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The relationship between gut microbiota and intestinal epithelial cells, particularly specialized enteroendocrine cells, such as GLP-1-secreting L-cells, is a critical aspect of gastrointestinal homeostasis and the gut-brain axis. This study delves into the molecular signalling pathways activated by the interaction between Bifidobacterium breve ncfb2258, a putative probiotic, and GLP-1-secreting L-cells. Bifidobacterium breve ncfb2258 produces conjugated linoleic acids (CLAs), which activate peroxisome proliferator-activated receptors (PPARs) and free fatty acid receptors (FFARs). This study aims to shed light on whether L-cells function as crossbarrier signal transducers and the mechanisms with which they do. Significant findings emerged through immunofluorescent labelling and calcium imaging, revealing differential expression patterns of these molecules in response to exposure to Bifidobacterium breve ncfb2258 supernatants. A significant response was observed when the human colorectal cancer cell line, NCI-H716 cells, a model of human GLP-1-secreting L-cells, were exposed to Bifidobacterium breve ncfb2258 supernatants (p = 0.003). PPARα and FFAR4 were both detected on NCI-H716 cells and while exposure to Bifidobacterium breve ncfb2258 supernatants increased the intensity of their cellular expression (p = <0.001; 0.042), expressiononly decreased with the addition of PPARα antagonist (p < 0.001) and remained elevated after the addition of FFAR4 antagonist (p = 0.999). These findings suggest the NCI-H716 cell line is suitable for exploring human GLP-1-secreting L-cells as potential cross-barrier signal transducers for the putative probiotic, B. Breve ncfb2258, and identify PPARα as potentially being involved in the molecular mechanisms of this interaction.

