Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases. The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of Lactobacillus spp. in fecal samples retrieved from a study of the probiotic strain L. reuteri ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained L. reuteri belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.

Die DNA-Sequenzierung nimmt in der molekulargenetischen Diagnostik seit vielen Jahren einen großen Stellenwert ein. Zeit- und kosteneffektivere Methoden wie die DHPLC wurden seither für verschiedene Gene etabliert. Ziel dieser Arbeit war die Etablierung der DHPLC für das ATRX-Gen bei Patienten mit Verdacht auf das ATRX-Syndrom. Nach erfolgreicher Etablierung der DHPLC sollten im Rahmen dieser Arbeit 38 Patientenproben des Instituts für Humangenetik der Universität Leipzig mit Verdacht auf das ATRX-Syndrom mittels DHPLC und Sequenzierung untersucht werden. Die Etablierung der DHPLC gelang in der vorliegenden Arbeit für alle - das ATRX-Gen vollständig umspannenden - 42 Fragmente. Jede der vorliegenden Sequenzvariationen konnte nach Abschluss der Arbeit detektiert werden. Unter Verwendung von Maxima® stellten sich initial 22 von 24 verschiedenen Sequenzvariationen zum Wildtyp different dar. Die verbleibenden zwei Mutationen p.R246C und p.A238P im Fragment 9 wurden unter Verwendung höherer Temperaturen, eines kürzeren Fragmentes oder anderer Polymeraseenzyme (AmpliTaq Gold® bzw. HighFidelity) detektiert. Nach Abschluss der Etablierung der DHPLC konnte eine Sensitivität und Spezifität von 100% erreicht werden. In den Patientenuntersuchungen des Instituts für Humangenetik der Universität Leipzig fanden sich bei 38 Patientenproben vier verschiedene als benigne beschriebene Polymorphismen bei insgesamt 19 Patienten, ein noch nicht veröffentlichter Polymorphismus im Intron 26 sowie eine bis dato nicht beschriebene und als pathogen einzustufende Mutation im Exon 34. In 100 Kontrollen der DNA-Bank des Instituts für Humangenetik der Universität Leipzig konnte der bisher nicht publizierte Polymorphismus im Intron 26 sieben Mal gefunden werden. Die bis dato nicht beschriebene Deletion p.2385_2395del im Exon 34 führt zu einem vorzeitigen Abbruch des ATRX-Proteins und ist somit als sicher pathogen einzustufen. Die Untersuchung der Mutter des Patienten konnte den Konduktorenstatus nachweisen. Für die Familie des betroffenen Patienten konnte mit der vorliegenden Arbeit die Diagnose des ATRX-Syndroms gesichert werden. Die DNA-Proben der Patienten bei denen keine Mutation nachgewiesen werden konnte, sollten bei weiterhin dringendem Verdacht auf das ATRX-Syndrom mittels qRT-PCR bzw. MLPA untersucht werden, um große Deletionen, Insertionen oder Duplikationen auszuschließen. Mithilfe dieser Arbeit gelang die Etablierung der DHPLC für das ATRX-Gen als ökonomische, sehr sensitive und effiziente Methode zur Diagnostik des ATRX-Syndroms. Die Entscheidung, in welcher Form und mit welcher Methode DNA-Proben bei Verdacht auf ATRX-Syndrom untersucht werden, bleibt jedoch eine individuelle Entscheidung jedes Instituts unter Betrachtung der jeweiligen Gegebenheiten vor Ort.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Extensive research on amino acids, and even other biochemical assays usually present in low concentration and volume face challenges using known analytical techniques for analysis of traces amounts. Some limiting factors are the achievable efficiency, sensitivity (resulting from instrument limit of detection and/or experimental methods), volume requirement, and total analysis time. Counterflow electrofocusing techniques combining forces of electrophoresis and bulk flow (pressure driven flow and/or electroosmotic flow) provides a basis for the development of alternative detection techniques geared towards improving peak efficiency, sensitivity and time. The work presented gives a vivid description of recently developed capillary counterflow techniques: gradient elution isotachophoresis (GEITP) using UV detection, GEITP coupled to Capillary Zonal Electrophoresis (GEITP-CZE), temperature gradient focusing (TGF), and temperature gradient denaturing focusing (TGDF). A first demonstration of GEITP using UV detection was applied to enrichment and separation of tyrosine and tryptophan under optimized conditions. Primarily, separation is achieved as the result of the difference in electrophoretic velocity of analytes in a discontinuous buffer system. First, a plug of sample is allowed to preconcentrate (or enrich) between high mobility