La synthèse des ARN messagers est un processus hautement régulé. Pendant l’initiation de la transcription, un nombre important de protéines est recruté au niveau du promoteur des gènes, comprenant l’ARN polymérase II, les facteurs généraux de transcription comme TFIIH, des co-activateurs et des remodeleurs de la chromatine. L’assemblage du complexe de pré-initiation sur les promoteurs est suivi par leur ouverture. Les modèles acceptés à ce jour suggéraient que la transcription des gènes de classe II nécessite les activités ATPase et hélicase de la sous-unité XPB de TFIIH afin d’ouvrir le promoteur. Or nous avons observé que l’expression des ARNm s’accommode de l’absence de XPB mais nécessite son activité ATPase. Ces observations concordent avec un modèle alternatif dans lequel l’activité ATPase de XPB est utilisée pour transloquer la protéine en amont du site d’initiation et lever un blocage, imposé par la présence de XPB, de l’ouverture du promoteur. De plus, nous avons découvert un nouveau rôle de TFIIH dans le remodelage de la chromatine lors de l’initiation de la transcription. Nous avons mis en évidence un lien étroit entre TFIIH et l’histone acétyltransférase KAT2A, permettant le contrôle de la structure de la chromatine et l’expression des gènes, apportant en outre de nouvelles informations sur le Xeroderma pigmentosum combiné au syndrome de Cockayne, une maladie de la réparation avec une prédisposition au cancer. / The synthesis of messenger RNA is a highly regulated process. During transcription initiation, a large number of proteins are recruited to gene promoter including RNA polymerase II, general transcription factors like TFIIH, co-activators and chromatin remodelers. The assembly of pre-initiation complex on promoters is followed by their opening. Accepted models to date suggested that transcription of class II genes requires TFIIH XPB subunit ATPase and helicase activities to actively open the promoter. However, we have observed that mRNA expression is compatible with the absence of XPB but requires its ATPase activity. These observations are consistent with an alternative model in which the ATPase activity of XPB is used to translocate the protein upstream of the initiation site, alleviating a block, imposed by the presence of XPB, of the promoter opening. Moreover, we found a new role for TFIIH in chromatin remodeling during transcription initiation. We highlighted a tight connection between TFIIH and the histone acetyltransferase KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in combined Xeroderma Pigmentosum and Cockayne syndrome, a cancer-prone DNA repair-deficient disorder.