Angiogenesis is essential for physiological processes including embryonic development, tissue regeneration, and reproduction. Under various pathological conditions the same angiogenic process contribute to the onset, development, and progression of many human diseases including cancer, diabetic complications, ocular disease, chronic inflammation and cardiovascular disease. Vascular endothelial growth factor (VEGF) is a key angiogenic factor for physiological and pathological angiogenesis. In addition to its strong angiogenic activity, VEGF also potently induces vascular permeability, often causing tissue edema in various pathological tissues. VEGF transduces its vascular signal through two tyrosine kinase receptors-VEGFR1 and VEGFR2, the latter being a functional receptor that mediates both angiogenic and vascular permeability effects. To study physiological and pathological functions of VEGF, we developed novel zebrafish disease models that permit us to study hypoxia-induced retinopathy and cancer metastasis processes. We have also administered anti-VEGF and anti-VEGFR specific antibodies to healthy mice to study the homeostatic role of VEGF in the maintenance of vascular integrity and its functions in various tissues and organs. Finally, using a zebrafish model, we evaluated if VEGF expression is regulated by circadian clock genes. In paper I, we developed protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1:EGFP zebrafish were placed in hypoxic water for 3-10 days with retinal neovascularization being analyzed using confocal microscopy. This model provides a unique opportunity to kinetically study the development of retinopathy in adult animals using non-invasive protocols and to assess the therapeutic efficacy of orally administered anti-angiogenic drugs. In paper II, we developed a zebrafish metastasis model to dissect the complex events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells were implanted into the perivitelline cavity of 48-hour-old zebrafish embryos, which were subsequently placed in hypoxic water for 3 days. Tumor cell invasion, metastasis and pathological angiogenesis were analyzed using fluorescent microscopy in the living fish. The average experimental time for this model is 7 days. Our protocol offers an opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis. In paper III, we show that systemic delivery of an anti-VEGF or an anti-VEGF receptor (VEGFR)-2 neutralizing antibody cause global vascular regression in mice. Among all examined tissues, the vasculature in endocrine glands, intestinal villi, and the uterus are most affected in response to VEGF or VEGFR-2 blockades. Pro-longed anti-VEGF treatment resulted in a significant decrease in the circulating levels of the predominant thyroid hormone, free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid function. These findings provide structural and functional bases of anti-VEGF-specific druginduced side effects in relation to vascular changes in healthy tissues. In paper IV, we show that disruption of the circadian clock by constant exposure to light coupled with genetic manipulation of key genes in the zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. These results offer mechanistic insights into the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis. Overall, the results in this thesis provide further insight to angiogenic mechanistic properties in tissues and suggest possible novel therapeutic targets for the treatment of various angiogenesis-dependent diseases. / Blodkärlsnybildning, så kallad angiogenes, är viktigt för fysiologiska processer vid embryonal utveckling, vävnadsregenerering och reproduktion. Samma angiogena process kan också under olika sjukdomstillstånd bidra till uppkomst, utveckling och progress av många sjukdomar, såsom cancer, diabeteskomplikationer, ögonsjukdomar, kronisk inflammation samt hjärtkärlsjukdom. Vascular endothelial growth factor (VEGF) är mycket viktig för fysiologisk och patologisk angiogenes. Utöver sin starka angiogena effekt inducerar VEGF även ökad kärlpermeabilitet, som ofta orsakar ödem. VEGF utövar sin effekt på kärlen via två tyrosinkinasreceptorer: VEGFR1 och VEGFR2, där den senare är en funktionell receptor som förmedlar både angiogena signaler och har effekter på vaskulär permeabilitet. För att öka möjlgheterna att studera fysiologiska och patologiska funktioner av VEGF, har vi utvecklat sjukdomsmodeller i zebrafisk - hypoxi-inducerad retinopati och metastasering av cancer. Vi har också givit anti-VEGF och anti-VEGFR-specifika antikroppar till friska möss för att utvärdera VEGFs roll vid stabiliseringen av kärlfunktionen i olika vävnader och organ. Slutligen,utvärderade vi om expressionen av VEGF regleras av dygnsrytmen genom så kallade klock-gener. I papper I utvecklade vi en modell för hypoxiinducerad retinopati hos vuxna zebrafiskar. Vuxna fli1:EGFP zebrafiskar placeras i syrefattigt vatten i 3-10 dagar, varpå retinal nybildning av kärl analyserades. Denna modell ger en unik icke-invasiv möjlighet att studera kinetiskt utveckling av retinopati och den möjliggör bedömning av terapeutiska effekter av oralt givna anti-angiogena läkemedel. I papper II utvecklade vi en zebrafiskmodell för utvärdering av cancermetastasering, som möjliggör studier av detaljerade delprocesser vid hypoxi-inducerad tumörcellsinvasion och metastasering i samband med angiogenes på encellig nivå. I denna modell användes fluorescerande Dil-märkta humana- eller mustumörceller som implanterades vid den perivitellina hålighet hos 48-h-gamla zebrafiskembryon placerade i syrefattigt vatten i 3 dagar. Tumörcellinvasion, metastasering och patologisk angiogenes analyserades med mikroskopi i levande fiskar. Vårt protokoll möjliggör studier av molekylära mekanismer bakom hypoxi-inducerad cancermetastasering. I papper III visas, att systemisk administration av anti-VEGF eller anti-VEGF-receptor (VEGFR)-2 neutraliserande antikroppar in en musmodell orsakar generell kärlregression. Bland alla undersökta vävnader påverkades endokrina körtlar, tarmslemhinna och uterus mest av VEGF eller VEGFR-2 blockad. Långvarig anti-VEGF behandling resulterade i en signifikant minskning av cirkulerande nivåer av det dominerande sköldkörtelhormonet, fritt tyroxin, men inte av trijodtyronin, vilket tyder på att kronisk anti-VEGF behandling försämrar sköldkörtelfunktionerna. Resultaten påvisar risken för biverkningar i friska vävnader av anti-VEGF behandling. I papper IV visar vi att störningar i dygnsrytm genom konstant exponering för ljus och genetisk manipulation av nyckelgener i zebrafisk ledde till nedsatt angiogenes under embryonal utveckling. En bmal1-specifik morfolino hämmade angiogenes i zebrafisk utan att orsaka andra kärl-oberoende fenotyper. Omvänt, en period2 morfolino accelererade angiogeneskärltillväxt, vilket tyder på att Bmal1 och Period2 utövar motsatta effekter påkärlstillväxt. Dessa resultat ger mekanistisk kunskap om den roll som dygnsrytmen har i regleringen av angiogenes, och resultat kan rimligen utvidgas till andra typer av fysiologisk eller patologisk angiogenes. Sammanfattningsvis ger resultaten i denna avhandling ytterligare kunskap om angiogenetiska mekanismer och pekar på möjliga nya terapeutiska mål för behandling av olika angiogenes-beroende sjukdomar.

