Les réseaux actuels et la prochaine génération des réseaux sans fil cellulaires (5G) doivent garantir, non seulement, les communications entre les gens (aussi connu sous le nom d'humain à humain - H2H), mais aussi à un déploiement massif de communication de type machine (MTC). MTC, ou encore Machine à Machine (M2M), peut être considérée comme des appareils qui peuvent établir des communications avec d’autres appareils sans aucune intervention humaine. M2M est aussi vue comme la pierre angulaire de la vision des objets connectés (IoT). Elle attire beaucoup d'attention, car elle peut être considérée comme une nouvelle opportunité pour les opérateurs de réseau et service IoT. Il existe aujourd’hui plusieurs types d’applications se basant sur MTC couvrant plusieurs domaines. On peut citer comme exemples les applications suivantes: la santé, les systèmes de transport intelligents (ITS), les compteurs intelligents et les réseaux intelligents, et la sécurité publique (PS). Le déploiement de ce type d'applications dans les réseaux mobiles cellulaires actuels, particulièrement Long Term Evolution (LTE) et LTE-Advanced (LTE-A) , ne peut être effectif sans surmonter les challenges posés par le déploiement d’un grand nombre d’équipement MTC dans la même cellule. En effet, le déploiement d'une myriade d'appareils MTC causera une congestion et une surcharge du système des réseaux d'accès radio (RAN) et du cœur de réseau (CN). Comme les appareils MTC sont équipés d'une batterie non rechargeable, la consommation d'énergie est aussi un défi. Dans cette thèse, nous allons étudier les problèmes de congestion et de consommation d'énergie dans le contexte des réseaux LTE et LTE-A en présence des appareils M2M. En ce qui concerne la congestion et la surcharge de système, nous nous concentrons sur la partie RAN, puisqu'elle peut être considérée comme la première ligne de défense pour le réseau cellulaire. Les contributions de cette thèse sont organisées sous les axes suivants: 1) Proposition d'un algorithme générique pour prédire le trafic entrant, de sorte que la congestion dans le réseau peut être facilement résolue, 2) Étude et proposition d'un modèle analytique générique de la procédure d'accès aléatoire au canal (RACH). Le modèle a pour but l’évaluation des méthodes de contrôle de congestion ciblant la partie RAN, 3) Approfondissement et proposition des méthodes permettant d'améliorer la méthode Pagination de Groupe (GP) approuvée par le 3GPP pour contrôler la congestion. / The current and next generation wireless cellular networks (5G) have to deal with not only communications between people (known as Human-to-Human - H2H), but also with a massive deployment of Machine-Type-Communication (MTC). MTC, or alternatively Machine-to-Machine (M2M), can be viewed as devices connected among them without any human intervention. M2M can be considered as the cornerstone of Internet-of-Things (IoT) vision. It attracts a lot of attention, since it can be considered as a new opportunity and business market. Nowadays, there is a vast number of MTC applications, covering a large number of fields. Some of these applications are Healthcare, Intelligent Transport System (ITS), smart metering and smart grids, public safety (PS), forming the so-called smart city. Deploying this type of applications in the current cellular mobile networks, especially Long Term Evolution (LTE) and LTE-Advanced (LTE-A), cannot be achieved before overcoming the accompanied challenges. Indeed, caused by the existence of a myriad of MTC devices, Radio Access Network (RAN) and Core Network (CN) congestion and system overload is one of these challenging issues. As the MTC devices are using non-rechargeable batteries, power consumption is also a challenge. In this thesis, we study the congestion and power consumption problems in the context of LTE and LTE-A networks featuring M2M communications. Regarding the congestion and system overload, the focus will be on the RAN part since it can be considered as the first defense line on the network. The contributions of the thesis are organized on the following axes: 1) Propose a general algorithm to predict the incoming traffic, so that the congestion in the network can be easily remedied, 2) Study and propose a general analytical model of the Random Access Channel (RACH) procedure. The model can help to evaluate the congestion control methods targeting the RAN part, 3) Depth study and propose methods improving the performance of Group Paging (GP) method, one of the methods approved by 3GPP to control the congestion.

