Influenza is a viral infection that affects global health and economy with its endemic and sometimes pandemic spread. Rapid detection of Influenza viruses enables antiviral use and can bring financial savings. It is also essential for the global surveillance of prevalent Influenza strains. RT-PCR is considered the most specific and sensitive method for detection of Influenza, but Influenza mutates at a high rate and it is therefore crucial that RT-PCR methods are updated regularly. In 2014, Cepheid released their Xpert Flu/RSV XC assay, which can detect Influenza A and B and RSV by multiplex RT-PCR in approximately one hour. The aim of this study was to evaluate this assay at Laboratoriemedicin Västernorrland by using the laboratory’s previous PCR assay for detection of Influenza viruses as reference method. Real-time RT-PCR was used to compare Xpert Flu/RSV XC to the reference method. A dilution series was performed to estimate the methods’ PCR efficiencies and precision was calculated from quadruplicates of a positive control sample. Clinical specimens (n=42) were used to evaluate the diagnostic sensitivity and specificity of Xpert Flu/RSV XC. Objective statistical analysis of PCR data was performed and discussed. The Xpert Flu/RSV XC was equivalent to the reference method and demonstrated high diagnostic sensitivity and specificity. Estimated PCR efficiencies were however low. With the introduction of Xpert Flu/RSV XC to the laboratory follows many potential benefits, primarily in form of a simplified pre analytical procedure and a shortened analysis time. The Xpert Flu/RSV XC assay enables fast diagnosis of Influenza infection.

La familia de transportadores heteroméricos de aminoácidos posee la característica única de estar compuestos por dos subunidades: una subunidad pesada y una subunidad ligera unidas por un puente disulfuro. Mutaciones en alguno de sus miembros son responsables de aminoacidurias hereditarias, como la lisinuria con intolerancia a proteínas (LPI) o la cistinuria. Al inicio de esta tesis, nos propusimos identificar alguna nueva subunidad ligera humana de esta familia. Mediante búsqueda por homología de secuencia se clonó el cDNA correspondiente a una nueva proteína altamente homóloga a una proteína de ratón asociada al sistema de transporte xc-. La caracterización funcional confirmó que xCT humano, en combinación con 4F2hc, induce una actividad de transporte de cistina y glutamato, con sodio-independencia (sistema xc-). Los perfiles de captación de los sustratos en función del pH del medio de transporte indican que tanto el L-glutamato como la L-cistina son transportados de forma aniónica, cada uno con una carga negativa neta. Por experimentos de salida de sustrato se determinó el carácter intercambiador de este transportador, con una estequiometría de 1:1.El patrón de expresión de xCT humano por Northern blot muestra una ligera banda presente únicamente en cerebro. Experimentos de RT-PCR también resultaron positivos para los islotes pancreáticos y líneas celulares correspondientes a líneas epiteliales y tumorales, mientras riñón e hígado no mostraron ninguna señal. El papel fisiológico de xCT parece estar implicado en la captación de cistina para la síntesis de glutatión y su mantenimiento, dándole un papel relevante en situaciones de estrés oxidativo. A continuación se abordó la determinación de la topología de xCT como modelo de subunidad ligera. Experimentos de inmunodetección evidencian la localización intracelular de los dos extremos de la proteína. Utilizando la estrategia de introducir cisteínas individuales en los distintos loops y testar su accesibilidad a reactivos tiol-específicos, la topología de xCT es compatible con un modelo de 12 dominios transmembrana. Los resultados obtenidos en la zona IL2-3, con dos residuos de accesibilidad exterior (el 110 y 112) flanqueados por residuos de accesibilidad interior (el 102, 109 y 116) en un rango de 15 aminoácidos nos hacen pensar en una estructura de reentrant loop, con el residuo H110 como ápice. El hecho que estas estructuras suelen ser zonas asociadas a la ruta de paso del sustrato del transportador, nos llevó a estudiar la implicación del residuo H110. En resumen, los resultados muestran que i) la biotinilación del residuo H110C se bloquea por los sustratos y el inhibidor no transportable 4SCPG; ii) la inactivación provocada por MTSES en el transporte de h110c también es protegida por