Der Typ I Plasminogenmangel ist eine seltene Multisystemerkrankung mit einer gestörten extravaskulären Fibrinolyse, die zur Ausbildung fibrinreicher Pseudomembranen auf Schleimhäuten führt. Kausale Therapien existieren bisher nicht, Fallberichte beschreiben jedoch eine Besserung der Symptomatik bei Patientinnen bei Einnahme oraler Kontrazeptiva. Östrogen wirkt im Körper über Rezeptoren durch Beeinflussung der Genexpression an bestimmten regulatorischen Elementen im Bereich der Promotoren (Estrogen responsive Elements (EREs)). Dies führte zu der Fragestellung, ob und durch welche Promotorelemente der Plasminogen Promotor durch Östrogen regulierbar und die Genexpression hierdurch modulierbar ist. Hierfür wurden verschiedene Promotorkonstrukte mit und ohne regulatorische Elemente kloniert und mittels Dual Luciferase Reporter Assay analysiert. In silico wurden 2 EREs (-11,5 kb und +4,2 kb relativ zur Transkriptionsstartstelle) identifiziert und anschließend ebenfalls in den Konstrukten getestet. Der kleinste „Plasminogenminimalpromotor“ war nicht durch Östrogen beeinflussbar. Proximale Promotorelemente wie die DNAse hypersensitive Region II mit einer beschriebenen Estrogen Responsive Unit sowie ein -2,4kb umfassendes Promotorkonstrukt wirkten unter Östrogenstimulation hemmend auf die Promotoraktivität. Ursächlich dafür sind wahrscheinlich weitere Interaktionen des Östrogen Rezeptors mit transkriptionsmodulierenden Proteinen, z.B. sind Interaktionen vermittelt über eine AP-3 Bindungsstelle denkbar. Die hemmenden Effekte konnten als Plasminogen-Gen-spezifisch und Leberzell-spezifisch demonstriert werden. Im Gegensatz dazu lösten beide vor die Minimalpromotoren klonierten EREs eine starke Stimulation aus, die sich auch in nichthepatischen Zelllinien - dort jedoch in geringerem Ausmaß ¬- zeigte. Somit sind diese EREs starke Enhancer, die eine Leberspezifität aufweisen. Die in der Summe komplexe Regulation der Östrogen-vermittelten Plasminogen Transkriptionskontrolle lässt auf eine Mitwirkung zusätzlicher Faktoren schließen. Um den in vitro Effekt der scheinbar widersprüchlichen Ergebnisse zu untersuchen, wurden humane Leberzellen einer Primärzellkultur mit Östrogen stimuliert und anschließend hinsichtlich der Plasminogen Expression untersucht. Diese Methode ließ sich jedoch aufgrund der Limitierung des Probenmaterials auf Leberbiopsien von Patienten mit gastrointestinalen Karzinomen mit Lebermetastasierung, damit einhergehend fehlender Östrogenrezeptorexpression, sowie fehlender Plasminogenexpression nicht etablieren, sodass hier der Nachweis des wirklichen Effektes des Östrogeneinflusses nicht gelang. In dieser Arbeit konnte gezeigt werden, dass sich der Plasminogen Promotor durch Östrogen regulieren lässt. Der genaue Mechanismus und der in vitro Effekt ließ sich jedoch nicht abschließend klären und bedarf weiterer Forschung. Eine vielversprechende Fortführung der Arbeit, besonders im Hinblick auf adäquate Therapieoptionen des Typ I-Plasminogenmangels, wäre die Etablierung eines geeigneten Zellmodells und die Erprobung weiterer Plasminogen modifizierender Substanzen.:I Bibliografische Beschreibung und Referat…………………………………………………………………………………………II II Abkürzungsverzeichnis……………………………………………………………………………………………………………………III 1. Einleitung und Hintergrund…………………………………………………………………………………………………………….1 1.1. Plasminogen und Typ I Plasminogenmangel……………………………………………………………………1 1.2. Organisation des Plasminogen Gens und des Plasminogen Promotors……………………..…….2 1.3. Östrogen und dessen Signaltransduktion…………………………………………………………………………3 2. Überleitung zur Originalpublikation – Fragestellung……………………………………………………………………….5 3. Originalpublikation………………………………………………………………………………………………………………….……..7 4. Diskussion und Ausblick………………………………………………………………………………………………………………..15 5. Zusammenfassung………………………………………………………………………………………………………………………..17 6. Literaturverzeichnis……………………………………………………………………………………………………………………...19 III Eigenständigkeitserklärung..………………………………………………………………………………………………………….IV IV Curriculum vitae…………………………………………………………………………………………………………………………….V V Liste der Publikationen…….…………………………………………………………………………………………………………….VI VI Danksagung………………………………………………………………………………………………………………………………….VII

AG to A transition at position -76 in the promoter region of the apoAI gene was previously identified, and the A-76 has been shown to be associated with high apoAI levels. The functional significance of the point mutation was assessed by analysing the DNA-protein binding and promoter activities of the different alleles. This data would suggest that the point mutation alters the function of the apoAI promoter as gel retention assays revealed that the G fragment (-140 to +10) formed an extra DNA-protein complex compared to the A fragment (-140 to +10). Concurrent with the altered DNA-protein interaction between the G and the A fragments, the transcriptional activities of the apoAI gene were found to also be altered. CAT assays have indicated a 1.91 fold increase in promoter activity of the A fragment as compared to the G fragment (-256 to +397). The difference in promoter activity was, however, highly dependent on the particular fragment used, as no difference was observed between the alleles when a fragment {-256 to +68) was used. In this study elements were identified in the region +68 to +397 that causes a reduction in the promoter activity of the G allele by 3.6 fold, whilst reducing the A allele activity by 2 fold. This data would suggest that the point mutation functionally alters the apoAI promoter activity via its interaction with other sequences especially in the region +68 to +397.

