A new detection reagent that could possibly augment or replace antibodies research and diagnosis methods are aptamers. Aptamers are ssDNA, RNA or polypeptide constructs that function like active antibodies. Antibodies and aptamers both specifically bind to selected target molecules, and as such they enable the detection or targeting of the presence or absence of a specific antigen. In order to ensure that ssDNA aptamers perform similarly to antibodies, anti-VEGF-A polyclonal antibody and anti-VEGF-A ssDNA aptamer were evaluated against vascular endothelial growth factor A (VEGF-A) using Surface Plasmon Resonance (SPR). It was hypothesized that the anti-VEGF-A aptamer had the same, if not better, binding kinetics than the anti-VEGF-A polyclonal antibody, and as such offers an ideal replacement for use in in field, real-time testing assays. SPR revealed that both the polyclonal antibody and ssDNA aptamer bound the target antigen, VEGF-A. Additionally, from the SPR kinetic analysis, the anti-VEGF-A aptamer had KD values of 20-28 nM and the anti-VEGF-A antibody had KD values of 16-127 uM. The binding efficacy of the aptamer was several orders of magnitude better than that of the antibody. The aptamer was also stable in solution for a longer amount of time than the antibody, which denatured in solution after two weeks.

Patients with classical vitiligo lose partially their protecting inherited pigment. The cause of the disease is still unknown. Despite massive epidermal oxidative / nitrative stress and signs for DNA-damage in the skin and in the plasma, these patients have no higher prevalence for sun induced non-melanoma skin cancer and increased photo-damage. Protection and DNA-repair have been attributed to a functioning up-regulated wild type p53 / p21 cascade in association with up-regulated p76 MDM2. As some patients with cutaneous melanoma develop depigmentations away from their primary tumour site post surgical excision, it became of our interest, whether this melanoma associated leucoderma (MAL) is the same as classical vitiligo. The purpose of this thesis was two-fold. In part I, we wanted to further substantiate the reasons behind the constantly up-regulated wild-type functioning p53 / p21 cascade in classical vitiligo utilising a panel of proteins with direct and / or indirect action on p53 regulation, including p21, p76MDM2, MDM4/MDM4phospho, SPARC, VEGF-A and TGF-β1. In part II, we wanted to characterize MAL and compare this peculiar leucoderma with classical vitiligo using the same protein panel and methodologies. To achieve our goals, we used in vivo FT-Raman spectroscopy, in vitro cell cultures, in vitro and in situ immuno-fluorescence labelling, Western blot, dot blot and computer modelling techniques. Our data showed distinct differences between classical vitiligo and MAL. Our results in MAL exhibited a concentration dependent protein expression gradient between the basal / suprabasl layers and the upper layers of the epidermal compartment using catalase, ONOO-, p53, p21, MDM4, p76MDM2, TGF-β1 and VEGF-A expression gradient. Moreover, we document for the first time the presence of a nitrated non-fuctional SPARC protein in classical vitiligo which is absent in MAL. Although we show in vivo considerable ROS / RNS- mediated stress in MAL and classical vitiligo documented by FT-Raman spectroscopy, Western blot and in situ immuno-fluorescence, our results prove that MAL and classical vitiligo are two distinct entities.

Dedifferentiation and proliferation of retinal pigment epithelial (RPE) cells are characteristics of retinal diseases. Dedifferentiation is likely associated with changes of inwardly rectifying potassium (Kir) channels. The roles of Kir4.2 channels in viability, and proliferation of cultured RPE cells were investigated. Gene expression levels were determined using qRT-PCR. RPE cells expressed Kir2.1, 2.2, 2.4, 3.2, 4.1, 4.2, 6.1, and 7.1 mRNA. Kir4.2 protein was verified by immunocytochemistry and Western blotting. Kir4.2 mRNA in cultured cells was upregulated by hypoxia (hypoxia mimetic CoCl2 or 0.2% O2) and extracellular hyperosmolarity (addition of high NaCl or sucrose). Kir4.2 mRNA was suppressed by vascular endothelial growth factor (VEGF), blood serum, and thrombin whereas platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and transforming growth factor-1 (TGF-1) increased it. Hyperosmotic Kir4.2 gene expression was mediated by TGF-1 receptor signaling while hypoxic gene transcription was dependent on PDGF receptor signaling. VEGF receptor-2 blockade increased Kir4.2 mRNA level under control, hyperosmotic, and hypoxic conditions. SiRNA-mediated knockdown of Kir4.2 decreased the cell viability and proliferation under control and hyperosmotic conditions. Kir4.2 channels play functional roles in maintaining the viability and proliferation of RPE cells. Downregulation of Kir4.2 by VEGF, via activation of VEGF receptor-2 and induction of blood-retinal barrier breakdown, may contribute to decreased viability of RPE cells under pathological conditions.

