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Syfte: Syftet med denna studie var att utvärdera ytkontamination (förorenade ytor) i behandlingsrum före och efter behandling på student- och specialistkliniken i Universitetstandvården Endodonti vid Malmö universitet. Material och metod: Provtagning utfördes vid Malmö Universitet, Universitetstandvården Endodonti, före och efter behandling på specialistkliniken och studentkliniken. Följande ytor utvärderades: tangentbord, mus, lamphandtag, behandlarens unit och assistentens unit. Totalt togs prover från 200 ytor. ATP-bioluminescence tillämpades för att utvärdera potentiell kontamination av mikroorganismer vid ett tröskelvärde på 250 RLU/100cm². Fluid thioglycollatemedium (FTM) tillämpades för bakterieodling.  Resultat: Tillväxtfrekvensen i behandlingsrummen var högre före behandling i förhållande till efter. Specialistkliniken hade högre tillväxt före behandling i jämförelse med studentkliniken. Efter behandling var tillväxten högre för studentkliniken. Högst frekvens av tillväxt påvisades på tangentbord och mus på båda klinikerna. ATP-bioluminescence visade en sensitivitet på 80 % och specificitet på 31 %.  Slutsats: Trots hygienrutiner och preventiva åtgärder som tillämpas förelåg det en svikt i desinfektion av ytor som tangentbord och mus då dessa hade högst frekvens av kontamination. Utifrån studien kan det konstateras att ATP-bioluminescence och FTM kan vara ett hjälpmedel vid kontroll av hygienrutiner. Det krävs fler studier inom tandvården som utvärderar ytkontamination och förekomst av vårdrelaterade infektioner för att uppnå en säkrare vårdmiljö. / Aim: The aim of this study was to evaluate surface contamination in treatment rooms before and after treatment at the student and specialist clinic in the Dental University, department of Endodontics at Malmö University. Materials and methods: Sampling was performed at Malmö University, Dental University, Endodontics, before and after treatment at the specialist clinic and student clinic. The following surfaces were evaluated: keyboard, mouse, lamphandle, the operator's unit and the assistant's unit. A total of 200 surfaces were sampled. ATP-bioluminescence was applied to evaluate potential contamination by microorganisms at a threshold value of 250 RLU/100cm². Fluid thioglycollatemedium (FTM) was used for bacterial cultivation. Results: The microbial growth rate in the treatment rooms was higher before treatment compared to after. The specialist clinic had a higher growth before treatment compared to the student clinic. After treatment, growth was higher at the student clinic. Highest frequency of growth was detected on keyboards and mouses in both clinics. ATP-bioluminescence showed a sensitivity of 80 % and specificity of 31 %. Conclusion: Despite hygiene routines and preventive measures that are applied, there is a failure in the disinfection of surfaces such as the keyboard and mouse as these had the highest frequency of contamination. From the study, it can be concluded that ATP-bioluminescence and FTM can be used as an evaluation tool in control of hygiene routines. More studies are required in dental care that evaluate surface contamination and the occurrence of healthcare-associated infections to achieve a safer healthcare environment.

Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, microbial aspects related to Salmonella as a food pathogen are investigated. A bioluminescent reporting system was developed for Salmonella to monitor the attachment and growth of the pathogen on food products. Twelve and eleven Salmonella strains from the broiler production continuum were tagged with bioluminescence by plasmid and integration of the lux operon into the chromosome, respectively. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, an attachment model using chicken skin was developed. Variables including washing and temperature were tested in the attachment model to determine the effects on attachment of Salmonella strains to chicken skin, a characteristic that enhances persistence during processing. Additionally, the invasion process for two serovars of Salmonella with differing host tropism was examined with emphasis on the initial establishment of the bacterium in the host. The major facilitator for invasion, type III secretion system, was inactivated through deletion mutation to evaluate invasion of human epithelial cell line by additional means. The difference in host tropism between the two subspecies of Salmonella was also taken into account when evaluating invasion. Results showed that invasion of human epithelial cells can be initiated despite inactivation of the type III secretion system. A serovar of Salmonella that is not typically associated with human illness was also shown to initiate invasion of human epithelial cells, a result that carries public health implication as this serovar has recently been shown to be multi-drug resistant.

