Diabetes or diabetes mellitus which is the correct medical term is a medical condition were the affected person lack the ability to regulate his or her blood glucose levels. This inability is directly due to the fact that the insulin producing cells, residing in the pancreas, can’t meet the body’s demand for insulin. It is estimated that close to 200 million people are suffering from diabetes today and this number is predicted to double within 20 years. Of the approximately 200 million people suffering from diabetes today approximately 20 million are in dependent on daily injections of insulin. Being dependent on exogenous insulin is not only an inconvenience it also increase the risk for several medical complications such as stroke, heart disorders, kidney failure, retinopathy, atherosclerosis and impaired wound healing. The major risk factor for all these complications is long periods of high blood sugar levels that is damaging to thin blood vessels and nerves.  Even in the best of situations the blood sugar levels of a diabetic with need for daily insulin injections can never be as well controlled as in a healthy individual. Increased understanding in the developmental processes behind the formation of the pancreas, and more specifically the insulin producing β-cells could result in new treatments for diabetics. By imitating the in vivo conditions generating pancreatic development scientist are now able to induce embryonic stem cells to differentiate into pancreatic progenitors as well as insulin producing β-cells in vitro. These in vitro generated pancreatic cells might in the future serve as a donor source for transplantations, thereby restoring the insulin producing capability of diabetic patients. An alternative approach to restore insulin production in diabetics is to influence cells in the pancreas to generate more insulin producing cells. To successfully achieve this, what cell types have the capacity to generate β-cells needs to be appreciated. In this thesis papers concerning in vitro differentiating of embryonic stem cells towards a pancreatic fate as well as in vivo studies in basic pancreas development are presented and discussed.

Fibrosis is a sequel of chronic inflammation and is defined as an excessive deposition of collagen that ultimately leads to organ dysfunction. To date there are no effective treatments for fibrosis. The main cell type involved in collagen deposition and organization is the myofibroblast. In the first study we examined how myofibroblasts differentiate in human fibrotic conditions and in experimental animal models. Human tissues were stained with antibodies that recognize integrin receptors and in addition we also stained for α-SMA, a myofibroblast marker. We found a co-localization between these two markers in stromal cells and hypothesized that integrin α1 is important for the acquisition of the myofibroblast phenotype. To tests this hypothesis we used knockout animals for this integrin subunit. These animals showed a reduction of α-SMA positive fibroblasts, indicating that the α1 integrin subunit is required for proper myofibroblast differentiation. In the second study we used a neuroblastoma tumor model to study tumour growth when a drug targeting the synthesis of cellular NAD was administered. In treated animals an expansion of the nonvascular stroma was observed compared to controls. Normalization of the vasculature was observed in treated tumors together with a decrease in hypoxia. Moreover, this was followed by a decrease in stromal PDGF-B and VEGF expression, suggesting a deactivation of the stroma. In the third study the effects of over-expression of the two pro-fibrotic growth factors TGF-β and PDGF-B in skin was evaluated. We observed that both growth factors induced fibrosis. Over time, a decrease in blood vessel density was observed in both treatment groups. Both factors also stimulated an expansion of the connective tissue cell population originating from the microvascular pericyte, but the phenotype of these cells differed in the different treatments with regards to expression of markers. Furthermore, in tissue over-expressing PDGF-B but not TGF-β, the fibrotic process was partially reversible.

Methods that enable interrogation of multiple genomic regions in parallel are very useful for efficient detection of genetic variation. Two different types of probes are described in this thesis that can be used for direct analysis or for sample preparation upstream of Next Generation Sequencing.  In addition to the development of molecular probing systems it also reports on the progress of two assay formats for biological experiments. The Selector probe enrich for genomic regions of interest by probe mediated specific circularization of target fragments. Amplification based enrichment of circles can be carried out using polymerase chain reaction, rolling-circle amplification or multiple displacement amplification. Enrichment of all exons in 28 genes known to be mutated in lung and/or colon cancer is demonstrated.  Selection and analysis by SOLiD Sequencing was performed on fresh frozen and formalin fixed paraffin embedded (FFPE) samples, and mutations previously detected by Sanger sequencing were detected.  The extractor probe is another probe variant that can be used for multiplex enrichment of DNA. It targets genomic fragments by using both ligation and sequence specific elongation for discrimination between on and off target sequences. A microfluidic platform fabricated by compact disc injection molding that can be used for biological assays is described.  Microchannel structures in thermoplastic material are coated with silicon dioxide by electron beam evaporation which facilitates closing of the structures by PDMS- glass bonding by ozone plasma. The platform’s utility for biological experiments is demonstrated by for detection of amplified single molecules (ASM), cell culturing and on-chip peristaltic pumping. The thesis also includes an exploratory study for the purpose of using a non-optical system for detection of ASM’s.  Optimizations were performed of the conditions needed in order to detect an increase in hydrodynamic size of magnetic particles, using a superconducting quantum interference device (SQUID), as they form complex with ASM’s.