El objetivo del presente trabajo de investigación es determinar los factores más influyentes en la calidad percibida del servicio de taxi asistido por aplicativo móvil, desde la percepción de los usuarios que viven o trabajan en Lima Moderna. Para ello, se utilizó la metodología del modelo Servqual y se realizó un estudio no experimental del tipo descriptivo. El instrumento de recolección fue una encuesta estructurada de 22 preguntas, tanto para las percepciones como para las expectativas. La diferencia entre ambas dio como resultado brechas negativas del servicio. Las preguntas se agruparon en cinco factores (fiabilidad, sensibilidad, seguridad, empatía y tangibilidad). La encuesta se aplicó a 388 usuarios, lo que permitió conocer el nivel de satisfacción por cada factor, por sexo y por empresa. Entre los resultados más relevantes de la investigación se pudo evidenciar que los usuarios se sienten insatisfechos con la calidad del servicio, especialmente los relacionados con los factores de seguridad y empatía. El trabajo también investiga la relación entre cada factor y la calidad percibida, estableciéndose una correlación moderada. Además, se usa la herramienta Net Promoter Score que mide la lealtad de los usuarios basándose en sus recomendaciones. Si bien en el Perú no existen estudios que aplican Servqual para medir la calidad de servicio en la industria del taxi por aplicativo, este es muy usado internacionalmente. Confiamos que esta investigación servirá de base para futuros estudios a nivel nacional. Nuestra investigación puede ser aprovechada por los directivos de las empresas de taxi por aplicativo para realizar acciones de mejora en su servicio. / The aim of this recent investigation is to determine the most significant aspects that influence in the quality of taxi service apps in Modern Lima. For this, it was used the methodology of Servqual Model and it was developed as a non-experimental research of descriptive design. The instrument to collect data was a structured survey of 22 questions for both, perceptions, as well as expectations. The difference between them result in negative gaps in the service. The questions were classified according to five aspects (reliability, responsiveness, assurance, empathy and tangibles). The survey was applied to 388 users, which allowed to know the level of satisfaction in each factor, by gender and company. Among the most relevant results of the investigation it was noticeable that user feel unsatisfied with the quality of the service, especially those related to factors of reliability and empathy. Likewise, the research attempts to stablish the relationship between each factor and the quality perceived, stablishing a moderate correlation. Furthermore, it was used the Net Promoter Score tool, which measures the users’ loyalty based on their recommendations. Although in Peru there has not been any research that apply Servqual to measure the quality of service in transportation network companies, it is a model used internationally. We trust that this research will be used as a reference for future local studies. This investigation can benefit managers of taxi companies in order to carry out improvements in their service. / Tesis

(ENGLISH) The bacterial cell needs to regulate its gene expression in response to changing environmental conditions. RNA polymerase (RNAP) is the pivotal enzyme of this process and its activity is controlled by a number of auxiliary factors. Here I focus on RNAP-associating factors involved in regulation of transcription in G+ bacteria:  factors, initiating nucleoside triphosphates (iNTPs), HelD, δ and small RNA Ms1. The main emphasis is on σ factors from Bacillus subtilis. σ factors allow RNAP to specifically recognize promoter DNA. In my first project I set up in vitro transcription systems with purified alternative σ factors, σB , σD , σH , σI from B. subtilis. Using these systems, I studied the effect of initiating NTP concentration ([iNTP]) on transcription initiation. I showed that promoters of alternative  factors are often regulated by [iNTP]. In the next project I comprehensively characterized one of the least explored alternative  factors from B. subtilis, I . I identified ~130 genes affected by I , though only 16 of them were directly affected. Moreover, I discovered that I is involved in iron metabolism. Finally, I showed that I binding requires not only the conserved -35 and -10 hexamers, but also extended -35 and -10 elements located in the spacer region. In collaboration with...

Biomass combustion can be seen as CO2 neutral, thereby biomass combustion plants can have negative CO2 emissions if retrofitted with post combustion capture (PCC) technology using liquid absorbents. Monoethanolamine (MEA) has been used for carbon capture in coal combustion plants but are not suitable for use in biomass combustion plants due to corrosion and high solvent regeneration cost. Instead, the hot potassium carbonate (HPC) process using potassium carbonate (K2CO3) as absorbent show better attributes in these aspects. Although, K2CO3 has slow reaction kinetics with CO2 which need to be improved using promoters. Piperazine is the most tested promoter but are hazardous to humans. Recent research has revealed promising alternatives, among these different amino acid salts such as glycine, proline, and isonipecotic acid which are chemically benign. Biomass flue gas composition vary depending on the biomass fuel characteristics. How this affects the degradation and potential formation of hazardous substances need to be studied further. Biomass combustion plants are generally equipped with flue gas condensation systems, making retrofitting more feasible due to increased system flexibility and energy recovery options. The operation costs of carbon capture and sequestration (CCS) in biomass combustion plants need to be monitored to optimize the plant revenue. To make implementation of HPC in biomass combustion plants a reality, piperazine should be used as promoter. Meanwhile, research should focus on improving the absorption rate in HPC process with more chemically safe promoters.

