Schmerz gehört zu den Kardinalsymptomen einer Entzündung. Im Wesentlichen kann die Entstehung von Schmerz am Ort des Entzündungsgeschehens auf das Einwandern (Diapedese) von Leukozyten aus dem peripheren Blut-strom in das Gewebe zurückgeführt werden. Dort findet sowohl die Produktion von Zytokinen und Chemokinen statt, welche weitere Entzündungszellen rekrutieren und die Entzündungsreaktion verstärken, als auch die Freisetzung von Opioidpeptiden, die schmerzlindernd wirken. In Vorarbeiten der Arbeitsgruppe konnte eine Opioidfreisetzung aus neutrophilen Granulozyten nach Stimulation mit bakteriellen Antigenen oder Chemokinen \(in\) \(vitro\) nachgewiesen werden. Diese führen \(in\) \(vivo\) eine Antinozizeption herbei. Für neutrophile Granulozyten wurden der Chemokinrezeptor CXCR1/2 sowie der Formylpep-tidrezeptor als Signal-transmittierende Rezeptoren identifiziert. Über den klassischen Mechanismus der Exozytose gelangt das Beta-Endorphin somit in das Gewebe und interagiert mit Opioidrezeptoren auf primär sensorischen Nervenendigungen. \(in\) \(vivo\) äußerte sich die Freisetzung des Opioidpeptids in einer Anhebung mechanischer Schmerzschwellen, die durch den Opioidrezeptorantagonisten Naloxon aufgehoben werden konnten. Die Bindung, vornehmlich an MOP, führt zur Erniedrigung des cAMP-Spiegels, zur Hyperpolarisation der Nervenzelle und zur Verminderung von Schmerzschwellen. Im Mittelpunkt dieser Arbeit stehen Monozyten als führende Zellpopulation der späten Entzündungsphase. Es sollte untersucht werden, welche Rezeptoren eine Opioidfreisetzung aus Monozyten vermitteln sowie welche intrazellulären Signalwege involviert sind. Humane Monozyten wurden isoliert und \(in\) \(vitro\) mit dem bakteriellen Antigen Lipopolysaccharide (LPS) stimuliert. Dieses steht exemplarisch für mikrobielles Infektgeschehen und Entzündung. In den Zellüberständen wurde mittels ELISA die Beta-Endorphin-Konzentration ermittelt. Weiterhin wurden Opioidgehalt und -freisetzung in der nicht-klassischen CD14+CD16+ Monozytensubpopulation im Vergleich zu klassischen CD14+CD16- Monozyten analysiert. Zur weiteren Aufklärung des Rezeptors, welcher die Opioidfreisetzung vermittelt, wurde der niedermolekulare TLR4-Antagonist TAK-242 genutzt. Wir fanden eine Zunahme der Beta-Endorphin-Freisetzung nach Stimulation mit LPS im Vergleich zur unstimulierten Kontrolle. Eine Zugabe des TLR4-Inhibitors reduzierte die Beta-Endorphin-Freisetzung signifikant. TLR4 agiert somit als PRR für die Opioidfreisetzung aus Monozyten. CD14+CD16+ Monozyten enthalten einen geringeren Anteil an Beta-Endorphin und setzten dementsprechend weniger frei. Ihre Rolle als pro-inflammatorisch und ihre Beteiligung an der Genese inflammatorischer Krankheitsbilder wird dadurch gestützt. Die Signalkaskade, über die diese Freisetzung erfolgt, konnte durch den Einsatz von Rezeptorinhibitoren dahingehend entschlüsselt werden, dass eine Beteiligung des IP3-Rezeptors sowie von intrazellulärem Calcium wichtig ist. Ferner wurde evident, dass auch eine basale Freisetzung existiert, die über denselben Weg verläuft. Durch die Behandlung mit dem TLR4-Antagonisten TAK-242, der die Freisetzung von Beta-Endorphin \(in\) \(vitro\) unterdrückt, wird auch die analgetische Wirkung von LPS \(in\) \(vivo\) aufgehoben. TLR4 Agonisten sind daher potentielle alternative Analgetika, welche die endogene Schmerzkontrolle unterstützen könnten. Jedoch fließen viele Wechselwirkungen wie z.B. proalgetische Wirkungen von TLR4 in das komplexe Gefüge der Immunzellantwort ein. Diese wurden nicht weiter untersucht. Vor einer klinischen Anwendung müssten solche Effekte näher betrachtet werden. / Endogenous opioids from monocytes mediate tonic endogenous antinociception in the late phase of inflammation. Monocytes expressing TLR4 dose-dependently released \(\beta\)-END after stimulation with lipopolysaccharide (LPS) dependent on intracellular calcium. \(in\) \(vitro\) \(\beta\)-endorphin (\(\beta\)-END) content increased during human monocyte differentiation as well as in anti-inflammatory CD14\(^+\)CD16\(^-\) monocytes. Peripheral TLR4 acts as a counter-regulatory mechanism for inflammatory pain \(in\) \(vivo\), and increases the release of opioid peptides from monocytes \(in\) \(vitro\). TLR4 antagonists as new treatments for sepsis and neuropathic pain might unexpectedly enhance pain by impairing peripheral opioid analgesia.