No description available.

No description available.

No description available.

No description available.

Cancer is still one of the most common causes of death world-wide and in parallel there is a need to update the repertoire of therapies that withstand resistance of recurrent cancers. Since the introduction of antibody therapies as anti-cancer pharmaceuticals, recognized as immunotherapy in health care, it has been an increasing field in cancer therapy, as a more targeted treatment compared to chemotherapy. Despite the great success, immunotherapy rely on parenteral administration, partly due to poor tissue penetration. If the treatment is administered intravenously, specialized personnel is required, in addition to that it can be inconvenient for the patient. Also, pharmaceuticals based on antibodies often require costly production steps which yields a high-priced treatment. To approach this problem, researchers have developed small affinity domains with the aim to increase tissue penetration while keeping a high specificity to its target. Albumin Binding Domain Derived Affinity Protein (ADAPT) is an example of a small affinity domain of only 7 kDa, which is based on albumin binding domain (ABD) from the streptococcal protein G. Recently, it was shown that the ADAPTs can be further engineered to bind albumin and another relevant target protein of interest simultaneously, which suggests a tolerable half-life in patient serum, alternative administration routes and lower production costs compared to antibody treatments. Furthermore, less side effects are expected due to higher specificity compared to chemotherapy. This work presents the characterization of novel ADAPT proteins that the target the cancer relatedproteins C-C motif ligand 7 (CCL7), vascular endothelial growth factor A (VEGF-A) and carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5). The new constructs were produced recombinantly in Escherichia coli (E. coli) and purified using affinity chromatography. Moreover, the results demonstrate bispecific binding with high affinity towards serum albumin and CCL7 and CEACAM5 respectively, while the ADAPT variants targeting VEGF-A remain to be further developed. Lastly, the importance of different amino acids for structural and binding properties of one CEACAM5 binder are stated. It reveals that the target binding relies on hydrophobic interactions which also can be connected to its poor structural attributes. Accordingly, this project adds new insights about the ADAPTs which can be useful in research towards future clinical applications aimed to improve cancer treatments.

Patients on peritoneal dialysis (PD) are reliant on the peritoneum to provide a semi-permeable barrier to allow for dialysis (solute clearance), salt and water removal (ultrafiltration). PD patients are at risk of developing peritoneal fibrosis and angiogenesis which can lead to a decline in peritoneal membrane function. Specifically, PD patients develop increased solute transport and decreased osmotic conductance leading to ultrafiltration failure. Peritoneal angiogenesis is the leading factor that results in augmented peritoneal membrane solute transport which is associated with worse outcomes – increased risk of mortality and PD technique failure. Transforming growth factor beta (TGFB) is one of the primary cytokines involved in inducing epithelial to mesenchymal transition (EMT) and fibrosis. We hypothesize that PD leads to injury of the epithelial lining of the peritoneum – the mesothelial cells. These cells undergo a transition process and transitioned mesothelium are a source for angiogenic and fibrogenic growth factors. Matrix Metalloproteinase (MMP) 9 is an angiogeneic factor and has been observed to correlate with increased expression of vascular endothelial growth factor (VEGF). MMP9 has the ability to cleave and activate membrane bound factors such as E-cadherin and b-catenin respectively. There is substantial evidence that the canonical WNT/b-catenin pathway is active during fibrosis, and angiogenesis in different biological contexts. Thus, we investigated the role of MMP9 and WNT signaling in peritoneal angiogenesis. Limited evidence exists describing the role of noncanonical WNT signaling but some reports suggest that non-canonical WNT signaling inhibits WNT/b-catenin signaling. Non-canonical WNT5A has differential effects based on receptor context and has been shown to block WNT/b-catenin signaling in the presence of Receptor Tyrosine Kinase Like Orphan Receptor 2 (Ror2). The overall hypothesis of this PhD thesis is that MMP9 and WNT signaling play a key role in inducing peritoneal angiogenesis and are associated with changes in peritoneal membrane function. We expect WNT5A and Ror2 to protect against peritoneal membrane injury. From the overnight effluent of stable PD patients, we cultured mesothelial cells and assayed these for expression of MMP and WNT related genes. MMP9 and WNT1 gene expression were observed to be strongly correlated with peritoneal membrane solute transport in patients on PD. WNT2 mRNA was also positively correlated with peritoneal solute transport. We overexpressed MMP9 in the mouse peritoneum to demonstrate its role in angiogenesis and confirmed these findings using MMP9 -/- mice. In addition to this, we have shown a novel mechanism by which MMP9 induces angiogenesis by E-cadherin cleavage and b-catenin mediated signaling. The observed cross-talk between MMP9 and b-catenin prompted investigation of the activation of canonical WNT/b-catenin signaling in development of peritoneal membrane injury. In an experimental model of TGFB induced pertioneal injury, we confirmed the activation of WNT/b-catenin signaling. In addition to this we, we blocked the WNT pathway and observed that WNT/b-catenin signaling is required to induce peritoneal angiogenesis. WNT5A mRNA was downregulated during TGFB induced injury suggesting a more protective role. Furthermore, several studies have demonstrated its ability to antagonize the WNT/b-catenin signaling pathway. We demonstrated that WNT5A protected against angiogenesis by blocking the canonical WNT pathway. WNT5A is thought to antagonize the WNT/b-catenin signaling pathway by signaling through receptor Ror2. In cell culture, we overexpressed TGFB and blocked Ror2. This resulted in elevated levels of VEGF and fibronectin suggesting that Ror2 is involved in mediating protection. Therefore, Ror2 possesses the ability to regulate VEGF and may be a potential candidate by which WNT5A mediates its protective effects. In conclusion, our findings identified MMP9 and WNT1 as potential biomarkers of increased peritoneal solute transport in patients that are on PD. We have also found a novel mechanism by which MMP9 interacts with b-catenin to induce peritoneal angiogenesis and have provided a first look at WNT/b-catenin signaling in peritoneal angiogenesis. Lastly, we have shown WNT5A to protect against peritoneal angiogenesis. Taken together, our findings are not only significant to the realm of PD research but hold wide applicability to research in the biomedical sciences. / Thesis / Doctor of Philosophy (PhD)