Nicotinic acetylcholine receptors (nAChR) are found widely throughout the body. Like all members of the cys-loop family of receptors, nAChRs are composed of five protein subunits, each with a large extra-cellular domain and four transmembrane domains. Together these subunits form a binding domain, transmembrane pore, and selectivity filter. Neuronal nicotinic acetylcholine receptors, formed exclusively from α2-10 and β2-4 subunits, can form in many arrangements and stoichiometries. Each arrangement can have varying binding affinities and channel kinetics, resulting in great modulatory control. α3 and β2 subunit mRNA is found in CA1 interneurons in the stratum radiatum and stratum oriens of the rat hippocampus, and in surprising expression frequency and ratios. Further study of α3 and β2 subunit mRNA injected into Xenopus laevis oocytes yields interesting results about the potential for two α3β2 subtypes. These results were in intriguing, and prompted further study to better characterize and screen the α3β2 nAChR. In order to do so, a model was needed where the α3β2 nAChR could be studied in a more physiologically relevant mammalian environment, with consistent control over α3 and β2 subunit expression ratios, and sufficient protein expression and functionality. To this end, we created a doxycycline inducible HEK-293 cell line, stably transfected with the genetic sequences for the α3 and β2 subunits and NACHO, a transmembrane protein of the neuronal endoplasmic reticulum, which has been shown to mediate the assembly of α3β2 and other nAChRs. This new model is able to induce expression various ratios between α3 and β2 subunits in a consistent, manner, proving to be valuable tool in the characterization and screening of the α3β2 nAChR.

Breast cancer resistance protein (BCRP/ ABCG2), an ATP-binding cassette half transporter, confers multidrug resistance (MDR) to a series of antitumor agents such as mitoxantrone, daunorubicin, SN-38, and topotecan, and often limits the efficacy of chemotherapy. Recent studies have indicated that a putative estrogen response element (ERE) is located in the promoter region of the BCRP gene. However, whether and how BCRP is regulated transcriptionally by toremifene (TOR) remains unknown. In the present study, two plasmid vectors have been designed to express the wild-Type full-length BCRP cDNA enforced driven by its endogenous promoter containing a functional ERE and a constitutive cytomegalovirus (CMV) promoter as control, respectively, which were transfected into estrogenresponsive MCF-7 and estrogen-independent MDA-MB-231 human breast adenocarcinoma cell lines. We showed that toremifene alone significantly downregulated BCRP mRNA and protein levels in estrogen receptor a (ERa)-positiveMCF- 7 cells in a dose-dependent manner, and the inhibitory effect was partially reversed by estrone (E1). Furthermore, gel shift assays demonstrated that specific binding of ERa to the ERE in the BCRP promoter is essential for transcriptional inhibition of BCRP by toremifene. Interestingly, toremifene alone increased the cellular accumulation of mitoxantrone inBCRPtransfected cells, suggesting that TOR indeed inhibits BCRPmediated drug efflux and overcome drug resistance. To the best of our knowledge, this is the first report describing a direct effect of toremifene on BCRP. Our results thus indicate that toremifene by itself downregulates BCRP expression to reverse BCRP-mediated atypical multidrug resistance via a novel transcriptionally mechanism, which might be involved inTOR-ERcomplexes binding to theEREofBCRP promoter to repress transcription of BCRP gene.