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Le cancer de la prostate est une maladie très hétérogène dont le développement est principalement médié par l'action du récepteur aux androgènes. Ceci a donc résulté en une émergence de thérapies ciblant spécifiquement la voie du récepteur aux androgènes afin de traiter la maladie. Toutefois, tous les patients traités avec ces stratégies thérapeutiques développeront une résistance à celle-ci par des mécanismes dépendants et indépendants du récepteur aux androgènes. Comme la réponse clinique peut seulement être évaluée trois mois après le début de la thérapie, il est primordial de développer un test permettant de prédire précocement la réponse aux traitements. Ceci permettrait d'orienter, le plus rapidement possible, les patients réfractaires vers des thérapies alternatives. L'objectif principal de cette thèse était donc de développer des outils moléculaires afin d'étudier la réponse thérapeutique et l'impact des différents mécanismes de résistance sur celle-ci. Mes travaux présentés dans le chapitre 1 ont démontré qu'il était possible de suivre dynamiquement par imagerie à bioluminescence la réponse aux traitements de cellules cancéreuses prostatiques en immobilisant les cellules isolées grâce à un biofilm de matrice extracellulaire. Afin de suivre la réponse aux anti-androgènes spécifiquement dans les cellules cancéreuses prostatiques, un premier biosenseur basé sur l'activité de deux promoteurs spécifiques a été développé dans le chapitre 2. Celui-ci intègre l'activité du promoteur PCA3, qui est spécifique au cancer de la prostate, et du promoteur PSEBC, qui est sensible aux androgènes, afin de générer un signal luminescent quantitatif et représentatif de la réponse androgénique. Grâce à ce système, la sensibilité de cellules cancéreuses prostatiques isolée de patients naïfs et résistants aux anti-androgènes a pu être corrélée avec la réponse clinique des patients. L'applicabilité de ce biosenseur est toutefois limitée aux cellules exprimant un récepteur aux androgènes transcriptionnellement actif. Un deuxième système exploitant les capacités de la Cre recombinase à décoder l'activité des promoteurs PCA3 et PSEBC en deux signaux bioluminescents spectralement distincts a donc été développé dans le chapitre 3. Ce deuxième système combine tous les avantages et capacités du premier, tout en permettant la détection de cellule n'ayant pas d'activité androgénique. Celui-ci permet donc de visualiser des mécanismes de résistance complexes qui peuvent être indépendants ou dépendants du récepteur aux androgènes et qui collaborent pour échapper aux effets inhibiteurs des anti-androgènes. Les outils développés peuvent cibler et détecter avec précision les cellules épithéliales de cancer de la prostate ainsi que leur réponse aux thérapies. Les travaux de cette thèse ont ainsi ouvert la voie à de nouvelles techniques et connaissances sur des méthodes de diagnostic et de pronostic pour le cancer de la prostate. Ultimement, cela permettra d'améliorer le choix des traitements disponibles afin d'augmenter la qualité de vie des patients. / Prostate cancer is a very heterogeneous disease and its growth is driven mainly by the androgen receptor transcriptional activity. This has led to the development of androgen deprivation therapy as the main treatment of this disease to prevent its progression. However, almost all patients receiving these therapies will develop resistance through the acquisition of androgen receptor dependent and independent mechanisms. The challenge of prostate cancer management is largely due to drug-refractory sub-populations with in heterogeneous tumors. Only few biomarkers have been validated for clinical use and none of them address complex anti-androgens response heterogeneity. The main objective of this thesis was to develop bioluminescence imaging systems to detect and monitor prostate cancer cells therapeutic response. In chapter 1, we have developed an extracellular matrix biofilm coating method to dynamically follow by bioluminescence imaging heterogenous treatment response of prostate cancer cells. In chapter 2, a first biosensor based on the activity of two specific promoters was developed to monitor antiandrogens response specifically in prostate cancer cells. This system integrates a transcriptional amplification system and the activities of the PCA3 and PSEBC gene promoters as a single output driving the firefly luciferase gene reporter. The PCA3 promoter provides the specificity to PCa cells, while the PSEBC promoter measures AR transcriptional activity. When tested on different cohorts of patients from primary PCa to mCRPC, our method could discriminate the overall response of a patient to antiandrogens. However, the applicability of this biosensor is limited to cells expressing a transcriptionally active androgen receptor. A second system exploiting the capacities of Cre recombinase and decoding PCA3 and PSEBC promoter activity in two spectrally distinct bioluminescent signals was therefore developed in chapter 3. This second system combines all the advantages and capacities of the first system, but also allows the detection of prostate cancer cells with no androgenic activity. This method visualizes complex androgen receptor independent and dependent resistance mechanisms and their interactions together to escape from antiandrogen inhibitory effects. The tools developed in this thesis can specifically target and detect prostate cancer cells and their response to therapies. Thus, they have the potential to play an important role in patients therapeutic management and therefore improve their overall survival.

