variants rares et des phénotypes sanguins. / Cardiovascular health, immune function, hemostasis and the response to other illnesses depend on the abundance and specific features of blood cells. Over the years, a considerable effort has been made to find which genetic variants, genes and regulatory mechanisms are involved in the creation of these cells. The inactivation of an allele, called a loss-of-function (LoF), is a type of coding variant we would like to associate with blood phenotypes. For obvious ethical reasons, these mutations cannot be artificially induced in human, so we fall back on natural occurrences and hope that large cohorts will provide enough samples to find statistically significant associations. The inactivation of both alleles, called a knockout (KO), may have stronger consequences than a simple loss-of-function. We also hope to find naturally occurring knockouts thanks to the size of a large cohort. The combination of two different LoF variants is called a compound heterozygote knockout. We are also interested in non-coding variants that affect the expression of genes that are involved in hematopoiesis. Some of these variants create or disrupt the binding sites of transcription factors (TF), the proteins that bind to specific DNA sequences and regulate gene expression. Transcription factors binding sites (TFBS) are found in gene promoters and cell type specific enhancers. While some of these mutations can be benign or even beneficial, the presence of a LoF or KO may be too detrimental for the individual to survive. The results of this study are limited by survival bias. Compared to a genome-wide association study, this study focuses on a smaller number of genetic variants to increase statistical power and give an interpretation to the statistically significant findings. The Trans-Omics for Precision Medicine (TOPMed) program collects and ensures the quality of the 45,000 whole-genome sequences we used in this study, as well as the corresponding complete blood counts. Thanks to this raw data, we were able to find several known and novel associations between rare variants and blood phenotypes.

Conducting polymers find variety of application in many areas in microelectronics. PPY and PT are among the most extensively studied intrinsically conducting polymers. The problem of poor adhesion of the electrochemically deposited conducting polymers was the main focus of this work. This easy peeling of the PPY layer from the oxide substrate was circumvented by design of novel adhesion promoters, which compatibilise the two incompatible surfaces- the polymer film and the oxide substrate. The first part of the research was focused on the synthesis of different classes of adhesion promoters. These monomers were based on monoheterocyclic derivative of 3-substituted pyrrole and a tricyclic derivative of 2,5-dithienylpyrrole. The synthesized monomers were bifunctional with a specified and defined task for each group. Pyrrole based monomers, 3-phenyl N-alkyl pyrrolyl phosphonic acids referred as C10PhP and C12PhP and the monomers based on 2,5-dithienylpyrrole were synthesized with different anchoring groups namely, -Si(OMe)3 and-PO3H2 and spacer groups (n= 4, 6, 10, 12, benzyl). SNSnP for [(2,5-dithiophen-2-yl-pyrrol-1-yl)-dodecyl]-phosphonic acid, SNSnTMS for [(2,5-dithiophen-2-yl-pyrrol-1-yl)-alkyl]-trimethoxysilane and ArP for 4-(2,5-di-thiophen-2-yl-pyrrol-1-yl)-1-yl)-benzyl]-phosphonic acid was used as acronym for further references. The adsorption and self-organisation process as well as the surface reactions of these adhesion promoters on different oxide surfaces have been investigated. The aim was to graft conducting polymer layers covalently on modified metal oxide surfaces. This novel molecule has the self-assembling property and can bind to the surface via acid-base interactions with the oxide surface. The pre-treated oxide substrates were modified by self-assembling technique from solution. Static contact angle gave the first indication of a successful adsorption. The kinetics of adsorption was monitored by Surface Plasmon Resonance Spectroscopy (SPR), Angle-Resolved X-Ray Photoelectron Spectroscopy (AR-XPS) confirms that the molecules are standing with the phosphonic acid group present in the inner part of the self-assembled monolayer (SAM), and the 2,5-dithienylpyrrole group is free on the top for further surface reactions. Such self-assembling molecules can find applications in grafting conducting polymer layers on metal oxide surfaces. The grafting process of conducting polymer was done either chemically or electrochemically with additional monomer. Thickness and morphology of the polymer films were studied by AFM and SEM. The film thickness could be adjusted between several hundreds of nanometer by varying the polymerization conditions. The tape-test confirms the strong adhesive bonding of the polymer to the modified substrate. Besides PPY, Poly(SNS) and Poly(SNSnP) were studied for their properties. The optical properties of Poly(SNS) were studied by UV-Vis spectroscopy. Electrochemically deposited polymer films of (Poly(SNS), Poly(SNSnTMS), Poly(ArP) and Poly(SNSnP)) was investigated by UV-Vis spectroscopy, XPS, grazing-angle FTIR spectroscopy, four-point conductivity and I-V and luminance - voltage characteristics. Grazing-angle FT-IR of the homopolymer, poly(SNSnP) showed interesting results. We observed the peaks due to P-OH at 1111 cm-1 indicating that the phosphonic acid is free on the top. This can find application as ion sensor because phosphonates are known to be good chelating agents. Comparison of the EIS and four-point conductivity measurements for the homopolymers of SNSnP and Poly(SNS) indicated a drop in the conductivity in case of substituted hompolymers. Poly(SNSnP) based LED device give white emission. EL spectra show broad bands, which cover the entire spectrum. They can be studied further for the development of light emitting diodes of different colours.