leading electrolyte (LE) and low mobility trailing electrolyte (TE) under controlled hydrodynamic pressure and continuous injection. This preconcentration is initiated outside the capillary in a conductivity bubble. Although analyte focus according to their electrophoretic velocity, the inclusion of spacer molecule in sample matrix was instrumental in achieving separation with tradeoff between analyte resolution and enrichment. Gradient produced results from reduction in pressure as sample is loaded on column. Separation using this technique is a one step process. A hybrid method marking the first successful coupling of GEITP to CZE with laser induced fluorescence detection was used for separation of six fluorescently labeled amino acids (which formulates the Mars-7). An eleven minute separation was achieved under optimized conditions. A proof-of-concept demonstration of TGF with LIF detection showed focusing and separation of fluorescein and carboxyfluorescein dye molecules, and carboxyfluorescein-labeled glutamate and aspartate. The generation of null focusing points along the thermal separation column (set between 80-20oC) was produced in collaboration with continuous sample injection, discontinuous buffer system and balancing of counterflows (electrophoresis and bulk flow). Preliminary results showed stability in instrument. The TGDF method carried out on a TGF apparatus is a modification to the temperature gradient gel electrophoresis and denaturing gradient gel electrophoresis methods. In principle, TGDF primarily achieves focusing and separation on a thermal separation column (set between 20 to 80 oC) as a result of conformational changes. It is currently being developed for the detection and simultaneous separation of single and double stranded DNA. Preliminary results show enrichment of wildtype and mutant synthetic DNA strands (containing twenty-four base pairs in sequence) in different buffer matrices. / Chemistry

A imunidade passiva surge como uma alternativa para a manipulação da fermentação ruminal e anticorpos de origem aviária contra bactérias específicas começam a ser pesquisados. Objetivou-se com este trabalho avaliar um preparado de anticorpos policlonais contra Streptococus bovis, Fusobacterium necrophorum e algumas cepas de bactérias proteolíticas (Peptostreptococcus anacrobius, Clostridium aminophilum e Clostridium sticklandii) sobre a diversidade microbiana ruminal e degradabilidade in situ de alguns alimentos. Foram utilizadas nove vacas mestiças portadoras de cânula ruminal. O delineamento experimental foi o quadrado latino 3 x 3 replicado 3 vezes, com arranjo fatorial de tratamentos 3 x 3 referente a 2 modificadores ruminais representados pela monensina (MON) e pelo preparado de anticorpos policlonais (PAP) mais o grupo controle e 3 fontes energéticas suplementadas na dieta, representadas pelo milho seco moído (MSM), silagem de grão úmido de milho (SGUM) e polpa cítrica (PC). Cada subperíodo experimental foi composto de 21 dias, sendo 16 dias para adaptação aos tratamentos e 5 para coleta de dados. A degradabilidade in situ dos alimentos testados foi mensurada através da técnica de saco de náilon. A coleta de amostras para a análise quantitativa de protozoários ocorreu no 21° dia de cada período, às 0 e 4 horas pós-alimentação, sendo estas coletadas por varredura do assoalho ruminal. O conteúdo ruminal foi coletado no 21° dia de cada período, às 4 h pós-alimentação, para análise da diversidade microbiana através de técnica de eletroforese em gel com gradiente de desnaturação. Observou-se que dietas contendo PC apresentaram aumento de 80,6%; 75,4% e 66,8% da degradabilidade efetiva da FDN da cana-de-açúcar para as taxas de passagem de 2, 5, 8%/h, respectivamente, em relação ao grupo tratado com SGUM, mas não em relação ao grupo com MSM. O tratamento com PAP demonstrou efeito sobre a fração solúvel (a) do amido do MSM, diminuindo esta em 45,26% e 45,37% em relação ao grupo CON e grupo MON, respectivamente. Observou-se que o tratamento com MON diminuiu em 16,14% o valor da fração potencialmente degradável (b) da MS da SGUM em relação ao grupo CON, mas não em relação ao grupo tratado com PAP. Já sobre a taxa de degradação (c), a MON aumentou o valor desta em 63,18% e 60,65% em relação ao grupo CON e PAP, respectivamente. Também a MON diminuiu a degradabilidade potencial (Dp) da MS da SGUM em 3,40% em relação ao grupo CON, mas não em relação ao grupo tratado com PAP. Observou-se que o PAP aumentou em 93,65% a contagem relativa