los sustratos con una IC50 similar a la Km para cada sustrato; iii) esta protección es independiente de temperatura, y por tanto no implica grandes cambios conformacionales; y iv) aunque los mutantes H110C y H110D no alteraron la Km del transportador ni su especificidad de sustrato, la sustitución por una lisina inactiva totalmente la función de xCT. Por tanto, tenemos evidencias de que H110 se encuentra cercano al lugar de unión al sustrato o ruta de paso del mismo. Estos resultados pueden encontrarse en las publicaciones siguientes: J Biol Chem (2004) vol. 279, 31228-36, J Biol Chem (2004) vol. 279, 11214-21, y Pflugers Arch. (2001) vol. 442 (2) 286-296. ENGLISH / The family of heteromeric amino acid transporters has the particular characteristic of being composed of two proteins: a light subunit (LSHAT) and a heavy subunit (HSHAT) linked by a disulfide bridge. Some of its members are associated with hereditary aminoacidurias. Characterisation of a new human LSHAT, responsible for transport system xc-, is reported here. Human xCT associates with 4F2 heavy chain and induces cystine/glutamate exchange with sodium independence and 1:1 molar ratio. Topology studies using immunodetection and substituted cysteine accessibility method reveal a model compatible with 12 transmembrane domains and intracellular N and C-terminus. The results obtained in the intracellular loop between 2 and 3 transmembrane domain (IL2-3), with two residues of extracellular accessibility (110 and 112) flanked by residues of intracellular accessibility (the 102, 109 and 116) in a rank of 15 amino acids, resembles the structure of a reentrant loop with apex in position H110. The fact that these structures are usually associated with the substrate pathway through the transporter, lead us to further studies with residue H110.In summary, the results on that position show that: i) biotinylation of H110C is blocked by its substrate and the non-transportable inhibitor 4-S-carboxiphenyl-glycine (4-S-CPG); (ii) the inactivation caused by MTSES in the transport of mutant H110C is also protected by each substrate with a IC50 similar to the Km; (iii) this protection is independent of temperature, and therefore it does not imply great conformational changes; and (iv) although mutants H110C and H110D did not alter the Km of the transporter nor their specificity of substrate, the substitution by a lysine inactivates the function of xCT totally. Therefore, we conclude that residue H110 resides near the binding site/translocation pathway of the substrates in the transporter xCT. These results can be found in the following publications: J Biol Chem (2004) vol. 279, 31228-36, J Biol Chem (2004) vol. 279, 11214-21, y Pflugers Arch. (2001) vol. 442 (2) 286-296.

No description available.

<p>Breast cancers are the most common source of metastases to bone of which cancer-induced bone pain is a frequent pathological feature. Cancer-induced bone pain is a unique pain state with a multiplicity of determinants that remains to be well understood and managed. Current standard treatments are limited by dose-dependent side effects that can depress the quality of life of patients. Glutamate is a neurotransmitter and bone cell-signalling molecule that has been found to be released <em>via</em> the system x_C^-cystine/glutamate antiporter on cancer cells of types that frequently metastasize to bone, including breast cancers. This project examines the hypothesis that limiting glutamate release from cancer cells metastasized to bone will reduce bone tissue disruption and cancer-induced bone pain. A mouse model of cancer-induced bone pain was established with intrafemoral human breast cancer cells (MDA-MB-231), and behavioural measurements were taken for weight bearing and induced paw withdrawal thresholds. The system x_C^- inhibitors sulfasalazine and (S)-4-carboxyphenylglycine both attenuated glutamate release from cancer cells in a dose-dependent manner <em>in vitro</em>. Treatment with sulfasalazine induced a moderate delay in the onset of behavioural indicators of pain in mouse models, and treatment with (S)-4-carboxyphenylglycine had no apparent results. This data suggests that the limitation of extracellular glutamate released from cancers in bone with sulfasalazine may provide some alleviation of the often severe and intractable pain associated with bone metastases.</p> / Master of Science (MSc)

No description available.