Multiphase flow is the simultaneous flow of two or several phases through a system such as a pipe. This common phenomenon can be found in the petroleum and chemical engineering industrial fields. Transport of sand particles in multiphase production has attracted considerable attention given sand production is a common problem especially to the oil and gas industry. The sand production causes loss of pipe wall thickness which can lead to expensive failures and loss of production time. Build-up of sand in the system can result in blockage and further hamper production. Monitoring of multiphase flow is a process that has been established over several decades. This thesis reports an assessment of the application of Acoustic Emission (AE) technology as an alternative online technique to monitoring of sand particles under multiphase flow conditions in a horizontal pipe. The research was conducted on a purpose built test rig with the purpose of establishing a relation between AE activity and sand concentration under different multiphase flow conditions. The investigation consisted of five experimental tests. The initial experiment was performed to provide a basis for the application of AE technology to detect sand particle impact prior to performing tests in multiphase flow conditions. Further investigations are reported on two phase air-sand, water-sand and air- water-sand three-phase flows in a horizontal pipe for different superficial gas velocities (VSG), superficial liquid velocities (VSL) and sand concentrations (SC). The experimental findings clearly showed a correlation exists between AE energy levels and multiphase flow parameters, such as superficial liquid velocity (VSL), superficial gas velocity (VSG), sand concentration and sand minimum transport condition (MTC).

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A research report submitted to the Faculty of Commerce, Law and Management, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree of Master of Commerce (Taxation). Johannesburg, 2015 / Tax treaties have a developed language of their own within the field of international law. They may include terms that are unknown in particular jurisdictions of domestic law or therein defined differently. Because the language of tax treaties and domestic law differ from each other, the definitions of certain terms and income type under a tax treaty and under different states’ domestic law are not necessary identical. Despite these differences, tax treaty definitions must be used for tax treaty classification purposes, and domestic law definitions must be used for domestic law classification purposes. The tax definition determines the type of the income for tax treaty purposes even though the income would qualify under another income category under the treaty states’ domestic law. Similarly, the domestic tax law definition determines the type of income for domestic law purposes (Helminen 2010). In most instances the treaty definitions of the various types of income refer back to domestic tax law, and where the domestic tax law definition deviates between the two treaty countries, this may lead to the application by these countries of different articles of the treaty. If this is caused by the application of the domestic law, this is referred to as a conflict of qualification in the Commentaries to the OECD Model Tax Convention. In general a conflict of qualification refers to a situation where identical facts are treated differently for tax purposes in different countries. Such a conflict may either concern the subject or the object of taxation. Key words: Tax treaties, OECD MTC, Double Tax Agreements, double taxation, conflicts of qualification, hybrid entities, partnerships, fiscally transparent, domestic law, Mutual Agreement Procedures, permanent establishment.

With a wide range of potential applications, the machine type communication (MTC) is gaining a tremendous interest among mobile network operators, system designers, MTC specialist companies, and research institutes. The idea of having electronic devices and systems automatically connected to each other without human intervention is one of the most significant objectives for future wireless communications. Low data rate transmission and the requirement for low energy consumption are two typical characteristics for MTC applications. In terms of supporting low cots MTC devices, industrial standards will be more efficient if designers can re-use many features of existing radio access technologies. This will yield a cost effective solution to support MTC in future communication systems. This thesis investigates efficient MTC waveform and receiver designs for superior signal transmission quality with low operational costs. In terms of the downlink receiver design, this thesis proposes a novel virtual carrier (VC) receiver system for MTC receivers, which aims to reduce the maximum bandwidth to improve the data processing efficiency and cost-efficiency by using analogue filters to extract only sub-carriers of interest. For the VC receiver systems, we thus reduce the sampling rate in order to reduce the number of subsequent processing operations, which significantly reduces the analogue-to-digital converter (ADC) cost and power consumption while providing high signal to interference noise ratio (SINR) and low bit to error rate (BER) to support low data rate MTC devices. Our theoretical equations account for the interference effect of aliasing on the sub-carrier location, and this helps the system designer to evaluate what kind of filters and receiver sampling rate can be used to balance the energy cost and detection performance. In terms of the uplink waveform design, considering the enhanced number of MTC devices in the future communication systems, i.e. the fifth generation (5G) communications, the same tight synchronisation as used in today appears not to be cost-effective or even possible. Synchronisation signals, which aim to provide a perfect time or frequency synchronisation in the current fourth generation (4G) communication systems (known as the long-term evolution, LTE), is much more costly for low data rate MTC transmissions. The system bandwidth will be significantly reduced if a base station tries to synchronise all received signals among hundreds or thousands MTC devices in one transmission time period. In terms of relaxing the synchronisation requirements, this thesis compares and analyses the side-lobe reduction performance for several candidate multi-carrier waveforms to avoid these problems. We also propose the infinite impulse response universal filtered multi-carrier (UFMC) system and the overlap and add UFMC system, which significantly reduce the processing complexity compared with the state of the art UFMC techniques. This thesis derives closed-form expressions for the interference caused by time offsets between adjacent unsynchronised MTC users. Our analytical equations can be used in both simple and complex time-offset transmission scenarios, and enable the system designer to evaluate the SINR, the theoretical Shannon capacity and the BER performance.