de Isotricha em relação ao grupo CON, mas não em relação ao grupo MON. Já a PC aumentou em 334,42% (0h) e 399,75% (4h) a contagem relativa de protozoários do gênero Isotricha em relação às dietas de MSM e SGUM. Observou-se que o tratamento com PC aumentou em 52% a contagem do número de bandas em DGGE para a comunidade Archaea, em relação ao grupo MSM, sem diferir do grupo SGUM. Em linhas gerais, no presente experimento, não foi possível atribuir um padrão na estrutura de amplificação das comunidades Bacteria ou Archaea do conteúdo ruminal de animais tratados com dois diferentes modificadores ruminais ou 3 fontes energéticas distintas. / Passive immunity arises as an alternative for ruminal fermentation manipulation and aviary antibodies against specific bacteria starts to be studied. The objective of the present study was to evaluate a polyclonal antibody preparation (PAP) against Streptococus bovis, Fusobacterium necrophorum and some proteolytic bacteria (Peptostreptococcus anacrobius, Clostridium aminophilum and Clostridium sticklandii) on ruminal microbial community diversity and in situ degradability of some feedstuffs. Nine ruminally fistulated cows were used in a latin square 3 x 3 replicated 3 times with factorial arrangement of treatments 3 x 3 regarding to two rumen modifiers [monensin (MON) and (PAP) plus a control group (CON)] and three energetic sources supplemented in the diet represented by the dry-grounded corn grain (CG), high moisture corn silage (HMCS) and citrus pulp (CiPu). Each trial lasted 21 days where 16 days were for treatments adaptation and 5 for data collection. In situ degradability of the experimental diets was measured by nylon bag in situ technique. The collection of samples for quantitative protozoa analysis occurred on day 21 of each trial at 0 and 4 h after feeding by scanning the ruminal floor. The ruminal content was collected in the day 21 of each trial at 0 and 4 h after feeding for the analysis of microbial ruminal diversity by the denaturing gradient gel electrophoresis. Diets with CiPu presented an increase of 80.6%; 75.4% and 66.8% in effective degradability of NDF of sugar cane for outflow rates of 0.02, 0.05, 0.08/h, respectively, in relation to group treated with HMCS but not to the group CG. The group treated with PAP showed effect on soluble fraction (a) of starch of CG decreasing it in 45.26% and 45.37% in relation to CON and MON group respectively. It was observed that the treatment with MON decreased in 16.14% the value of potentially soluble fraction (b) of DM from HMCS in relation to CON group but not in relation to PAP. For the degradation rate (c) the MON increased it values in 63.18% and 60.65% in relation to CON and PAP group respectively. Also, MON treatment decreased potential degradability (Pd) of DM of HMCS in 3.40% in relation to CON group but not to PAP. It was observed that PAP treatment increased in 93.65% the relative counting of Isotricha in relation to CON group but not in relation to MON group. It was observed that CiPu diet increased in 334.42% (0h) and 399.75% (4h) the relative counting of Isotricha in relation to CG and HMCS. It was observed that CiPu increased in 52% the counting of the numbers of bands in DGGE for Archaea community in relation to CG without difference to HMCS group. In general lines, in the present experiment, it was not possible to assign that there was a pattern in the structures of amplification by Bacteria and Archaea communities of the ruminal content of animals treated with two different rumen modifiers or three distinct energetic sources.

Ziel der Arbeit war die Nutzbarmachung von Bioethanol zur Wandlung in Strom und Wärme in einer Hochtemperaturbrennstoffzelle. Dazu waren neben der Entwicklung eines langzeitstabilen, effektiven Katalysators zur Synthesegaserzeugung und dessen Testung sowie der Übertragung gewonnener Erkenntnisse auf in einem Reformer einsetzbare Konzepte die Verifizierung kommerzieller Katalysatorsysteme für die partielle Oxidation von Ethanol notwendig. Außerdem ist für die Entwicklung eines ethanolbetriebenen SOFC-Systems eine pulsations- und ablagerungsfreie Verdampfung von unvergälltem und vergälltem Ethanol – welche nicht Stand der Technik ist – erforderlich, für die ein geeignetes Verdampferkonzept entwickelt und getestet wurde. Experimentell konnte die Betreibbarkeit eines SOFC-Systems mit Ethanol an einem für den Betrieb mit LPG ausgelegten System nachgewiesen werden.

In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.