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In vitro differentiation of stem cells in biocompatible scaffolds in a bioreactor is a promising method for creating functional engineered tissue replacements suitable for implantation. Basic studies have shown that mechanical, chemical, and pharmaceutical stimuli enhance biological functionality of the replacement as often defined by parameters such as cell viability, gene expression, and protein accumulation. Most of the assays to evaluate these parameters require damage or destruction of the cell-scaffold construct. Therefore, these methods are not suitable for monitoring the development of a functional tissue replacement in a spatial and temporal manner prior to implantation. Bioluminescence imaging is a technique that has been utilized to monitor cell viability and gene expression in various in vivo applications. However, it has never been applied in an in vitro setting for the specific purpose of evaluating a cell-scaffold construct. This research describes the design of flow perfusion bioreactor system suitable for bioluminescence imaging. In the first experimental chapter, the system was tested using MC3T3-E1 cells transfected with a constitutive bioluminescent reporter. It was found that bioluminescence imaging was possible with this system. In the second experimental chapter, MC3T3-E1 cells transfected with BMP-2 linked bioluminescence reporter were cultured by flow perfusion for a period of 11 days. Bioluminescence was detectable from the cells starting at day 4, while peaking in intensity between days 7 and 9. Further, it was also found that bioluminescence occurred in distinct regions within the scaffold. These results indicate that these strategies may yield information not available with current assays. / Master of Science

Myriad small molecule compounds targeting metabotropic glutamate receptors (mGluRs) have been investigated for the treatment of various neuropsychiatric diseases and displayed promise in preclinical studies. At the clinical level, many of these compounds have been well tolerated by human subjects but have eluded success as promising therapeutics. There are eight subtypes of mGluRs, which express as constitutive dimers. This dimerization can occur between identical (homodimerization) or different (heterodimerization) mGluR protomer subtypes, which are subject to pairing-specific signaling mechanisms. Subtype expression of mGluRs is heterogenous between brain regions and cell types, yielding probable cell-specific homo- and heterodimer combinations that respond differently to certain drugs. While G protein recruitment to active mGluR dimers has been studied extensively, little is known about arrestin recruitment to these receptors. I used bioluminescence resonance energy transfer (BRET) assays, which provide a quantitative measure of protein-protein proximity, to observe and quantify arrestin recruitment to specific mGluR subtype pairings upon ligand administration in heterologous cells. I studied how select allosteric ligands affect communication between protomers to enhance arrestin recruitment to dimers. My findings indicate that arrestin recruitment occurs only at select mGluR homodimers upon orthosteric stimulation but is frequently stimulated or enhanced by administration of activating allosteric ligands. Additionally, I found that trans-protomer communication is highly specific to mGluR protomer subtype pairings, the ligand administered,a nd inter-protomer signal direction. Lastly, my findings reveal a cooperative effect of mGluR2 and 3 heterodimerization on arrestin recruitment that is dependent on the functional ability of each protomer to bind orthosteric agonist and responds distinctively from homodimers to stimulation by certain allosteric ligands. Taken together, this work shows that mGluR signaling can be tuned using strategic pharmacology and energizes hope for future clinical success of mGluR-targeting ligands.

Salmonellosis, a significant foodborne illness in humans, is caused by Salmonella, with poultry and poultry products acting as significant reservoirs and sources of human infection. Salmonella enterica subspecies enterica serotype Reading has recently emerged as a notable foodborne pathogen responsible for extensive multistate human outbreaks in North America. This study focused on evaluating the capacity of the emerged serotype to colonize broiler breeder reproductive tissues and potentially contaminate eggs, indicating the potential for vertical transmission. For this investigation, two Salmonella Reading strains were utilized, one associated with outbreaks and another non-outbreak strain. Both strains were initially modified with bioluminescent marker genes to facilitate tracking post-experimental infection in broiler breeders. The results indicated that both strains could colonize the reproductive tract of infected hens and be transmitted vertically through the eggs. This finding enhances our understanding of the colonization and vertical transmission capabilities of this serotype in broiler breeders.