Cytokine induced transcription of the germline immunoglobulin heavy chain gene directs isotype switch recombination to that gene. Therefore, understanding the regulation of germline transcription is an important first step in understanding the class switching process. Treatment of human B cells with IL-4 results in germline epsilon transcription. Subsequent activation of a second signal is necessary for these cells to undergo class switch recombination and express surface IgE. In contrast, treatment of splenic murine B-cells with IL-4 alone does not induce germline epsilon transcription. However, treatment with IL-4 plus LPS does induces germline epsilon transcription, followed by class switching to the IgE isotype. In both human and mouse, IL-4 is absolutely required for induction of germline transcripts and expression of IgE. Therefore, IL-4 is considered to be an IgE switch factor. The murine B lymphoma line, I.29μ is an IgM+ B cell line which can be induced to switch to the IgE isotype by treatment with IL-4 plus LPS. In these cells, germline epsilon transcription is constitutive and can be further induced 5-20 fold with IL-4, whereas LPS has no effect at the RNA level. Thus, the I.29μ cell line provides a model system to study the regulatory effects of IL-4 on the murine germline epsilon promoter. The aim of this thesis is to characterize the murine germline epsilon promoter and identify the minimal DNA elements necessary and sufficient for IL-4 induction. To identify the promoter elements, two kb of the 5' flanking region to the first exon (Iε) of the germline epsilon transcript was cloned into a Luciferase reporter plasmid and assayed for promoter activity. Assay of successive 5' deletion mutations by transfections into two B cell lines, I.29μ and M12.4.1, identified the 213 bp promoter construct, -162Luc, as containing sufficient sequence to confer full promoter function. Assay of the linker scanning mutations in the -162Luc plasmid localized the IL-4 responsive effect to a 46 bp region of the promoter. This region contains three nuclear factor binding elements: a C/EBP site, a recently identified NF-IL-4 site and a NFкB/p50 site. In order to detect protein complexes that specifically interact with this active region of DNA, electrophoretic mobility shift assays were performed using double stranded, oligonucleotide probes of this IL-4 responsive region. An IL-4 inducible complex was identified in nuclear extracts of I.29μ as well as murine splenic B-cells. Competition experiments with mutant probes mapped this inducible complex to the NF-IL-4 site. Constitutive binding of both C/EBP and NFкB/p50 was demonstrated by cold competition and supershift experiments. Transfection experiments using a series of linker scanning mutations allowed identification of DNA elements necessary for IL-4 induction. In order to test if these elements are sufficent for IL-4 induction, double stranded oligonucleotides containing these elements were transfered to a minimal fos promoter plasmid and assayed for IL-4 responsiveness. A 27 bp fragment containing two DNA elements, a C/EBP and a NF-IL-4 site were sufficient to confer IL-4 inducibility to a minimal c-fos promoter. This study defined a different IL-4 response element in the murine germline epsilon promoter from that previously published. This IL-4 response element is identical to the IL-4 response element in the human germline epsilon promoter. The NF-IL-4 site is also present in the promoter of the IL-4 responsive gene, CD23b (FcεRII), and this element binds an IL-4 inducible complex present in the human monocytic cell line U937. Various reports demonstrate the presence of an IL-4 inducible complex by gel shift assays and indeed the binding activity of NF-IL-4 has been mapped to a 9 bp consensus sequence within a 19 bp fragment. However, the transfer of IL-4 inducibility has not been reported using fragments smaller than 123 bp, the importance of which is underscored by the fact that more than one factor is involved in this induction. The contribution of this thesis to the understanding of transcriptional induction by IL-4 is in the delineation of the factors involved - namely, a member of the C/EBP family and NF-IL-4 are required for IL-4 induction and NFкB/p5O modulates this induction.

El presente trabajo de investigación evalúa la viabilidad y rentabilidad de la implementación de una solución integral omnicanal de atención al cliente. Este trabajo se centra en el rubro de las empresas de telecomunicaciones que ofrecen sus servicios en Latinoamérica, enfocada en aquellas con operaciones en Perú. Nos encontramos en un mundo donde, a pesar de los avances tecnológicos, todo servicio o producto necesita atención posterior de calidad, un servicio que brinde soluciones ágiles y rápidas a consultas simples y complejas. En el primer capítulo detallamos las bases teóricas, términos y herramientas utilizadas a nivel mundial para el procesamiento de información, además de la metodología de trabajo que facilita la implementación de una solución de autogestión en la atención al cliente. En el segundo capítulo presentamos la situación actual de la empresa, la misma que ha realizado varios intentos por bajar los costos operativos sin deteriorar la calidad de su servicio, implementando diversos canales nuevos de atención digital. Se presentan los principales indicadores de atención (NPS, satisfacción) y la usabilidad de los diversos canales de atención ofrecidos a sus clientes. En el tercer capítulo elaboramos la evaluación y análisis financieros que impactará esta solución integral de omnicanal, comparándola con un escenario BAU (business as usual) o sin cambios en la estrategia de atención de la compañía. Los resultados obtenidos nos muestran una solución rentable y beneficiosa para la compañía. Finalmente, la propuesta de valor que ofrece esta solución integral impactará en la fidelización, retención y captación de clientes. / This research evaluates de viability and rentability of the implementation of an integrated omnichannel solution for the after sales and customer support. This research focus in telecommunication companies which offers their services in Latin American countries, especially the ones that operates in Perú. Nowadays, we live in a world where, despite of the technological breakthrough and advances, every service or product after sales attention is in need of special attention to seek quality and to offer agile and fast solutions to simple and complex inquiries from their customers. In the first chapter we present, in detail, the terms, tools and methodology used on a worldwide level, in information processing and implementation of self-management solution tools for customer service. In the second chapter we studied the current situation of the company, which tried, several times, with no success, to lower their operational costs without compromising the quality of their service. We present Net Promoter Scores (NPS) and the usability of the different channels offered for the customer service between their users. In the third chapter, we carry out the financial evaluation of the project, and measure the impact of the omnichannel solution, comparing it with the business-as-usual scenario (the actual company situation). The results obtained from this evaluation show us a profitable and beneficial solution for the company. Finally, the project that offers a value proposal to implement an omnichannel solution self-management will impact the customer loyalty and customer acquisition in the near- and long-term future. / Trabajo de Investigación