A imunidade passiva surge como uma alternativa para a manipulação da fermentação ruminal e anticorpos de origem aviária contra bactérias específicas começam a ser pesquisados. Objetivou-se com este trabalho avaliar um preparado de anticorpos policlonais contra Streptococus bovis, Fusobacterium necrophorum e algumas cepas de bactérias proteolíticas (Peptostreptococcus anacrobius, Clostridium aminophilum e Clostridium sticklandii) sobre a diversidade microbiana ruminal e degradabilidade in situ de alguns alimentos. Foram utilizadas nove vacas mestiças portadoras de cânula ruminal. O delineamento experimental foi o quadrado latino 3 x 3 replicado 3 vezes, com arranjo fatorial de tratamentos 3 x 3 referente a 2 modificadores ruminais representados pela monensina (MON) e pelo preparado de anticorpos policlonais (PAP) mais o grupo controle e 3 fontes energéticas suplementadas na dieta, representadas pelo milho seco moído (MSM), silagem de grão úmido de milho (SGUM) e polpa cítrica (PC). Cada subperíodo experimental foi composto de 21 dias, sendo 16 dias para adaptação aos tratamentos e 5 para coleta de dados. A degradabilidade in situ dos alimentos testados foi mensurada através da técnica de saco de náilon. A coleta de amostras para a análise quantitativa de protozoários ocorreu no 21° dia de cada período, às 0 e 4 horas pós-alimentação, sendo estas coletadas por varredura do assoalho ruminal. O conteúdo ruminal foi coletado no 21° dia de cada período, às 4 h pós-alimentação, para análise da diversidade microbiana através de técnica de eletroforese em gel com gradiente de desnaturação. Observou-se que dietas contendo PC apresentaram aumento de 80,6%; 75,4% e 66,8% da degradabilidade efetiva da FDN da cana-de-açúcar para as taxas de passagem de 2, 5, 8%/h, respectivamente, em relação ao grupo tratado com SGUM, mas não em relação ao grupo com MSM. O tratamento com PAP demonstrou efeito sobre a fração solúvel (a) do amido do MSM, diminuindo esta em 45,26% e 45,37% em relação ao grupo CON e grupo MON, respectivamente. Observou-se que o tratamento com MON diminuiu em 16,14% o valor da fração potencialmente degradável (b) da MS da SGUM em relação ao grupo CON, mas não em relação ao grupo tratado com PAP. Já sobre a taxa de degradação (c), a MON aumentou o valor desta em 63,18% e 60,65% em relação ao grupo CON e PAP, respectivamente. Também a MON diminuiu a degradabilidade potencial (Dp) da MS da SGUM em 3,40% em relação ao grupo CON, mas não em relação ao grupo tratado com PAP. Observou-se que o PAP aumentou em 93,65% a contagem relativa de Isotricha em relação ao grupo CON, mas não em relação ao grupo MON. Já a PC aumentou em 334,42% (0h) e 399,75% (4h) a contagem relativa de protozoários do gênero Isotricha em relação às dietas de MSM e SGUM. Observou-se que o tratamento com PC aumentou em 52% a contagem do número de bandas em DGGE para a comunidade Archaea, em relação ao grupo MSM, sem diferir do grupo SGUM. Em linhas gerais, no presente experimento, não foi possível atribuir um padrão na estrutura de amplificação das comunidades Bacteria ou Archaea do conteúdo ruminal de animais tratados com dois diferentes modificadores ruminais ou 3 fontes energéticas distintas. / Passive immunity arises as an alternative for ruminal fermentation manipulation and aviary antibodies against specific bacteria starts to be studied. The objective of the present study was to evaluate a polyclonal antibody preparation (PAP) against Streptococus bovis, Fusobacterium necrophorum and some proteolytic bacteria (Peptostreptococcus anacrobius, Clostridium aminophilum and Clostridium sticklandii) on ruminal microbial community diversity and in situ degradability of some feedstuffs. Nine ruminally fistulated cows were used in a latin square 3 x 3 replicated 3 times with factorial arrangement of treatments 3 x 3 regarding to two rumen modifiers [monensin (MON) and (PAP) plus a control group (CON)] and three energetic sources supplemented in the diet represented by the dry-grounded corn grain (CG), high moisture corn silage (HMCS) and citrus pulp (CiPu). Each trial lasted 21 days where 16 days were for treatments adaptation and 5 for data collection. In situ degradability of the experimental diets was measured by nylon bag in situ technique. The collection of samples for quantitative protozoa analysis occurred on day 21 of each trial at 0 and 4 h after feeding by scanning the ruminal floor. The ruminal content was collected in the day 21 of each trial at 0 and 4 h after feeding for the analysis of microbial ruminal diversity by the denaturing gradient gel electrophoresis. Diets with CiPu presented an increase of 80.6%; 75.4% and 66.8% in effective degradability of NDF of sugar cane for outflow rates of 0.02, 0.05, 0.08/h, respectively, in relation to group treated with HMCS but not to the group CG. The group treated with PAP showed effect on soluble fraction (a) of starch of CG decreasing it in 45.26% and 45.37% in relation to CON and MON group respectively. It was observed that the treatment with