Angiogenesis is the formation of new blood vessels from pre-existing ones. Unregulated angiogenesis is associated with several diseases such as diabetic retinopathy and tumour growth. Many signal transduction pathways have been implicated in the regulation of angiogenesis such as p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K), extracellular signal-related kinase 1/2 (Erk1/2) and of particular interest the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. Inhibition of calcineurin activity by the drug cyclopsorin A (CsA) has been shown to inhibit processes required for successful angiogenesis such as in vitro cell migration, tube formation and additionally attenuates corneal angiogenesis in vivo. CsA is associated with severe side effects and therefore the identification of an endogenous regulator of this pathway would be beneficial. One possibility is the plasma membrane calcium ATPases (PMCAs). These high affinity calcium extrusion pumps have been shown to interact with calcineurin in mammalian cells and cardiomyocytes and down-regulate the calcineurin/NFAT pathway. This is hypothesised to be due to the interaction between the two proteins which maintains calcineurin in a low calcium micro-environment generated by the calcium removal function of the pump. Interestingly, PMCA4 has been shown to interact with calcineurin in endothelial cells. The aim of our study was to further our understanding of PMCA4s regulation of the calcineurin/NFAT pathway specifically in endothelial cells and establish if PMCA4 has a role in the regulation of angiogenesis. ‘Gain of function’ by adenoviral over-expression of PMCA4 and ‘loss of function’ by either si-RNA mediated knockdown of PMCA4 or isolation of PMCA4-/- MLEC were used as models. Over-expression of PMCA4 in HUVEC resulted in inhibition of the calcineurin/NFAT pathway with the opposite result occurring in the case of the knockout of PMCA4, identifying PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells. Over-expression of PMCA4 significantly attenuated VEGF-induced protein and mRNA expression of the pro-angiogenic proteins RCAN1.4 and Cox-2, endothelial cell migration and in vitro and in vivo tube formation with the opposite result occurring in knockdown or knockout studies, confirming PMCA4 as a down-regulator of angiogenesis. Interestingly, over-expression or knockdown of PMCA4 had no effect on VEGF-induced HUVEC proliferation or Erk1/2 phopshorylation proposing PMCA4 may be a potential inhibitor of angiogenesis without compromising cell survival. Disruption of the interaction between PMCA4 and calcineurin by generation and ectopic expression of an adenovirus encoding the region of PMCA4 that interacts with calcineurin (428-651) (Ad-ID4) resulted in an increase in NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify the mechanism of PMCA4s inhibitory effect of the calcineurin/NFAT pathway and consequently angiogenesis is a result of the interaction between the two proteins. The novel findings of this study establish PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells and angiogenesis. These results are far reaching and highlight a potential role for PMCA4 as a therapeutic target in a variety of diseases that are associated with pathological angiogenesis.