Cette thèse a comme objectif l'optimisation temporelle au cours du processus de synthèse des réseaux programmables à base de LUT, en particulier ceux de la famille Flex8000 d'Altera et de la famille XC4000 de Xilinx. L'optimisation temporelle consiste en deux étapes essentielles: la détection de la zone critique et la décomposition technologique orientée vitesse dans cette zone. Les nouvelles notions de zone sensible et de zone critique sont utilisées pour rechercher la zone dont l'optimisation temporelle devrait satisfaire les demandes de l'utilisateur. Un modèle prédictif réaliste du délai des chemins dans un réseau à base de LUT a été proposé. La fonction de coût proposée, exprimant bien la relation entre les entrées/sorties critiques, non critiques ou sensibles, permet à la décomposition technologique orientée vitesse d'optimiser efficacement la zone nécessaire, en augmentant très peu la surface. De plus, les réinjections contrôlées des sous-fonctions booléennes appliquées dans la zone critique permettent d'obtenir des zones de travail, destinées à une resynthèse, avec une taille convenant à la technologie considérée. L'approche proposée pour la décomposition technologique orientée vitesse en tenant compte des contraintes de l'utilisateur s'est avérée rapide et donne de bons résultats. Le gain en vitesse de l'optimisation temporelle par rapport à l'option surface est en moyenne de 32% avec une augmentation de surface de 30% seulement. Ce travail a fait l'objet d'un transfert technologique vers un système industriel de CAO, appelé ASYL+. La qualité des résultats a été démontrée en comparaison avec des outils commerciaux tels que Exemplar, Maxplus2 de la société Altera et XACT de Xilinx. Les tests sur de nombreux benchmarks donnent un gain moyen en performance temporelle d'ASYL+ sur Exemplar, Maxplus2 et XACT respectivement de l'ordre de 28%, 18% et 17%

Despite the lack of robust clinical response, treatment strategies for cancer-induced depression (CID) are currently limited to those developed for non-cancer-related depression. The work presented in this dissertation conceptualizes CID as a pathophysiologically distinct form of depression. To investigate CID at the most basic level, we first developed a preclinical model that was validated by comparison to an established model of stress-induced depressive-like behaviours. The positive control model was developed by chronically treating female BALB/c mice with oral corticosterone (CORT). The CID model was developed using subcutaneous inoculation with 4T1 mammary carcinoma cells. Anhedonia, behavioural despair, and dendritic atrophy in the medial prefrontal cortex (mPFC) were observed in both models. Similar to many human cancer cell lines, 4T1 cells were shown to secrete significant amounts of glutamate, which was markedly attenuated using the system xc- inhibitor sulfasalazine (SSZ). In CID mice, oral treatment with SSZ was at least as effective as fluoxetine, a popular clinical antidepressant, at preventing depressive-like behaviours. This effect was primarily attributable to intact SSZ, rather than its anti-inflammatory metabolite. RNA-sequencing was performed on hippocampal samples from CID and CORT animals. Analysis of differential expressed genes (DEGs) revealed significant overlap between the two models. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological process gene ontologies (GO:BP) terms related to ion homeostasis and neuronal communication were enriched for both models. CID was associated with additional DEGs that were not identified in the CORT model. These DEGs were enriched in KEGG pathways and GO:BP terms related to neuronal development, intracellular signalling cascade, learning, and memory. These studies suggest that CID may involve a distinct aetiology, and that glutamate secretion by cancer cells presents a viable target for antidepressant treatment. The development of mechanism-based therapeutics for CID will dramatically improve the quality of life for cancer patients. / Thesis / Doctor of Philosophy (PhD) / Cancer patients are at a high risk of developing depression. In addition to the psychological stress caused by a cancer diagnosis, there is evidence that cancer causes depression through biological pathways. To investigate these pathways, a mouse model of cancer-induced depression (CID) was developed. This model showed comparable behavioural and structural brain deficits to those observed in a stress model of depression. Cancer cells secrete elevated levels of glutamate, a signalling molecule that is involved in depression. In CID mice, inhibiting glutamate release had an antidepressant effect similar to that of fluoxetine, a standard clinical antidepressant. A genetic analysis on brain samples from the CID model revealed significant overlap with the stress model of depression. CID mice had additional changes relevant to learning, memory, and brain cell development that were not detected in the stress model. A better understanding of CID will lead to better treatment strategies developed specifically for cancer patients.