Sammanfattning Bakgrund Läraryrket är röstkrävande och lärare har en högre förekomst av röstbesvär jämfört med andra yrkesgrupper. Röstbesvär har även visats vanliga hos lärarstudenter. På Umeå universitet erbjuds en preventiv behandling för lärarstudenter som är viktig för att förbättra rösthälsan i läraryrket. Syfte Denna studie syftar till att undersöka förekomst av upplevda röstbesvär hos lärarstudenter vid Umeå universitet. Vi vill bidra med information om faktorer som kan påverka studenternas upplevda röstbesvär samt utveckla den preventiva behandlingsinsats som finns i dagsläget. Metod Autentisk data från lärarstudenter vid Umeå universitet har samlats in under 20 år i samband med ett förebyggande röstmoment i lärarutbildningen. Genom en databasforskning och en enkätundersökning för röstlogopeder i Västerbotten har upplevda röstbesvär och faktorer som kan påverka dessa undersökts. Resultat Aktuell studie har visat att av alla deltagande lärarstudenter under en 10-årsperiod var det 18,4% som upplevde röstbesvär. Det vanligaste symtomet bland studenterna var ansträngd eller trött röst. När inverkan på studenternas upplevda röstbesvär undersöktes i samband med verksamhetsförlagd utbildning syntes en signifikant ökning av upplevda röstbesvär hos hela gruppen. Röstlogopederna ansåg att röstbelastning var en betydande faktor gällande individers medvetna röstbesvär. Röstkrävande fritidsaktiviteter var även den enda protokollfaktor som var procentuellt högre hos studenter med upplevda röstbesvär. Slutsats Resultatet av denna studie har visat att verksamhetsförlagd utbildning har en inverkan på upplevda röstbesvär och styrker även tidigare teorier om detta. Aktuell studie vill därmed belysa vikten av den preventiva behandlingens placering då den med fördel bör ske i samband med att studenterna ökar sin röstbelastning under VFU-perioderna.

Chronic lymphocytic leukemia (CLL) cells rapidly die when put in culture implying that microenvironmental signals delivered by accessory cells confer CLL cells with a growth advantage. Recent findings show that CLL cells are antigen experienced and antigen binding play a critical role in the pathogenesis of the disease. The overall aim of this thesis was to study the influence of the microenvironment and antigen binding in CLL. In paper I, we studied the influence of the small redox-regulatory molecule thioredoxin (Trx) on CLL cell survival and proliferation. We found Trx to be highly expressed in CLL lymph nodes (LNs), secreted from stromal cells surrounding proliferating CLL cells in proliferation centers, indicating growth promoting properties. Secreted Trx was also shown to protect CLL cells from apoptosis. In paper II, oxidized LDL was added to subset #1 CLL cells. However, in contrast to our hypothesis, we could not observe activation and proliferation of CLL cells. Instead subset #1 CLL cells were unresponsive/anergic through the B cell receptor (BcR). This anergic state could however be overcome by “wash out” of bound antigen or addition of toll-like receptor 9 stimulation in some patients. Gene expression profiles differ between groups of CLL patients and in peripheral blood (PB) and LN compartment, due to different microenvironments. However, it is not known whether these differences also apply for DNA methylation. In paper III, we identified various genes that were alternatively methylated between IGHV mutated (M) and unmutated (UM) groups. For example prognostic genes, CLLU1 and LPL, genes involved in B cell signaling, IBTK, as well as numerous TGF-β and NF-κB/TNF pathway genes. The intensity and duration of BcR signals are fine-tuned by enhancing or inhibitory coreceptors. SHP-1 inhibits BcR-signals by dephosphorylation. In paper IV, we compared the expression and activity of SHP-1 in CLL cells from LN with matched PB samples. However, in contrast to our hypothesis, SHP-1 activity/phosphorylation status in PB and LN, did not differ significantly. This thesis, add another piece to the puzzle, on how the microenvironment and antigens influence CLL pathogenesis. Since great variations among individuals are seen, further studies in different groups of patients are necessary to elucidate the importance of antigen for the development of CLL.