Multiphase flow is the simultaneous flow of two or several phases through a system such as a pipe. This common phenomenon can be found in the petroleum and chemical engineering industrial fields. Transport of sand particles in multiphase production has attracted considerable attention given sand production is a common problem especially to the oil and gas industry. The sand production causes loss of pipe wall thickness which can lead to expensive failures and loss of production time. Build-up of sand in the system can result in blockage and further hamper production. Monitoring of multiphase flow is a process that has been established over several decades. This thesis reports an assessment of the application of Acoustic Emission (AE) technology as an alternative online technique to monitoring of sand particles under multiphase flow conditions in a horizontal pipe. The research was conducted on a purpose built test rig with the purpose of establishing a relation between AE activity and sand concentration under different multiphase flow conditions. The investigation consisted of five experimental tests. The initial experiment was performed to provide a basis for the application of AE technology to detect sand particle impact prior to performing tests in multiphase flow conditions. Further investigations are reported on two phase air-sand, water-sand and air- water-sand three-phase flows in a horizontal pipe for different superficial gas velocities (VSG), superficial liquid velocities (VSL) and sand concentrations (SC). The experimental findings clearly showed a correlation exists between AE energy levels and multiphase flow parameters, such as superficial liquid velocity (VSL), superficial gas velocity (VSG), sand concentration and sand minimum transport condition (MTC).

This thesis is dealing with the problem of MTC (Machine Type Communication) devices accessing LTE (Long Term Evolution) network. First part of work contains theoretical description of MTC, LTE network and possibility of using QoS (Quality of Service) for this type of communication. Influence of simultaneous access of large number of MTC devices into LTE network is examined. A solution is proposed -- usage of MTCG. In simulation enviroment NS-3 (Network Simulator 3), two scenarios are created for simulation with and without MTCG. The results of these simulations are then compared.

Die Rindertuberkulose ist eine chronische Erkrankung, die von Mycobacterium (M.) bovis und M. caprae, Mitgliedern des Mycobacterium-tuberculosis-Komplex (MTC), ausgelöst wird. Tuberkulose-Erregern werden sowohl mittels kultureller als auch molekulare Untersuchungsmethoden nachgewiesen. Ziel der vorliegenden Studie war es, die Sensitivität des DNA-Nachweises von Tuberkulose-Erregern zu steigern. Dafür wurden drei Fragestellung im Bereich der molekularen Mykobakterien-Diagnostik bearbeitet. I) Zur Verbesserung der Lyse der mykobakteriellen Zellwand als Voraussetzung für eine Zunahme der Freisetzung von DNA wurden im Vergleich zu einer standardisierten DNA-Isolierungsmethode vier verschiedene Lyseprotokolle (thermische, enzymatische, thermo-enzymatische und mechanische Lyse) entwickelt und mit M. bovis BCG durchgeführt. Die Verbesserung wurde anhand der cycle threshold (Ct)-Werte einer MTC-spezifischen Real-Time (rt) Polymerase-Kettenreaktion (PCR) geprüft. Zwei Lyseprotokolle (thermische und mechanische Lyse) wurden bei zehn Gewebeproben (Lymphknoten, Leber und Lunge) von zehn Tieren (acht Rinder, ein Lama und ein Luchs) mit nachgewiesener Tuberkulose ange-wendet. II) Ausserdem, wurde eine rt-PCR mit dem 16S rRNA Gen als Zielgen (16S-rt-PCR) für den direkten Nachweis von Erregern der Gattung Mycobacterium im Gewebe entwickelt. III) Ein neu entwickelter Spoligotyping-Microarray wurde mit der konventionellen Spoligoty-ping-Methode verglichen, um die neue Methode in Bezug die Sensitivität des Nachweises und