Metastatic bone cancers cause severe symptoms including pain that compromises patient functional status, quality of life, and survival. Current treatment strategies have limited efficacy and dose-limiting side effects. Cancer-induced bone pain (CIBP) is a unique pain state that shares features with but is distinct from the pathology of neuropathic and inflammatory pain. This dissertation investigates how CIBP is generated and maintained by the direct effects of cancer cells on their metastatic microenvironment and the peripheral nervous system, including unique signaling properties and gene expression changes. In particular, we found that genetic knockdown of the functional subunit xCT of the system xC- cystine/glutamate antiporter can reduce CIBP, further elucidating this as a therapeutic of interest. We found that the neuroprotective voltage-gated calcium channel inhibitors progesterone and pregabalin markedly reduce mechanical hypersensitivity and excitability in sensory neurons of the dorsal root ganglion (DRG) in male rat models of neuropathic pain, but that these effects and less pronounced in females. In cancer pain, these sex differences are reversed, with females but not males demonstrating a delay in time-to-onset of mechanical hypersensitivity. We also analyzed gene expression at the DRG by RNA-Sequencing of rat models of CIBP. Our findings uncovered differential gene expression between CIBP and sham controls and between ipsilateral and contralateral DRGs in CIBP model rats. These studies have identified several promising avenues for therapeutic research for CIBP. / Dissertation / Doctor of Philosophy (PhD) / The tools we have right now to manage severe and chronic pain are insufficient. Patients with advanced cancers including bone cancer can suffer from very severe pain. This pain is generated in a number of ways including by the tumour itself releasing chemicals that activate pain-sensing nerves, by the destruction of the bone in and around the tumour, and by the sensitization of the nervous system, which can make pain worse and longer lasting. We have taken three approaches to researching cancer pain and to investigating new treatments. We have found that by reducing the amount of glutamate that cancer cells can release into their environment, we can reduce cancer pain in mice. We also found that treating rats with pregabalin and progesterone can change nerve signaling and reduce neuropathic pain, but that this effect is most pronounced in male rats with neuopathic pain and smaller in female rats with neuropathic pain, and even smaller in rats with cancer pain. We also analyzed expression of all the protein-coding genes in dorsal root ganglia from rats with cancer pain and found that there are many differences from rats without pain. Some of these differences may be promising new research targets. Going forward this research has provided important evidence necessary for next steps to develop new therapies and research strategies for cancer pain.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and is the most aggressive malignant primary brain tumor in adults. Without treatment, patients are expected to survive an average of three months. Conversely, current treatment regimens only extend survival to 12-14 months. Characteristically, GBM tumors are highly proliferative, invasive and stop responding to treatments relatively fast due to therapy resistance. Interestingly, GBM also exhibits high metabolic activity but manages to maintain a low level of reactive oxygen species (ROS). These ROS neutralization capabilities are sustained by system Xc–, a sodium-independent, electro neutral transporter that is found in the plasma membrane of GBM cells. System Xc– is composed of a regulatory heavy subunit (4F2hc) linked to a 12 transmembrane domain catalytic light chain subunit (xCT) that mediates the uptake of L-cystine into the cell, and L-glutamate out of the cell, at a 1:1 ratio. Imported cystine is quickly reduced to L- cysteine, the rate limiting substrate in glutathione (GSH) synthesis. Glutathione is a major antioxidant in the central nervous system that is responsible for maintaining intracellular redox homeostasis by neutralizing ROS by direct and indirect methods. The function of chemo and radiation therapy is to generate significant levels of ROS that tigger the cell to undergo apoptosis. High intracellular GSH levels in cancer cells are associated with drug resistance and detoxification of alkylating agents such as temozolomide (TMZ). Therefore, system Xc– represents a potential target to reduce glioma cell survival and reduce tumor progression. Sulfasalazine is an FDA approved drug in the treatment of arthritis and Crohne’s disease and has been shown to inhibit system Xc–. In vitro SASP studies demonstrated a strong antitumor potential in preclinical mouse models of malignant glioma. However, two clinical trials using sulfasalazine with standard chemo and radiation therapy to treat GBM patients were terminated due to off-target effects. Both results showed high toxicity and no change in the overall survival of patients. These studies demonstrate the need for a more effective inhibitor of system Xc–. To further elucidate the role of system Xc– in GBM survival, stable xCT knock-down and over-expressing U251 glioma cells were generated. These lines were characterized for survival, proliferation, apoptosis and resistance to oxidative and genotoxic insult. As expected xCT-knockdown cells exhibited lower GSH levels, increased intracellular ROS and markers for apoptosis after oxidative and genotoxic insult. The xCT-over-expressing cells displayed higher levels of GSH, increased resistance to hydrogen peroxide and various chemotherapy drugs including TMZ. An interesting unforeseen result of xCT over-expression in glioma cells was an increase in the metabolic activity as a result of increased mitochondria. Using xCT-modified glioma lines stably, we demonstrate for the first time that system XC– over-expression not only promotes survival under oxidative stress but may also decreases sensitivity to chemotherapy treatment and increase metabolic properties. Therefore, therapeutic manipulation of this transporter either alone or in combination with other treatments may improve clinical outcome in patients diagnosed with GBM.