IRAK2 besitzt eine Schlüsselrolle im Signalweg des TLR4. Fehlregulationen dieses Signalwegs führen zu fehlgeleiteten Immunreaktionen, die auch die Entstehung und Progression von Krebserkrankungen fördern. Bevor IRAK2 als therapeutisches Ziel in Frage kommen kann, muss erst noch weitere Klarheit über die grundsätzliche Funktionsweise dieses Proteins bestehen. So ist für IRAK2 aufgrund der Substitution einer Aminosäure in der Kinase-Domäne im Vergleich zu IRAK1 noch nicht abschließend geklärt, ob es sich um eine aktive Kinase oder eine Pseudokinase handelt und ob diese Veränderung eine Erhöhung oder eine Erniedrigung der Funktion im TLR4-Signalweg nach sich zieht. Um diese Fragen anzugehen, wurde in dieser Arbeit Asparagin im vermeintlich aktiven Zentrum (Aminosäure 333) wieder zur Asparaginsäure [N333D] revertiert und damit versucht die Phosphorylierungsaktivität zu steigern bzw. vergleichbar zu IRAK1 wiederherzustellen. Das Einbringen der Mutation in IRAK2 erfolgte mittels ortsspezifischer Mutagenese. Mit dieser und anderen Mutanten und mit wildtypischem IRAK2 wurden durch die CRISPR/Cas9-Methode generierte IRAK2-defiziente 264.7 Makrophagen rekonstituiert und damit ein System etabliert, mit dem der Einfluss der Mutation auf den Signalweg des TLR4 nach Stimulation mit LPS quantitativ analysiert werden konnte. Sowohl die indirekte NF-κB-Messung über CD40-Expression als auch die direkte NF-κB-Messung über die NF-κB-getriebene Expression eines Reportergens (cyan fluorescent protein) ergab, dass IRAK2[N333D] die LPS-abhängige NF-κB-Aktivierung über den TLR4 Signalweg schlechter ermöglicht als IRAK2. Insgesamt deuten die Ergebnisse darauf hin, dass die in der Entwicklungsgeschichte aufgetretene Veränderung des aktiven Zentrums von IRAK2 im Vergleich zu IRAK1 zu einer besseren Aktivierung der MyD88-abhängigen NF-κB-Aktivität führte und somit eine erhöhte und länger anhaltende Signalleitung ermöglichte. Diese Erkenntnis kann als weiterer Schritt hin zu einem besseren Verständnis der Funktion des IRAK2-Proteins und zu einer möglichen zukünftigen Verwendung von IRAK2 als Ziel therapeutischer Behandlungen gesehen werden. / Toll-like receptors are fundamental for many immune cells. The protein IRAK2 plays a key role in the signaling pathway downstream of TLR4 and other TLRs. Due to a change at amino acid position 333 of the protein from aspartate (D) to asparagine (N) in the presumed active center of the kinase it is unclear if IRAK2 is an active kinase. In this research the mutation IRAK2-N333D was investigated thereby trying to reconstitute the evolutionarily original version of the protein. Interestingly, overexpression of IRAK2-N333D in IRAK2-deficient RAW 264.7 cells obtained by using the CRISPR/Cas9-system was less effective in activating LPS-induced CD40 expression and NF-κB activity as compared to wild type IRAK2. Our results suggest that the evolutionary alteration in the "catalytic center" of IRAK2 led to improved signal transduction thereby allowing longer-lasting activation of the cells. This knowledge might help to better target IRAK2 in therapies.