Inflammatory bowel disease (IBD) comprises diseases that are characterized by chronic or relapsing inflammation of the gastrointestinal tract. Primary sclerosing cholangitis (PSC) is an extraintestinal manifestation in IBD. Immunoreactivity against an autoantigen that is expressed both in the gastrointestinal tract and the biliary tract could be the link between these diseases. A possible source of such an antigen is goblet cells. Immunostainings of normal human tissues using IBD patient sera showed goblet cell immunoreactivity against goblet cells in all parts of the gastrointestinal tract. The most frequent immunostaining was found against goblet cells in the appendix against which 84% (42/50) of IBD patients compared to 8% (4/50) of healthy blood donors showed immunoreactivity. To identify the corresponding antigen we used three different approaches, investigation of immunoreactivity to different candidate proteins compared to IBD sera, immunoscreening of an appendiceal cDNA library, and immunoprecipitation of protein lysates from mucin producing cells followed by SDS-PAGE and 2D gel electrophoresis. These approaches led to the identification of several candidate autoantigens of which complement C3 is the most promising. A novel staining pattern with strong immunoreactivity to granules and the apical membrane of biliary epithelial cells was identified with 35% (12/34) of PSC sera compared to none of healthy controls (n=28). Screening of a cDNA library from normal human choledochus identified PDZ domain containing 1 (Pdzk1) and Glutathion S transferase theta 1 (GSTT1) as potential candidates. Pdzk1 is an interesting candidate which is expressed in the intestinal tract and bile ducts. GSTT1 antibodies were not specific for PSC and are thought to develop as an alloimmune response in patients with the GSTT1-null genotype. In conclusion, we have identified specific immunoreactivity to goblet cells and biliary epithelial cells using sera from patients with IBD and PSC respectively. We have also identified several potential autoantigens.

Targeted radiotherapy (TRT) of cancer is a promising approach that enables selective treatment of tumour cells, while sparing normal tissue. The humanized monoclonal antibody A33 (huA33) is a potential targeting agent for TRT of colorectal cancer, since its antigen is expressed in more than 95 % of all colorectal carcinomas. The aim of this thesis was to evaluate the therapeutic potential of the two huA33-based TRT-conjugates, 177Lu-huA33, and 211At-huA33. The conjugates 177Lu-huA33, and 211At-huA33, bound specifically to colorectal cancer cells, both in vitro and in vivo. A dose dependent cytotoxic effect of 211At-huA33 was also demonstrated in vitro. From a therapeutic perspective, both conjugates had a favourable biodistribution in tumour-bearing nude mice, with high tumour uptake and a low uptake in normal organs (with the exception of an expected thyroid uptake of 211At). After injection of 211At-huA33, the blood absorbed a slightly higher dose than the tumour, but for 177Lu-huA33, the tumour received a 12 times higher dose than blood. Two days after intravenous injection of 177Lu-huA33 in tumour-bearing mice, the tumours could be clearly visualised by gamma camera imaging, with very low interference from normal tissue radioactivity. In an experimental therapy study, also performed in tumour-bearing mice, there was an excellent therapeutic effect of 177Lu-huA33. About 50 % of the treated animals were tumour free 140 days after injection of 177Lu-huA33, while none of the non-radioactive controls survived beyond 20 days after injection of treatment substances. In conclusion, this thesis demonstrates that the therapeutic conjugates 177Lu-huA33, and 211At-huA33, are promising targeting agents that might help improve therapy of colorectal cancer.