Die Zahl demenzerkrankter Patienten in Akutkrankenhäusern wird zukünftig ansteigen. Ein Aufenthalt in einer fremden Krankenhausumgebung bedeutet für diese Patientengruppe psychisch eine extrem belastende Situation. Die Folgen sind für die Betroffenen, das Personal und die Kliniken schwerwiegend. Seitens der Patienten sind besonders die Einbußen des kognitiven und physischen Status sowie ein Verlust an Selbstständigkeit zu nennen. Für das Pflegepersonal ist vor allem die hohe Arbeitsbelastung aufzuführen. Lösungsansätze einer besseren Versorgung schenken einer demenzsensiblen Gestaltung der gebauten Umwelt in Deutschland bislang zu wenig Beachtung. Das Ziel dieser Arbeit besteht daher in der Entwicklung eines Katalogs demenzfreundlicher Planungskriterien für Akutkrankenhäuser. Die Zusammenfassung der bisherigen, wissenschaftlich belegten positiven Auswirkungen einer demenzsensiblen Architektur in Altenpflegeeinrichtungen dient als Basis für die Überlegungen zu einer Übertragbarkeit dieser Planungskriterien. Hier beeinflussen beispielsweise eine segregative Betreuung, klare Grundrissstrukturen mit einprägsamen Referenzpunkten oder eine milieutherapeutische, wohnlich gestaltete Umwelt mit Gemeinschaftsräumen die Bewohner positiv. Kriterien, die speziell die Einbußen der kognitiven und funktionellen Fähigkeiten von demenziell erkrankten Menschen berücksichtigen, sind das Kaschieren von Türen und die Umsetzung visueller Barrieren. Auch ein durchgehend gleichmäßig gestalteter Bodenbelag, der dem Wahrnehmen von Stufen oder Abgründen und damit Stürzen vorbeugt, ist bei der Planung von demenzsensiblen Gebäuden wichtig. Weiterhin liefert neben der Analyse international bereits realisierter Konzepte, wie die Einrichtung von Spezialstationen oder Tagesbetreuungsräumen, ein Interview mit dem Pflegepersonal des Diakonissenkrankenhauses Dresden wichtige Ansatzpunkte für den erarbeiteten Kriterienkatalog. Viele der evidenzbasierten Handlungsanweisungen zur Planung von stationären Altenpflegeeinrichtungen sind auf die Architektur von Akutkrankenhäusern anwendbar wie zum Beispiel Maßnahmen, die die Orientierung erleichtern. Diese führen dort im Sinne eines Design for all bzw. Universal Designs für alle Menschen zu einer leichteren Nutzbarkeit. Gemeinschaftsräume für die Einnahme der Mahlzeiten oder tagesstrukturierende Angebote sind in der Planung von Akutkrankenhäusern nicht vorgesehen. Dies stellt einen wesentlichen Unterschied zum Raumprogramm stationärer Altenpflegeeinrichtungen dar. Für demenzerkrankte Patienten könnte jedoch so dem Verlust der Selbstständigkeit und kognitiver Fähigkeiten vorgebeugt werden. Die Übertragung einer wohnlichen Atmosphäre muss in Bezug auf Machbarkeit und Sinnhaftigkeit kritisch diskutiert werden. Die demenzsensible Anpassung des Eingangsbereichs und der Notaufnahme am konkreten Beispiel des Diakonissenkrankenhauses Dresden zeigt die Anwendbarkeit des Kriterienkatalogs. Dieser gliedert sich in die Punkte Orientierung, räumliche Organisation, Sicherheit und milieutherapeutische Gestaltung. Weiterhin wird hier die Einrichtung einer Spezialstation als Anbau an das Bestandsgebäude vorgeschlagen.