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Normal function of all epithelia relies on the correct polarized localization of ion transport proteins, and malfunctions in the trafficking of these proteins to the plasma membrane contribute to numerous diseases. Na+, HCO3− cotransporter NBCn1 (SLC4A7) is a major regulator of pHi in most cell types with important functions in the cardiovascular system, the kidneys, and other organ systems. The dysfunction of NBCn1 is implicated in several diseases, includig hypertension and cancer. In particular, NBCn1 plays a central role in breast cancer. Despite the importance of NBCn1 in physiology and disease, very little is known about the regulation and roles of NBCn1. Specifically, there is a near-complete lack of knowledge regarding NBCn1 biosynthesis, trafficking, and degradation pathways. Prior to this study, our lab performed mass spectrometry analysis of interaction partners of, revealing several putative C-tail interactions candidates. In this study, we performed co-IP to validate these suggested interactions partners. We found that NBCn1 interacts with γ-adaptin, suggesting NBCn1 sorting to the membrane via AP-1 clathrin-coated vesicles. Morover, we foung that NBCn1 interacts with lateral polaity proteins LLGL-1 and DLG, suggesting a lateral targeting pattern for NBCb1. Lastly, we found that scaffold protein RACK1 interacts with NBCn1 and that the proximal 22 amino acids of the C-tail are sufficient for RACK1 interaction.

Bone fractures are associated with morbidity and mortality and constitutes a major economic burden for societies. It is estimated that 43.300 lives were lost as a result of incident fractures in EU in 2010. In recent years, there has been an increasing appreciation of the therapeutic potentials of purinergic signalling as a toolkit for tissue repair. Nucleotides are released into the extracellular environment due to tissue stress, and here they are found to serve as a damage signal by acting on purinergic receptors. Purinergic receptors are found on the cell surface of almost all cell types where they propagate downstream signalling. Although purinergic signalling has been linked to very central roles in bone biology, such as differentiation of mesenchymal and hematopoietic stem cell into osteoblast and osteoclast, bone and cartilage mineralisation and biomechanical properties, the involvement of P2 receptors in fracture healing remains to be elucidated. We investigated the association of P2X4R, P2X7R and P2Y2R gene expression to fracture healing. A closed unilateral fracture model of mid-diaphysal tibia, was applied to 7 month old female Sprague Dawley (SD) rats. The rats were terminated for expression analysis using RT-PCR on day 1, 2, 3, 7 and 21 and for bone property analysis by x-ray and Dual-energy x-ray absorptiometry on day 7 and 21. External callus formation and mineralisation was seen on day 21 by x-ray and DEXA. BMD was increased from 0.165 g/cm2 on day 7 to 0.182 g/cm2 on day 21 (p=0.021, fig 12 B). BMC was increase from 0.060 g on day 7 to 0.106 g on day 21 (p=0.021, fig 12 C). Bone area was increased from 0.378 mm2 on day 7, to 0.565 mm2 on day 21 (p=0.021, fig 12 D).RNA extraction from bone is complicated by the hard nature of bone tissue. A protocol for homogenization and RNA extraction from bone tissue was successfully developed. Bone tissue was homogenized and lysed using a bead system containing matrix A (Biomedicals), QIAzol (Qiagen) and Fastprep 120. RNA was extracted with RNeasy (Qiagen). Primers were designed and tested on hepatic tissue lysate, which has been reported to have a high expression of target receptors. Specificity to gene expression was achieved with primers that specifically annealed to cDNA deriving from spliced mRNA expression of Actb, P2X4R, P2X7R and P2Y2R gene. Expression of P2X4 was seen on day 1 in RNA extracts from fractured and non- fractured tibias. Due to technical challenges along the way and the limitation in time, analysis of gene expression in the fracture samples are still pending.