Improved methods are needed to interrogate the genome and the proteome. Methods with high selectivity, wide dynamic range, and excellent precision, capable of simultaneously analyzing many biomolecules are required to decipher cellular function. This thesis describes a molecular and microfluidic toolbox designed with those criteria in mind. It also presents a tool for graphical representation of nucleic acid sequences. Proximity ligation is a novel protein detection method that requires dual and proximate binding of two oligonucleotide-tagged affinity reagents to a protein or protein complex in order to elicit a signal. The responses from such recognition reactions are the formation of specific nucleic acid reporter molecules that are subsequently amplified and quantitatively detected. A scalable microfluidic platform suitable for fluorescence detection, cell culture, and actuation is also described. The platform uses rapid injection molding to produce microstructures in thermoplastic materials. By applying a thin layer of silica to the structures, a lid made of silicone rubber coated onto a thermoplastic support can be covalently bonded to generate enclosed channels. A method is presented for precise biomolecule counting, termed “amplified single-molecule detection”. The method preserves the discrete nature of biomolecules, converting specific molecular recognition events to fluorescence-labeled micrometer-sized objects that are enumerated in microfluidic channels. I also present a novel microarray-based detection method. To attain high selectivity and a wide dynamic range, the method is based on dual recognition with enzymatic discrimination and amplification. Upon target recognition in solution, DNA probes are subjected to thousand-fold amplification in solution, followed by selective detection on arrays and another hundred-fold amplification of reporter molecule created from the first amplification reaction. Lastly, I describe a novel graphical representation of nucleic acid sequences using TrueType fonts that can be of value for visual inspection of DNA sequences and for teaching purposes

Methylglyoxal (MG), a highly reactive dicarbonyl accumulates after myocardial infarction (MI), causing adverse remodelling and cardiac dysfunction. We hypothesized that therapy using bone marrow cells (BMCs) overexpressing glyoxalase1 (Glo1), the main enzyme that metabolizes MG, injected into mouse MI model would translate into better survival of transplanted cells and improve their therapeutic effect. We found that Glo1 expression is significantly reduced at 7 days post-MI. Glo1 BMCs exposed to MG in vitro displayed greater angiogenic potential and reduced reactive oxygen species production compared to wild type (WT) BMCs. However, in the mouse MI model, Glo1 BMCs did not improve cardiac function or vascularity or reduce scar formation compared to WT BMCs and saline treatments. In conclusion, Glo1 overexpression in BMCs does not confer superior therapeutic efficacy for treating MI under the conditions tested.

No description available.

Myocardial infarction (MI) persists as one of the leading causes of death worldwide. Often patients whom survive the initial injury will develop heart failure characterized by a dilated and functionally incompetent heart. Heart failure (HF) carries a worse prognosis than most cancers, and the only curative therapy to date is heart transplantation. A better understanding of the repair and remodeling processes post-MI, and the development of novel therapies are required to combat this burgeoning medical challenge. This thesis research sought to identify a novel mediator of the impaired cardiac remodeling that often occurs post-MI, and to characterize a biomaterial hydrogel therapy as a novel treatment. We investigated the role of methylglyoxal (MG), an important precursor to advanced glycation end-products (AGE), using a transgenic mouse model to over-express glyoxalase 1 (GLO1). GLO1 is the primary enzyme involved in metabolizing MG and preventing its accumulation. The role for MG and AGEs in MI and HF had been alluded to in the literature, yet no study to date has causally linked them with the loss of function and impaired remodeling of the post-MI heart. We also assessed an injectable hydrogel for the treatment of MI using a mouse model and evaluated the impact of delivery timing on its therapeutic efficacy. In this thesis, we confirmed that MG derived AGEs accumulate post-MI (Chapter 3.1). We show that preventing their accumulation, through GLO1 over-expression, mitigates the loss of function post-MI and positively influences remodeling through reducing final infarct sizes and end-systolic volumes. We demonstrate that this may possibly occur through improving the bone marrow response post-MI by restoring ECM-cell signaling. In Chapter 3.2, we present results of a study assessing the efficacy of a collagen based injectable hydrogel for the treatment of MI, and assessing the role that timing plays into the benefits associated with this therapy by studying 3 separate timepoints including 3 hours, 7 days and 14 days post-MI. We found that the injectable hydrogel preserved cardiac function and reduced infarct sizes. It also positively interacted with the host repair response by reducing chronic inflammation and cell death. The benefits of the therapy depended on when the material was delivered, and we found that the earliest timepoint (3 hours post-MI) proved most beneficial. In Chapter 3.3, we combined the knowledge gained from Chapters 3.1 and 3.2 and functionalized our hydrogel with a flavonoid, Fisetin, that has been shown to scavenge MG and increase the activity of GLO1. We show that this novel functionalized material may be able to restore some function in MI, particularly in settings of low baseline cardiac function. Taken together, the results of this thesis demonstrate that MG accumulates as a result of the ischemia and contributes to the impaired repair resolution and remodeling processes post-MI. This identifies MG as a possible novel target for the treatment of MI. Indeed, we also confirm the role that delivery timing plays into injectable hydrogels post-MI, and present promising results for a functionalized material design to intervene on MG production.

No description available.