des diskriminatorischen Potenzials direkt bei infizierten Gewebeproben zu analysieren. Bei der konventionellen Methode erfolgt die Hybridisierung des PCR-Produktes auf einer Nylon Membran, auf der spezifische Oligonukleotide fixiert sind. Bei der Microarray-Methode sind diese auf einem Microarray-Chip fixiert. Die Ergebnisse der Untersuchungen (I) zur Lyse der Zellwand bei M. bovis BCG zeigten, dass bei der mechanischen Lyse eine Zunahme um 14 % und bei der thermischen Lyse eine Zunahme an PCR-Produkt von 6 % im Vergleich zur Standardlyse erbrachte. Bei beiden Lyseprotokollen wurde eine statistische Signifikanz von α = 1 % (Mann-Whitney-Test) im Vergleich zur Standardlyse errechnet. Bei den tuberkulösen Gewebeproben wurde bei der mechanischen Lyse eine durchschnitliche Zunahme an PCR-Produkt um circa 9 % im Vergleich zur Standardlyse erzielt. Dieser Unterschied war jedoch auf Grund der geringen Probeanzahl nicht statistisch signifikant. II) Bei der Untersuchung von 43 Mykobakterien-Spezies, sechs Mitgliedern des MTC (unter anderen M. bovis BCG) und 37 Non Tuberculous Mycobacteria (NTM) Spezies, konnten alle mit der entwickelten Real-Time PCR (16S-rt-PCR) nachgewiesen werden. DNA-Extrakte von acht nicht zur Gattung Mycobacterium gehörenden Spezies wurden mit der 16S-rt-PCR nicht erfasst. Ein Erreger der Gattung Gordonia und ei-ner der Gattung Rhodococcus wurden auf Grund ihres engen Verwandtschaftsgrades jedoch ebenfalls mit der 16S-rt-PCR detektiert. Die oben erwähnten mittels MTC-spezifischer rt-PCR (Zielgen IS 1081) als infiziert identifizierten zehn Gewebeproben, wurden mittels 16S-rt-PCR untersucht. Die Ergebnisse zeigten, dass beiden rt-PCR Systeme eine vergleichbare Sensitivität aufwiesen. III) Bei dem Vergleich zwischen den Spoligotyping-Methoden zeigte sich die neue Methode um einen Faktor von 100 bei der M. bovis BCG-Reinkultur und um einen Fak-tor von 10 bei DNA-Extrakten aus tuberkulösen Gewebeproben sensitiver als die konventionelle Methode. Im Rahmen dieser Arbeit hat sich der Einsatz der mechanischen Lyse für die Verbesserung der Freisetzung von mykobakterieller DNA als routinefähig erwiesen. Die entwickelte 16S-rt-PCR erwies sich als brauchbare Methode für den Nachweis von Erregern der Gattung Mycobacterium. Die Microarray-Methode stellte sich wesentlich einfacher, sensitiver und schneller dar als die konventionelle Methode. Zusammenfassend kann gesagt werden, dass alle drei Ansätze dieser Arbeit einen Beitrag zur Verbesserung der molekularen Labordiagnostik der Tuberkulose leisten. / Bovine tuberculosis is a chronic disease, that results from infection of Mycobacterium (M.) bovis and M. caprae, members of Mycobacterium tuberculosis complex (MTC), respectively. The laboratory diagnosis of bovine tuberculosis is possible with culture as well as considerate fast molecular methods. The aim of this study was to improve the sensitivity of DNA detec-tion of tuberculosis-causing pathogens. Therefore, three different issues were addressed in the complex molecular procedure targeted. I) four different lytic protocols (thermal, enzymatic, thermo-enzymatic and mechanical lysis) were developed and compared to a standardized DNA isolation protocol that was performed on pure culture of Mycobacterium (M.) bovis BCG in order to improve the mycobacterial cell wall lysis leading to an increase of DNA release. The efficiencies of the lysis protocols were assessed by the resulting cycle threshold (Ct) values of a MTC-specific real time (rt) Polymerase Chain Reaction (PCR). Two lysis protocols (thermal and mechanical) were selected to fur-ther testing of ten tuberculosis-infected tissue samples (lymph nodes, liver and lungs) from ten animals (eight cattle, one lama and one lynx). II) In addition, a real time PCR using the 16S rRNA gene as target sequence was developed, which is also suitable to detect pathogens of Genus Mycobacterium on tissue