Apoptosis is one type of programmed cell death, important during tissue development and to maintain the tissue homeostasis. Apoptosis comprises a complex network of internal signaling pathways, and an important part of this signaling network is the action of voltage‐gated ion channels. The aim of this thesis was to explore the role of ion channels and the role of intracellular metal ions during apoptosis in Xenopus laevis oocytes. The reasons for using these oocytes are that they are large, robust, easy to handle, and easy to study electrophysiologically. Apoptosis was induced either chemically by incubation of the oocytes in staurosporine (STS) or mechanically by centrifugation of the oocytes. Ion currents were measured by a two‐electrode voltage clamp technique, intracellular ion concentrations were measured either directly by in‐house developed K+‐selective microelectrodes or indirectly by the electrophysiological technique, and apoptosis was measured by caspase‐3 activation. Paper I describes that the intracellular K+ concentration was reduced by about 30 % during STS‐induced apoptosis. However, this reduction was prevented by excessive expression of exogenous ion channels. Despite the magnitude of the intracellular K+ concentration, either normal or reduced level, the oocytes displayed normal signs of apoptosis, suggesting that the intracellular K+ reduction was not required for the apoptotic process. Because the intracellular K+ concentration was not critical for apoptosis we searched for other ion fluxes by exploring the electrophysiological properties of X. laevis oocytes. Paper II, describes a non‐inactivating Na+ current activated at positive membrane voltages that was upregulated by a factor of five during STS‐induced apoptosis. By preventing influx of Na+, the apoptotic signaling network involving capsase‐3 was prevented. To molecularly identify this voltage‐gated Na channel, the X. tropicalis genome and conserved regions of the human SCNA genes were used as a map. Paper III, shows that the voltage‐gated Na channel corresponds to the SCN2A gene ortholog and that supression of this SCN2A ortholog using miRNA prevented cell death. In conclusion, this thesis work demonstrated that a voltage‐gated Na channel is critical for the apoptotic process in X. laevis oocytes by increasing the intracellular Na+ concentration.

Samverkan i team vid akutmottagningar är essentiellt för att främja en god, patientsäker vård. En fungerande teamsamverkan påverkas av flera faktorer. Syftet med litteraturstudien var att sammanställa faktorer som påverkar teamsamverkan vid akutmottagningar. En integrerad litteraturöversikt genomfördes och två frågeställningar formulerades, vilka faktorer främjar respektive motverkar teamsamverkan. 13 vetenskapliga artiklar med kvalitativ och kvantitativ ansats analyserades. I resultatet framkom fem grupper med faktorer som främjade teamsamverkan, god kommunikation, tydligt ledarskap, respekt och förtroende, positiv inställning och teamträning. Fyra grupper med faktorer som motverkade teamsamverkan framkom, bristande kommunikation, hierarki, bristande kompetens och personalbrist. Slutsatsen drogs att kommunikation är nyckeln för en fungerande teamsamverkan och denna kan stödjas genom teamträning. Vikten av fortsatt forskning inom området teamsamverkan anses betydande för att öka vårdkvalitet och patientsäkerhet.

Abstract Background Decision-making is a process that the dentist does every day, and which is based on experience and the patients status. Many factors influence the decision of the therapy regarding extraction or endodontic treatment such as stress, economy, lack of time, acutepain and tooth status. This means that the choice of treatment does not become obvious when choosing between these two therapies. Aim The purpose of the study is to investigate whether the dentists perform adequate treatments regarding extraction versus endodontics, based on the data provided to the participants. Methods Nine different cases are sent to nine different clinics in Västerbotten with access to patient information. Patient material in the form of X-rays is deidentified. Each patient case was assigned a code. Information letters, the questionnaire and the cases were sent digitally via e-mail. Result 20 dentists answered the survey out of 98 dentists who were included in the study. One of the questionnaires was excluded due to incomplete answers which resulted in a total of 19 complete responses. 35% of the answers chose extraction treatment, 33% endodontic treatment and 32% of the answers could not suggest a treatment. Conclusion In consultation with an endodontic specialist, one third of the dentists in Västerbotten do not make adequate therapy decisions. However, it is not possible to draw any general conclusions due to the large dropout.

Macrotroponin, formed by the complexation of endogenous cardiac troponin autoantibodies with circulating cardiac troponin, poses a challenge in high-sensitive assays generating high values without cardiac damage. Treatment of blood samples with polyethylene glycol (PEG) precipitates biomacromolecules, including macrotroponin, thereby freeing up smaller molecules, such as monomeric troponin in solution. While previous studies have predominantly examined the impact on recovery of PEG-precipitation on troponin I assays, this pilot study sought to explore its effects on routine samples using a troponin T assay and how the PEG-precipitation method affects the recovery of troponins and other proteins of varying sizes, including ferritin, albumin, TSH, cystatin C, prolactin, IgA, IgM, and IgG. PEG-precipitation was employed as the separation method and conducted on 20 different troponin T samples, with the yield of troponin and other proteins examined, and also on a series of dilutions of one and the same sample with initially high troponin concentration. To estimate the robustness of the PEG precipitation method, all samples were measured as duplicates.  For both the 20 individual samples and the diluted sample, the recovery of troponin decreased significantly with increasing troponin concentrations. This has not been previously documented in the literature. The finding implies that when employing PEG-precipitation to identify or rule out antibody-induced interference in troponin assays, it is imperative to account for the total concentration to evaluate the exchange rate. Furthermore, the PEG-precipitation method seems quite robust according to the low coefficient of variation for duplicate measurement.