Der probiotische Stamm E. coli Nissle 1917 ist ein Fäkalisolat, das in der Medizin traditionell zur Behandlung verschiedener gastrointestinaler Erkrankungen eingesetzt wird. Durch erfolgversprechende klinische Studien zur Remissionserhaltung bei Colitis ulcerosa, bei denen EcN als therapeutische Alternative zur Standardmedikation eingesetzt wird, ist das Interesse an den Wirkmechanismen von Probiotika stark gestiegen. EcN gehört derzeit zu den am besten untersuchten Probiotika. Einige Wirkmechanismen konnten dadurch schon aufgeklärt werden. So sind vermutlich Strukturkomponenten und stammspezifische Syntheseleistungen an der Ausprägung des probiotischen Phänotyps von EcN beteiligt. Schlüssige Konzepte, die über Gene, Genprodukte und molekulare Mechanismen den probiotischen Effekt von EcN erklären, fehlen bislang. Im Rahmen dieser Arbeit wird das Genom von EcN analysiert und auf der Basis der Genomsequenz mit anderen E. coli-Stämmen verglichen. Mit Hilfe einer Promotor-Reporter-Fusionsbibliothek (Promotorbank) werden intestinal in vivo regulierte Gene identifiziert und dadurch neue Ansätze zur Untersuchung der probiotischen Eigenschaften von EcN geschaffen. Die Grundlage für die molekulare Analyse von EcN ist die manuelle Nachannotation seines sequenzierten Genoms. Die EcN-Sequenz wird mit 13 weiteren annotierten E. coli-Sequenzen verglichen. Nach dieser Analyse kodiert EcN derzeit 121 stammspezifische Gene. Die Genomstruktur ist mit den enthaltenen genomischen Inseln und Prophagen dem Genom des uropathogenen E. coli CFT073 sehr ähnlich. Mit wenigen Ausnahmen kodiert EcN alle in E. coli CFT073 vorhandenen Virulenz- und Fitnessfaktoren, so dass auf der Nukleotidebene die nahe Verwandschaft dieser beiden Stämme bestätigt werden kann. Zudem kann gezeigt werden, dass EcN in artifiziellen Systemen wie der Zellkultur oder gnotobiotischen Mäusen ein pathogenes Potenzial hat, obgleich die Kolonisierungsfähigkeit pathogener Bakterien durch Inkubation mit EcN herabgesetzt wird. Eine wichtige Rolle bei der Besiedlung des Intestinaltrakts und der Immunstimulation von Darmepithelzellen spielt auch die globale Regulation der Genaktivität bei EcN durch den alternativen Sigma-Faktor RpoS, der im Gegensatz zu rpoS-Deletionsmutanten zu einer gesteigerten mRNA-Expression des Tight-junction Proteins ZO-1 führt. Des Weiteren führte die Untersuchung von EcN-Deletionsmutanten zu der Schlussfolgerung, dass einige genomische Inseln für Eigenschaften, die das probiotische Verhalten erklären können, eine Rolle spielen. Durch den Einsatz einer Promotorbank von EcN in konventionellen und gnotobiotischen Mäusen werden erstmalig Sequenzen von intestinal in vivo aktiven Promotoren identifiziert. Der Aufbau eines Promotor-Reportergen-Assays mit dem Biolumineszenz erzeugenden luxCDABE-Operon ermöglichte die Untersuchung ausgewählter Promotoren in vitro. Mit einem In Vivo Imaging System (IVIS) kann in weiteren Experimenten die Aktivität dieser Promotoren in lebenden Mäusen untersucht werden. Im Rahmen dieser Arbeit wird gezeigt, dass EcN kein vollkommen harmloser probiotischer Stamm ist. Weitere Informationen über EcN sind dehalb wichtig für eine optimierte Anwendung als Therapeutikum. Die molekulare Analyse ist somit eine unbedingt notwendige Grundlage für weiterführende Untersuchungen der Eigenschaften von EcN, die für seinen probiotischen Charakter verantwortlich sind. / The probiotic E. coli Nissle 1917 is a fecal isolate which is traditionally used for treatment of various gastrointestinal disorders. In clinical trials where EcN was used as therapeutic alternative for remission maintenance of ulcerative colitis compared to standard medication, promising results led to an increased interest in probiotics. Today, EcN is one of the best studied probiotics. Therefore, several mechanisms of action could be enlightened. Structural components and strain-specific products are responsible for its probiotic effects. But conclusive concepts about genes, gene products and molecular mechanisms that really contribute to the probiotic character of EcN have not been offered so far. In order to create new possibilities to elucidate the probiotic traits of EcN the genome is analysed by taking this as a basis for comparison to other E. coli genomes and identification of intestinal in vivo regulated genes using a promoter-trap-library. The sequenced EcN genome is annotated and compared to 13 other so far annotated E. coli genomes. Concerning these analyses EcN encodes 121 strain-specific genes. The genome structure including the genomic islands and prophages is highly homolog to the uropathogenic E. coli CFT073. EcN encodes most of the virulence and fitness factors that are present in E. coli CFT073. Therefore, the close relationship of these two strains is confirmed at nucleotide level. Furthermore, it is shown that in artificial systems like cell culture assays and gnotobiotic mice EcN reveals a pathogenic potential although EcN is able to decrease colonization efficiency of pathogenic bacteria. The alternative sigma factor RpoS that is responsible for global regulation and activity of several genes seems to play an important role during colonization of EcN in the intestine and its immunostimulatory effects on intestinal epithelial cells. Investigation of EcN-deletion mutants lacking genomic islands and prophages lead to the conclusion that some genomic islands may play a role for specific probiotic traits. This is the first time where a promoter-trap-library was used in conventional and gnotobiotic mice for collection of intestinal in vivo active promoters. Constructing and establishing a promoter-reporter gene assay with the bioluminescent luxCDABE operon made the investigation of selected promoters in vitro possible as well as establishing a bioluminescence assay using an In Vivo Imaging System (IVIS) for investigation of promoter activity in living mice. In this research project was shown that EcN is not a completely harmless probiotic. The genome structure and regulatory mechanisms of gene expression are the strain’s molecular traits that lead to probiotic activity and immunostimulatory effects. Therefore, the molecular analyses presented here, together with the complete genome sequence, are a basis for further investigations of mechanisms that are responsible for the probiotic effects of EcN.