samples. III) A comparison of a newly developed microarray and the conventional spoligotyping method was realised, to analyse the applicability of the microarray in relation of the sensitivity of the method and to analyse discriminatory potential directly from infected tissue samples. During the conventional spoligotyping method, the PCR product is hybridized with specific oligonucleotides, fixed on a nylon membrane. Using the newly developed method these oligonucleotides are fixed on a microarray-chip. The results I) of the mycobacterial cell wall lysis experiment with pure culture of M. bovis BCG showed an increase of 14 % by using mechanical lysis and an increase by 6 % of the PCR product by using thermal lysis compared to the standard protocol. Using the mechanical lysis as well as the thermal lysis a statistically significant (α = 1 % (Mann-Whitney-Test)) im-provement compared to the standard lysis was achieved. Mechanical lysis was performed on tuberculous tissue samples and the results of the lysis were improved by 9 % compared to the standard lysis. However, the difference between mechanical and standard lysis was not statistically significant due the small sample number. II) Forty-three different mycobacterial species, six members of MTC (among them M. bovis BCG) and 37 Non Tuberculous Mycobacteria (NTM), were detected using the newly developed real time PCR (16S-rt-PCR). Eight non-mycobacterial species were not detected using this rt-PCR, whereas one of genus Rhodococcus and one of genus Gordonia were detected by the 16S-rt-PCR due to their close genetic similarity to the genus Mycobacterium. The ten tuberculosis-infected tissue samples (see above) testing positive using a MTC-specific real time rt-PCR (target gene IS 1081) were sub-jected to the 16S-rt-PCR. Both rt-PCR systems showed a comparable sensitivity. III) By comparing the two spoligotyping-methods, the ArrayStrip™–format method was more sensitive than the conventional method by a factor of 100 applied to pure culture and by a factor of 10 when applied to DNA extracts from infected tissue samples. In conclusion, the mechanical lysis proved to be a practical method to liberate mycobacterial DNA. The newly developed rt-PCR was suitable to detect members of the genus Mycobacterium. The spoligotyping ArrayStrip™–format method appeared to be substantially easier to perform, more sensitive, and less time-consuming than the conventional method. The three methods described were suitable to improve the molecular laboratory diagnosis of tuberculosis.

The following article conveys a critical revision of the concessions renewal regimen as a matter of public telecommunication services of the Peruvian normative. For that, the author will focus on analyze the existing renewal forms of telecommunication in the Peruvian legislation, as well as the principles that rule the renewal processes of the telecommunication concessions. In addition, the author offers a new perspective about the authorities that are able to perform the process mentioned before, to finally show us the probability to reform the used methodology of the expiration dates of concessions based on certain principles,and bestow a modification proposal. / El presente artículo trata sobre una revisión crítica del régimen de renovación de concesiones en materia de servicios públicos de Telecomunicaciones en la normativa peruana. Para ello, el autor se centrará en analizar las formas de renovación existente en la legislación peruana, así como los principios que rigen el proceso de renovación de las concesiones en Telecomunicaciones. Además, el autor nos brinda una visión sobre las autoridades competentes para realizar el mencionado proceso, para, finalmente, mostrarnos una probabilidad de reforma en la metodología de renovación de plazos de concesión basada en determinados principios, así como una otorgarnos una propuesta de modificación.