Grado de magíster en fisiología / La obesidad, condición caracterizada por la presencia de un estado inflamatorio crónico, está estrechamente relacionada al desarrollo de diabetes tipo 2 (DM2), patología de alta prevalencia en individuos sedentarios. Se sabe que la actividad física regular es una de las medidas más eficaces para evitar los efectos deletéreos asociados a la obesidad y DM2, ya que además de reducir la actividad proinflamatoria, induce un ambiente antiinflamatorio. Proponemos en nuestra investigación que el entrenamiento de fortalecimiento muscular generará una disminución de la respuesta inflamatoria en la que participa el receptor de respuesta inmune TLR9, en un modelo de ratas obesas y DM2. Para tales efectos 8 ratas Wistar, macho, adultas, obesas y diabéticas fueron sometidas a un programa de entrenamiento de fortalecimiento muscular por 12 semanas (grupo entrenado (GE)), mediante el agarre, en sentido vertical, a una rejilla, por un tiempo predeterminado y con una carga externa sujeta a sus colas. 7 ratas de iguales características, no sometidas al entrenamiento, sirvieron como grupo control (GC). Al término del programa, se evaluó la fuerza muscular (tiempo máximo de agarre (segundos) sin carga externa) en todas las ratas. Luego, estas fueron sometidas a eutanasia y se cuantificó en el bazo la expresión del mRNA de genes implicados en la cascada de señalización de TLR9 y en la inducción de citokinas pro y antiinflamatorias. En el suero sanguíneo, se valoró la actividad inflamatoria sistémica por medio de la cuantificación de citokinas pro y antiinflamatorias y además, se midió la presencia de cfDNA (DNA de doble cadena, libre), ligando endógeno de TLR9. Adicionalmente, se midió masa corporal y grasa, parámetros de control glicémico y lipídico. En relación al GC, el GE mostró mayor fuerza muscular (47[16-139] segundos v/s 18 [10-22] segundos; p=0,01). Del total de ratas del GE, aquella mitad con valores de fuerza por sobre la mediana del grupo, las denominamos "respondedoras" (GR) al entrenamiento, mientras que al resto, las denominamos "subrespondedoras"(GSR). El GR mostró mayor fuerza respecto del GC y del GSR, presentando también una disminución significativa de su masa corporal respecto a los otros grupos. Además, el GR presentó menores valores en la expresión del mRNA de las citokinas proinflamatorias, TNF-α e IL-6, y de la proteína TNF-α valorada en el suero sanguíneo. No observamos modificaciones en la expresión del mRNA y proteína de la citokina antiinflamatoria IL-10. Por otra parte, se encontró en el GR una disminución significativa de la expresión de mRNA de las moléculas implicadas en la vía de señalización del receptor TLR9, así como menores concentraciones de cfDNA. Asociado a estos hallazgos, se observó en este grupo, menores valores en los parámetros metabólicos de control glicémico y mejoras en su perfil lipídico. En resumen, ratas obesas y diabéticas tratadas con un programa de entrenamiento de fortalecimiento muscular y que logran un aumento significativo en su fuerza muscular, presentan cambios también significativos en su composición corporal, marcadores metabólicos y de inflamación sistémica, así como una regulación negativa de la vía de señalización del receptor TLR9. Esto sugiere la necesidad de considerar las diferencias individuales en la capacidad de respuesta al entrenamiento de fortalecimiento muscular, al momento de su prescripción en pacientes obesos y diabéticos. / Obesity, a condition characterized by the presence of a chronic inflammatory state, is closely related to the development of type 2 diabetes (T2D), a pathology of high prevalence in sedentary individuals. It is known that regular physical activity is one of the most effective measures to avoid the deleterious effects associated with obesity and T2D, since in addition to reducing proinflammatory activity, it induces an anti-inflammatory environment. We propose in our research that muscle strengthening training will generate a decrease in the inflammatory response in which the immune response receptor TLR9 participates, in a model of obese rats and T2D. For this purpose 8 Wistar rats, male, adult, obese and diabetic rats were subjected to a muscle strengthening training program for 12 weeks (trained group (GE)), by gripping vertically to a grid, for a predetermined time and with an external load attached to their tails. 7 rats with the same characteristics, not subjected to training, served as a control group (CG). At the end of the program, muscle strength (maximum holding time (seconds) without external load) was evaluated in all rats. Then, they were euthanized and quantified in spleen the mRNA expression of genes involved in the TLR9 signaling cascade and induction of pro and anti-inflammatory cytokines. In the blood serum, the systemic inflammatory activity was assessed by means of the quantification of pro and anti-inflammatory cytokines and, in addition, the presence of cfDNA (free double-stranded DNA), endogenous ligand of TLR9, was measured. Additionally, body mass and fat, glycemic and lipid control parameters were measured. In relation to GC, the GE showed greater muscle strength (47 [16-139] seconds v / s 18 [10-22] seconds, p = 0.006). Of all the GE rats, which half with force values above the group median, we call them “responders” (GR) to training, while the rest, we call them "subresponders" (GSR). The GR showed greater strength with respect to the GC and the GSR, also presenting a significant decrease in their body mass with respect to the other groups. In addition, the GR