MON decreased in 16.14% the value of potentially soluble fraction (b) of DM from HMCS in relation to CON group but not in relation to PAP. For the degradation rate (c) the MON increased it values in 63.18% and 60.65% in relation to CON and PAP group respectively. Also, MON treatment decreased potential degradability (Pd) of DM of HMCS in 3.40% in relation to CON group but not to PAP. It was observed that PAP treatment increased in 93.65% the relative counting of Isotricha in relation to CON group but not in relation to MON group. It was observed that CiPu diet increased in 334.42% (0h) and 399.75% (4h) the relative counting of Isotricha in relation to CG and HMCS. It was observed that CiPu increased in 52% the counting of the numbers of bands in DGGE for Archaea community in relation to CG without difference to HMCS group. In general lines, in the present experiment, it was not possible to assign that there was a pattern in the structures of amplification by Bacteria and Archaea communities of the ruminal content of animals treated with two different rumen modifiers or three distinct energetic sources.

Tumors are one of the leading causes of death worldwide. Generally, the prognosis is better if the treatment begins at an early stage. Nowadays, the conventional chemotherapy treatment of cancer, known for its limited efficacy and side effects, is being gradually replaced by targeted biological treatment, which is used when specific genetic mutations are found. A part of the treatment is a detection of a potential progression, which is mainly based on the tumor biomarkers monitoring. Currently, further investigation of a so-called liquid biopsy method are ongoing, on which this thesis is focused. The main aim of this work was the experimental development and validation of the method for detection of the ctDNA in the plasma samples based on the somatic mutations presence. For the development and optimization of the system on the principle of denaturation capillary electrophoresis, the samples of cancer patients with KRAS mutation were used. Subsequently, a clinical part of the research was performed on a pilot set of 21 plasma samples. Finally, the method was optimized for the detection of BRAF and EGFR markers. A partial objective was to improve the detection sensitivity and increase the capture of the ctDNA in patients with advanced stage of the disease. The results of this work suggest the...

El presente trabajo de investigación tiene como objetivo principal analizar la problemática detectada en el Instituto Nacional de Radio y Televisión del Perú – IRTP respecto de la interposición de demandas judiciales en materia de desnaturalización de contratos, con el fin de proponer la implementación de mecanismos que permitan disminuir la incidencia de ese tipo de demandas en el futuro. La importancia de esta investigación radica en la posibilidad de demostrar que el uso de las órdenes de servicio para contratar personas que brinden servicios a favor de la entidad, así como las acciones realizadas por las diversas áreas usuarias de dichos servicios generan que los juzgados laborales acojan las pretensiones de los demandantes y por ende se declare la existencia de un contrato de trabajo, lo que se traduce en el pago de cuantiosas sumas de dinero por concepto de pago de sentencias judiciales. Para realizar el presente trabajo, se consultó literatura con el objeto de presentar los elementos teóricos del contrato de trabajo, así como la desnaturalización de contratos. Asimismo, se procedió a revisar procesos judiciales sobre la materia analizada notificados en los años 2018 y 2019. Con los resultados obtenidos se propone establecer lineamientos para mejorar el uso de las órdenes de servicio, así como un proceso donde se pueda verificar el cumplimiento de los mismos. / The main objective of this research work is to analyze the problem detected in the National Institute of Radio and Television of Peru - IRTP regarding the filing of legal claims regarding the denaturing of contracts, in order to propose the implementation of mechanisms that allow reduce the incidence of such lawsuits in the future. The importance of this research lies in the possibility of demonstrating that the use of service orders to hire people who provide services in favor of the entity, as well as the actions carried out by the various user areas of said services, generate that the labor courts accept the claims of the plaintiffs and therefore the existence of an employment contract is declared, which translates into the payment of large sums of money for the payment of court judgments. To carry out this work, literature was consulted in order to present the theoretical elements of the employment contract, as well as the denaturing of contracts. Likewise, we proceeded to review judicial processes on the analyzed matter notified in 2018 and 2019. With the results obtained, it is proposed to establish guidelines to improve the use of service orders, as well as a process where they can be verified. / Tesis