Die Rindertuberkulose ist eine chronische Erkrankung, die von Mycobacterium (M.) bovis und M. caprae, Mitgliedern des Mycobacterium-tuberculosis-Komplex (MTC), ausgelöst wird. Tuberkulose-Erregern werden sowohl mittels kultureller als auch molekulare Untersuchungsmethoden nachgewiesen. Ziel der vorliegenden Studie war es, die Sensitivität des DNA-Nachweises von Tuberkulose-Erregern zu steigern. Dafür wurden drei Fragestellung im Bereich der molekularen Mykobakterien-Diagnostik bearbeitet. I) Zur Verbesserung der Lyse der mykobakteriellen Zellwand als Voraussetzung für eine Zunahme der Freisetzung von DNA wurden im Vergleich zu einer standardisierten DNA-Isolierungsmethode vier verschiedene Lyseprotokolle (thermische, enzymatische, thermo-enzymatische und mechanische Lyse) entwickelt und mit M. bovis BCG durchgeführt. Die Verbesserung wurde anhand der cycle threshold (Ct)-Werte einer MTC-spezifischen Real-Time (rt) Polymerase-Kettenreaktion (PCR) geprüft. Zwei Lyseprotokolle (thermische und mechanische Lyse) wurden bei zehn Gewebeproben (Lymphknoten, Leber und Lunge) von zehn Tieren (acht Rinder, ein Lama und ein Luchs) mit nachgewiesener Tuberkulose ange-wendet. II) Ausserdem, wurde eine rt-PCR mit dem 16S rRNA Gen als Zielgen (16S-rt-PCR) für den direkten Nachweis von Erregern der Gattung Mycobacterium im Gewebe entwickelt. III) Ein neu entwickelter Spoligotyping-Microarray wurde mit der konventionellen Spoligoty-ping-Methode verglichen, um die neue Methode in Bezug die Sensitivität des Nachweises und des diskriminatorischen Potenzials direkt bei infizierten Gewebeproben zu analysieren. Bei der konventionellen Methode erfolgt die Hybridisierung des PCR-Produktes auf einer Nylon Membran, auf der spezifische Oligonukleotide fixiert sind. Bei der Microarray-Methode sind diese auf einem Microarray-Chip fixiert. Die Ergebnisse der Untersuchungen (I) zur Lyse der Zellwand bei M. bovis BCG zeigten, dass bei der mechanischen Lyse eine Zunahme um 14 % und bei der thermischen Lyse eine Zunahme an PCR-Produkt von 6 % im Vergleich zur Standardlyse erbrachte. Bei beiden Lyseprotokollen wurde eine statistische Signifikanz von α = 1 % (Mann-Whitney-Test) im Vergleich zur Standardlyse errechnet. Bei den tuberkulösen Gewebeproben wurde bei der mechanischen Lyse eine durchschnitliche Zunahme an PCR-Produkt um circa 9 % im Vergleich zur Standardlyse erzielt. Dieser Unterschied war jedoch auf Grund der geringen Probeanzahl nicht statistisch signifikant. II) Bei der Untersuchung von 43 Mykobakterien-Spezies, sechs Mitgliedern des MTC (unter anderen M. bovis BCG) und 37 Non Tuberculous Mycobacteria (NTM) Spezies, konnten alle mit der entwickelten Real-Time PCR (16S-rt-PCR) nachgewiesen werden. DNA-Extrakte von acht nicht zur Gattung Mycobacterium gehörenden Spezies wurden mit der 16S-rt-PCR nicht erfasst. Ein Erreger der Gattung Gordonia und ei-ner der Gattung Rhodococcus wurden auf Grund ihres engen Verwandtschaftsgrades jedoch ebenfalls mit der 16S-rt-PCR detektiert. Die oben erwähnten mittels MTC-spezifischer rt-PCR (Zielgen IS 1081) als infiziert identifizierten zehn Gewebeproben, wurden mittels 16S-rt-PCR untersucht. Die Ergebnisse zeigten, dass beiden rt-PCR Systeme eine vergleichbare Sensitivität aufwiesen. III) Bei dem Vergleich zwischen den Spoligotyping-Methoden zeigte sich die neue Methode um einen Faktor von 100 bei der M. bovis BCG-Reinkultur und um einen Fak-tor von 10 bei DNA-Extrakten aus tuberkulösen Gewebeproben sensitiver als die konventionelle Methode. Im Rahmen dieser Arbeit hat sich der Einsatz der mechanischen Lyse für die Verbesserung der Freisetzung von mykobakterieller DNA als routinefähig erwiesen. Die entwickelte 16S-rt-PCR erwies sich als brauchbare Methode für den Nachweis von Erregern der Gattung Mycobacterium. Die Microarray-Methode stellte sich wesentlich einfacher, sensitiver und schneller dar als die konventionelle Methode. Zusammenfassend kann gesagt werden, dass alle drei Ansätze dieser Arbeit einen Beitrag