presented lower values in the mRNA expression of the proinflammatory cytokines, TNF-αand IL-6, and TNF-α protein valued in the blood serum. We did not observe modifications in the mRNA and protein expression of the anti-inflammatory cytokine IL-10. On the other hand, a significant decrease in the mRNA expression of the molecules involved in the TLR9 signaling pathway was found in the GR, as well as lower concentrations of cfDNA. Associated with these findings, we observed in this group, lower values in the metabolic parameters of glycemic control and improvements in their lipid profile. In summary, obese and diabetic rats treated with a muscular strengthening training program that achieve a significant increase in their muscular strength, present also significant changes in their body composition, metabolic markers and systemic inflammation, as well as a negative regulation of the signaling path of the TLR9 receiver. This suggests the need to consider individual differences in the capacity to respond to muscle strengthening training, at the time of prescription in obese and diabetic patients.

<p><strong>Aim:</strong></p><p>The purpose of this study is to explore the Vietnamese market for Electronic Road Tolls. Company X is a world wide supplier of electronic toll systems. They are focusing on systems that will reduce traffic congestion in cities or congestion created by road charge toll systems. Company X has been active in the South East Asia for over 10 years. Now they want to explore new markets in this region. The company is interested in developing countries where road infrastructure is expanding. Since Vietnam is one of the fastest growing economies in Asia Company X is interested in if it is a market they should enter.</p><p> </p><p>Research questions: </p><p><em>Should Company X enter the Vietnamese market?</em></p><p><em>If yes, which entry strategy should be used entering this market?</em></p><p> </p><p><strong>Method:</strong></p><p>To analyze Company X’s internal and external environment I have used different theoretical methods, PEST analysis, Micro / Macro environmental analysis and with the SWOT framework I have summarized recommendations based on the facts I have gathered. I have also investigated different entry strategies to see what is most suitable for a Company X. I have collected data via interviews, literature and internet sources.</p><p> </p><p><strong>Result & Conclusions: </strong></p><p>Short term Company X has the opportunity to come in with their products into Vietnam with a co-operation with Competitor A. They will set up a test system at one toll plaza between the airport and Hanoi with two lane dedicated for EFC and Company X is their first choice if they are competitive. This will give them the opportunity to be first on this market to implement microwave equipment in Vietnam and an opportunity to set the standard in the country for the future. </p><p><strong>Suggestions for future research: </strong></p><p>The empirical data in this thesis is mainly gathered from secondary sources. The information would have been more accurate with more primary data.</p><p><strong> </strong></p><p><strong>Contribution of the thesis: </strong></p><p>This thesis shows that large investments in road infrastructure are coming up in Vietnam. This thesis also shows that currently there is low activity from competitors using the same technology and this was not previously known by Company X.</p>

Aim: The purpose of this study is to explore the Vietnamese market for Electronic Road Tolls. Company X is a world wide supplier of electronic toll systems. They are focusing on systems that will reduce traffic congestion in cities or congestion created by road charge toll systems. Company X has been active in the South East Asia for over 10 years. Now they want to explore new markets in this region. The company is interested in developing countries where road infrastructure is expanding. Since Vietnam is one of the fastest growing economies in Asia Company X is interested in if it is a market they should enter.   Research questions:  Should Company X enter the Vietnamese market? If yes, which entry strategy should be used entering this market?   Method: To analyze Company X’s internal and external environment I have used different theoretical methods, PEST analysis, Micro / Macro environmental analysis and with the SWOT framework I have summarized recommendations based on the facts I have gathered. I have also investigated different entry strategies to see what is most suitable for a Company X. I have collected data via interviews, literature and internet sources.   Result &amp; Conclusions: Short term Company X has the opportunity to come in with their products into Vietnam with a co-operation with Competitor A. They will set up a test system at one toll plaza between the airport and Hanoi with two lane dedicated for EFC and Company X is their first choice if they are competitive. This will give them the opportunity to be first on this market to implement microwave equipment in Vietnam and an opportunity to set the standard in the country for the future.  Suggestions for future research: The empirical data in this thesis is mainly gathered from secondary sources. The information would have been more accurate with more primary data.   Contribution of the thesis: This thesis shows that large investments in road infrastructure are coming up in Vietnam. This thesis also shows that currently there is low activity from competitors using the same technology and this was not previously known by Company X.