Lithium has been used for several decades as a drug for treatment of bipolar diseases. It still results to be a mainstay in the treatment of manic-depressive disorders, even though the precise molecular mechanisms underlying its therapeutic function are not yet fully elucidated. There is evidence that chronic lithium treatment has resulted in kidney disease such as polyuria. In this research it is shown that polyuria has probably developed as result of lithium induced cell cycle arrest. The toxic effect of lithium was tested in A6 cell line which represents tight epithelial cell culture from the distal part of the kidney. Tight junctions, present in this type of epithelium, seal paracellular spaces in between cells making the epithelium less permeable to water and ions, which in turn contributes in adjustment of urine composition. A6 cell line is often used as a model system for hormonal regulation of ion and water transport in the distal part of the kidney. The proliferation rate of A6 cells cultured in 24 well microtiter plates, was investigated for 6 days in control and cells exposed to 1, 10 and 20 mM of lithium. The proliferation rate was also investigated after 6 days of exposure, where media was replaced with lithium free media. Microscope analyses by ImageJ showed that cells exposed to 10 and 20 mM of lithium were strongly inhibited after 24h of exposure, while 1 mM treated cells developed the confluent layer the same way as control, after 3-4 days, showing no signs of inhibition. After lithium removal, cells exposed to two highest concentrations were able to grow and reestablish cell to cell contact, and form the confluent epithelia after 4 days. Measurements showed no sign of cell death. The experiment was designed the same way, and cell number was measured by Coulter counter. Results showed again dose- and time-dependent inhibition in 10 and 20 mM treated cells, the reversible effect of lithium was evident in this case too, indicating lithium as an inhibitor of cell cycle. Analyses by Coulter counter also carried out in Caco2 cells, a tight epithelial colorectal cell line. The results totally agreed with those of experiment in A6 cell. In order to investigate where in cell cycle the A6 cells were stopped, cell cycle analyses with flowcytometry by staining DNA content with propidium iodide and DAPI staining of DNA content was performed. Flowcytometric analyses showed that10 mM Li induced an accumulation of cells in the G0/G1 phase after 48 h and 72 h treatment and 20 mM Li induced an accumulation of cells the S phase after 24 h and 48 h treatment. No DNA fragmentation and the absence of apoptotic bodies due to Li exposure were also shown by flowcytometry analyses. DAPI staining analyses, apart from increase in amount of cells in S phase in 20 mM Li after 24, 48 and 72 h, showed also an increase in subpopulation of cells at G2/M phase after 24, 48 and 72 h of exposure. Assessment of GSK-3β was made by western blotting, to show whether it represents a target of Li in A6 cells. Li interference with organization of primary cilia was also investigated by performing immunofluorescence microscope analysis.

At kunne opbevare biologisk materiale, såsom celler, i længere tid uden for organismen, uden tab af viabilitet, er et vigtigt element i sygdomsbehandling. En ofte anvendt metode i denne sammenhæng er kryopræservering, selvom der er en række problemer forbundet med denne metode. Under kryopræservering af biologisk materiale udsættes cellerne for en række skadelige processer. En af disse processer er en dynamisk omstrukturering af iskrystallerne kaldet rekrystallisation. Antifryseproteiner (AFP), der indgår som et adaptivt forsvar mod isdannelse i en række forskellige organismer, har vist sig at modvirke rekrystallisation. Det er derfor blevet undersøgt i fryseforsøg, om proteinerne i kraft af denne egenskab, kan forbedre cellers overlevelsesmuligheder ved kryopræservering. Indtil videre er kun AFP fra fisk blevet undersøgt i denne sammenhæng, og disse har vist sig kun at være fordelagtige ved lave koncentrationer. Ved høje koncentrationer forårsager proteinerne skadelig spikulær isvækst, hvilket skader cellerne, og nedsætter overlevelsen kraftigt. AFP fra insekter binder til is på en anden måde, og forårsager ikke denne skadelige form for isvækst. I dette projekt blev der undersøgt, om AFP fra insekter kunne anvendes til kryopræservering med større succes. Suspenderede nyreceller af cellelinjen A6 blev kortvarigt kryopræserveret i ringermedie med 10 % DMSO ved -196 °C. Mediet var tilsat forskellige proteinopløsninger, herunder AFP fra billen Rhagium mordax, kontrolproteinerne Glutathion-S-Transferase (GST) eller albumin (BSA) eller en blanding af både GST og AFP. Efter kryopræserveringen blev cellernes viabilitet bestemt via farvning med trypan blå. I forsøgene blev parametre, der er særligt vigtige for cellernes overlevelsesmuligheder, kontrolleret, og nogle af disse blev varieret forsøgene imellem, for at undersøge hvilke skadelige processer AFP havde indvirkning på. Resultaterne viste, at tilsætning af AFP medførte enten en klar forbedring af overlevelsen eller en klar forringelse. Opfølgende forsøg viste, at denne observation sandsynligvis kunne relateres til en varierende grad af underafkøling, der skete i prøverne, før isdannelsen indtræf. Underafkøling associeres med en forbedring af overlevelsen, mens manglen på samme medfører en forringelse. En modsat tendens blev observeret for kontrolproteinerne. Dette tyder på at AFPs effekt er relateret til dynamikken omkring isdannelsen, og at denne effekt er proteinspecifik. Resultaterne viste også, at prøver med AFP beholdte en stabil overlevelsesrate, selvom varmeraten blev sænket, og omfanget af skadelig rekrystallisation normalt ville øges. I prøver uden AFP havde en lav varmerate større konsekvens for overlevelsesraten. Det blev også påvist at høje koncentrationer af AFP kunne med fordel anvendes, og bekræfter altså hypotesen om, at man ved brug af AFP fra insekter, kan undgå den skadelige ismorfologi, der følger med høje koncentrationer af AFP fra fisk. Udfordringen er nu at kunne styre den umiddelbare tilfældige opdeling af overlevelsesraterne, som disse forsøg viser, der følger med brugen af AFP fra insekter. Hvis dette lykkedes, kan AFP fra insekter med fordel anvendes til kryopræservering frem for AFP fra fisk. Forsøgene viste også, at GST, som blot var tiltænkt at fungere som kontrolprotein, havde en stabil positiv effekt på cellernes overlevelsesrater. Proteinets funktion er at neutralisere reaktive elektrofile komponenter, og den positive effekt, der blev observeret i kryopræserveringen, er sandsynligvis forbundet med dette. GST yder dog ikke beskyttelse mod rekrystallisation i samme grad som AFP, så disse to proteiner kan med fordel kombineres. GST og AFP har altså her vist, at kunne forbedre kryopræserveringen af celler på forskellige måder, og det må forventes at flere andre proteiner ligeledes kan have en gavnlig effekt. At inddrage specifikke proteiner i kryomedier ser derfor ud til at være en god mulighed for at forbedre fremtidige præserveringer.