zur Verbesserung der molekularen Labordiagnostik der Tuberkulose leisten. / Bovine tuberculosis is a chronic disease, that results from infection of Mycobacterium (M.) bovis and M. caprae, members of Mycobacterium tuberculosis complex (MTC), respectively. The laboratory diagnosis of bovine tuberculosis is possible with culture as well as considerate fast molecular methods. The aim of this study was to improve the sensitivity of DNA detec-tion of tuberculosis-causing pathogens. Therefore, three different issues were addressed in the complex molecular procedure targeted. I) four different lytic protocols (thermal, enzymatic, thermo-enzymatic and mechanical lysis) were developed and compared to a standardized DNA isolation protocol that was performed on pure culture of Mycobacterium (M.) bovis BCG in order to improve the mycobacterial cell wall lysis leading to an increase of DNA release. The efficiencies of the lysis protocols were assessed by the resulting cycle threshold (Ct) values of a MTC-specific real time (rt) Polymerase Chain Reaction (PCR). Two lysis protocols (thermal and mechanical) were selected to fur-ther testing of ten tuberculosis-infected tissue samples (lymph nodes, liver and lungs) from ten animals (eight cattle, one lama and one lynx). II) In addition, a real time PCR using the 16S rRNA gene as target sequence was developed, which is also suitable to detect pathogens of Genus Mycobacterium on tissue samples. III) A comparison of a newly developed microarray and the conventional spoligotyping method was realised, to analyse the applicability of the microarray in relation of the sensitivity of the method and to analyse discriminatory potential directly from infected tissue samples. During the conventional spoligotyping method, the PCR product is hybridized with specific oligonucleotides, fixed on a nylon membrane. Using the newly developed method these oligonucleotides are fixed on a microarray-chip. The results I) of the mycobacterial cell wall lysis experiment with pure culture of M. bovis BCG showed an increase of 14 % by using mechanical lysis and an increase by 6 % of the PCR product by using thermal lysis compared to the standard protocol. Using the mechanical lysis as well as the thermal lysis a statistically significant (α = 1 % (Mann-Whitney-Test)) im-provement compared to the standard lysis was achieved. Mechanical lysis was performed on tuberculous tissue samples and the results of the lysis were improved by 9 % compared to the standard lysis. However, the difference between mechanical and standard lysis was not statistically significant due the small sample number. II) Forty-three different mycobacterial species, six members of MTC (among them M. bovis BCG) and 37 Non Tuberculous Mycobacteria (NTM), were detected using the newly developed real time PCR (16S-rt-PCR). Eight non-mycobacterial species were not detected using this rt-PCR, whereas one of genus Rhodococcus and one of genus Gordonia were detected by the 16S-rt-PCR due to their close genetic similarity to the genus Mycobacterium. The ten tuberculosis-infected tissue samples (see above) testing positive using a MTC-specific real time rt-PCR (target gene IS 1081) were sub-jected to the 16S-rt-PCR. Both rt-PCR systems showed a comparable sensitivity. III) By comparing the two spoligotyping-methods, the ArrayStrip™–format method was more sensitive than the conventional method by a factor of 100 applied to pure culture and by a factor of 10 when applied to DNA extracts from infected tissue samples. In conclusion, the mechanical lysis proved to be a practical method to liberate mycobacterial DNA. The newly developed rt-PCR was suitable to detect members of the genus Mycobacterium. The spoligotyping ArrayStrip™–format method appeared to be substantially easier to perform, more sensitive, and less time-consuming than the conventional method. The three methods described were suitable to improve the molecular laboratory diagnosis of tuberculosis.