Value pricing strategies are beginning to be considered for future improvements in suburban areas that currently do not experience significant congestion but are expected to become congested in the future. This is a significant departure from implementing these strategies in congested urban areas as is commonly done now. Therefore, traveler reaction in these suburban areas is unknown. To plan and design value pricing projects most effectively, it will be necessary to gain an understanding of suburban travelers' potential reaction to value pricing. Responses to a survey of travelers using the eastern and western segments of Interstate 10 (I-10) outside of San Antonio were used to study differences in response to value pricing by suburban population groups. These surveys collected information on travelers' socioeconomic and trip characteristics as well as their attitudes towards value pricing in the form of potential Express Toll Lanes (ETLS). Stated preference scenarios presented to survey respondents were used to develop mode choice models. These models were used to determine characteristics that may impact the decision to choose to travel on the general purpose lanes (GPLs) or the ETLs. This research suggests that the implementation of value pricing strategies on suburban corridors may pose a challenge from a policy standpoint. The populations using these corridors appear to be more varied in their responses toward value pricing than populations using congested urban corridors. Overall, it was found that the majority of travelers on I-10E and I-10W are not favorable to the implementation of value pricing for the future expansion of these corridors. However, I-10W travelers seem to be more willing to pay for travel time savings. This is likely due to the fact that travelers on I-10W earn higher average incomes, are more likely to use I-10W on a regular basis for commute purposes, and are more often exposed to some traffic congestion. Conversely, travelers on I-10E are more likely to use I-10E less frequently for non-commute trips, travel longer distances, and probably do not have an intuitive sense of the value they would place on travel time savings since they do not regularly experience congestion.

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Chemokines triggered by Toll-like receptors (TLRs) are small chemoattractant proteins, which mainly regulate leukocyte trafficking in inflammatory reactions via interaction with G protein-coupled receptors. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this study, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to identify systematically chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. The TLR family represents evolutionarily conserved components of the patternrecognizing receptors (PRRs) of the innate immune system that recognize specific pathogen-associated molecular patterns (PAMPs) through their ectodomains (ECDs). TLR's ECDs contain 19 to 25 tandem copies of leucine-rich repeat (LRR) motifs. TLRs play important roles in the activation of pro-inflammatory cytokines, chemokines and modulation of antigen-specific adaptive immune responses. To date, nine TLRs have been reported in chicken, along with a non-functional TLR8. Two non-mammalian TLRs, TLR21 and TLR22, have been identified in pufferfish and zebrafish. The objectives of this study were to determine if there is the existence of chicken genes homologous to fish-specific TLRs, and if possible ligands of these receptors exist. After searching the chicken genome sequence and EST database, a novel chicken TLR homologous to fish TLR21 was identified. Phylogenetic analysis indicated that the identified chicken TLR is the orthologue of TLR21 in fish. Bioinformatic analysis of potential PAMP binding sites within LRR insertions showed that CpG DNA is the putative ligand of this receptor.

Reproduction de : Thèse de doctorat : Immunologie : Lille 2 : 2008. / Résumé en français et en anglais. Titre provenant de l'écran-titre. Bibliogr. p. 113-137.

The purpose of this research is to provide a theoretical framework for future commercial vehicle user-charging using real-time vehicle weight and configuration information collected using weigh-in-motion (WIM) systems. This work provides an extensive review of both mechanisms and technologies employed for commercial and passenger vehicle user-charging worldwide. Existing commercial vehicle-user charging structures use only broad vehicle classifications to distinguish between vehicles for the pricing of user-fees. The methodology proposed in this study employs highway cost allocation methods for development of an “Axle-Load” toll structure. A theoretical case study, based on information from Texas State Highway 130, is performed to explore the equity improvements that could be achieved through implementation of this proposed structure. Some sensitivity analysis is also performed to examine the potential revenue impacts due to uncertainties in different data inputs under existing and proposed structures. / text