Understanding the mechanisms of transcriptional regulation and the constraints that operate in gene promoter sequences is the key step in understanding the evolutionary conservation of transcriptional regulation. It is well known that regulatory regions with the same expression outputs do not have to share the sequence similarity. The most important elements in regulatory sequences are transcription factor binding sites and their position relocation does not usually influence the expression output. The least complex transcriptional regulation is characteristic for housekeeping genes. For their expression they require only basal core promoter elements (sometimes only CpG islands are sufficient) and general transcription factors, so they can be transcribed easily and immediately whenever they are needed. In this study we focused on transcriptional regulation of invertebrate amphioxus (Branchiostoma floridae) housekeeping genes in vertebrate systems. We prepared a set of constructs with amphioxus regulatory regions for testing their activity in different mammalian cell lines and a set of constructs with the same amphioxus regulatory regions for observing their spatial recognition in developing medaka fish embryo. We found that half of investigated amphioxus regulatory regions are recognized by...

Philosophiae Doctor - PhD / This chapter outlines basic gene structure and how gene structure is related to promoter structure in both prokaryotes and eukaryotes and their transcription machinery. An in-depth discussion is given on variations types of the promoters among both prokaryotes and eukaryotes and as well as among three prokaryotic organisms namely, E.coli, B.subtilis and Mycobacteria with emphasis on Mituberculosis. The simplest definition that can be given for a promoter is: It is a segment of Deoxyribonucleic Acid (DNA) sequence located upstream of the 5' end of the gene where the RNA Polymerase enzyme binds prior to transcription (synthesis of RNA chain representative of one strand of the duplex DNA). However, promoters are more complex than defined above. For example, not all sequences upstream of genes can function as promoters even though they may have features similar to some known promoters (from section 1.2). Promoters are therefore specific sections of DNA sequences that are also recognized by specific proteins and therefore differ from other sections of DNA sequences that are transcribed or translated. The information for directing RNA polymerase to the promoter has to be in section of DNA sequence defining the promoter region. Transcription in prokaryotes is initiated when the enzyme RNA polymerase forms a complex with sigma factors at the promoter site. Before transcription, RNA polymerase must form a tight complex with the sigma/transcription factor(s) (figure 1.1). The 'tight complex' is then converted into an 'open complex' by melting of a short region of DNA within the sequence involved in the complex formation. The final step in transcription initiation involves joining of first two nucleotides in a phosphodiester linkage (nascent RNA) followed by the release of sigma/transcription factors. RNA polymerase then continues with the transcription by making a transition from initiation to elongation of the nascent transcript.

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