Formålet med ”Kropslugte – Lugte som biomarkører for cancer”, har været at finde en sammenhæng mellem flygtige forbindelser, der udskilles fra kræftpatienter, og molekylære sygdomsprocesser, der er relateret til cancer. To forklaringer på ændret lugt hos kræftpatienter kan være: 1. kræftcellers metabolisme, herunder forøget ”oxidativt stress” og lipid peroxidation. 2. Ændringer i immunforsvaret (”tumor escape”), herunder ”major histocompability complex-”/(MHC)-molekyler, der præsenterer antigener på celleoverfladen. Analyser med gaskromatografi-massespektrometri (GC-MS) og mønstergenkendelsesprogrammer har resulteret i en række forslag til markører for cancer, f.eks.: alkaner, methylerede alkaner, aldehyder, isopren, toluen, benzen, m.fl. Indikation for særlige lugte af forskellige kræfttyper (”signaturlugte”) er givet med både GC/MS, ”elektroniske næser” (”gas sensor array”) og ved brug af detektorhunde (”cancer detector dogs”).

Previous studies have demonstrated that classical benzodiazepines decrease hypothalamic–pituitary–adrenocortical cortex (HPA) axis activity. Paradoxically, high doses of enzodiazepines also stimulate basal circulating corticosterone levels in some conditions. Because benzodiazepine agonists display little selectivity to any of the α subtypes of the γ-amino butyric acid GABAA receptor to which they bind, we propose that the unequivocal results are due to an a subtype-dependent modulation of the hypothalamic–pituitary–adrenocortical cortex axis output. To test this, basal hormonal output and induction of FOS in the hypothalamic paraventricular nucleus were measured after administration of various benzodiazepine ligands in mice. Zolpidem, a selective α1 subtype agonist, produced a very strong increase in plasma adrenocorticotropic hormone and corticosterone. The non-selective full agonist’s diazepam and zopiclone induced a lower increase in circulating corticosterone than after zolpidem. In contrast, the α2,3,5 selective benzodiazepine agonist and α1 antagonist L-838,417 had no effect on corticosterone levels. Strong induction of FOS in the paraventricular nucleus was found in response to zolpidem, diazepam, and zopiclone, but not L-838,417. Finally, pre-administration of L-838,417 prior to zolpidem strongly inhibited the effect of zolpidem on corticosterone. Likewise, the non-selective agonist’s diazepam and zopiclone at a dose that alone had no effect on corticosterone also inhibited the effect of zolpidem. Taken together, these results suggest that benzodiazepine ligands modulate the hypothalamic–pituitary–adrenocortical axis through partly opposite mechanisms; and that the net effect is dependent on the composition of the GABAA receptor subunits to which they bind. In order to further explain the biological mechanism by which the different benzodiazepins activate the hypothalamic–pituitary–adrenocortical axis, we performed in situ hybridization to clarify the GABAA subunit composition in the paraventricular nucleus and sub paraventricular area, we show in agreement with the available literature that the paraventricular nucleus is comprised of neurons with the α2 phenotype. We also find that the sub paraventricular nucleus is made up of neurons with the α1 phenotype this has not been shown elsewhere. In this study we also conclude that the neurons in the paraventricular nucleus that are activated after zolpidem administration do not co-express oxytocin to any significant degree. The study also makes an attempt to map afferent connection to the paraventricular nucleus, this is done using Cholera toxin subunit B as a retrograde tracer. We focuses the projections origination in the bed nucleus stria terminalis, and in agreement with the available literature find that the bed nucleus stria terminalis has numerous projections to the paraventricular nucleus, we also find that most of these projections originate in either the lateral division or in the medial division, anterior part of the bed nucleus stria terminalis.