Molecular basis of the kidney filtration barrier : Role of the nephrin protein complex

The kidney filtration barrier consists of fenestrated endothelial cell layer, glomerular basement membrane and slit diaphragm (SD), the specialized junction between glomerular viscelar epithelial cells (podocytes). Podocyte injury is associated with the development of proteinuria, and if not reversed the injury will lead to permanent deterioration of the glomerular filter. The early events are characterized by disruption of the integrity of the SD, but the molecular pathways involved are not fully understood. Congenital nephrotic syndrome of the Finnish type (CNF) is caused by mutations in NPHS1, the gene encoding the SD protein nephrin. Lack of nephrin results in loss of the SD and massive proteinuria beginning before birth. Furthermore, nephrin expression is decreased in acquired human kidney diseases including diabetic nephropathy. This highlights the importance of nephrin and consequently SD in regulating the kidney filtration function. However, the precise molecular mechanism of how nephrin is involved in the formation of the SD is unknown. This thesis work aimed at clarifying the role of nephrin and its interaction partners in the formation of the SD. The purpose was to identify novel proteins that associate with nephrin in order to define the essential molecular complex required for the establishment of the SD. The aim was also to decipher the role of novel nephrin interacting proteins in podocytes. Nephrin binds to nephrin-like proteins Neph1 and Neph2, and to adherens junction protein P-cadherin. These interactions have been suggested to play a role in the formation of the SD. In this thesis work, we identified densin as a novel interaction partner for nephrin. Densin was localized to the SD and it was shown to bind to adherens junction protein beta-catenin. Furthermore, densin was shown to behave in a similar fashion as adherens junction proteins in cell-cell contacts. These results indicate that densin may play a role in cell adhesion and, therefore, may contribute to the formation of the SD together with nephrin and adherens junction proteins. Nephrin was also shown to bind to Neph3, which has been previously localized to the SD. Neph3 and Neph1 were shown to induce cell adhesion alone, whereas nephrin needed to trans-interact with Neph1 or Neph3 from the opposite cell surface in order to make cell-cell contacts. This was associated with the decreased tyrosine phosphorylation of nephrin. These data extend the current knowledge of the molecular composition of the nephrin protein complex at the SD and also provide novel insights of how the SD may be formed. This thesis work also showed that densin was up-regulated in the podocytes of CNF patients. Neph3 was up-regulated in nephrin deficient mouse kidneys, which share similar podocyte alterations and lack of the SD as observed in CNF patients podocytes. These data suggest that densin and Neph3 may have a role in the formation of morphological alterations in podocytes detected in CNF patients. Furthermore, this thesis work showed that deletion of beta-catenin specifically from adult mouse podocytes protected the mice from the development of adriamycin-induced podocyte injury and proteinuria compared to wild-type mice. These results show that beta-catenin play a role in the adriamycin induced podocyte injury. Podocyte injury is a hallmark in many kidney diseases and the changes observed in the podocytes of CNF patient share characteristics with injured podocytes observed in chronic kidney diseases. Therefore, the results obtained in this thesis work suggest that densin, Neph3 and beta-catenin participate in the molecular pathways which result in morphological alterations commonly detected in injured podocytes in kidney diseases.

Copper Diffusion Barrier Deposition on Integrated Circuit Devices by Atomic Layer Deposition Technique

Transfer from aluminum to copper metallization and decreasing feature size of integrated circuit devices generated a need for new diffusion barrier process. Copper metallization comprised entirely new process flow with new materials such as low-k insulators and etch stoppers, which made the diffusion barrier integration demanding. Atomic Layer Deposition technique was seen as one of the most promising techniques to deposit copper diffusion barrier for future devices. Atomic Layer Deposition technique was utilized to deposit titanium nitride, tungsten nitride, and tungsten nitride carbide diffusion barriers. Titanium nitride was deposited with a conventional process, and also with new in situ reduction process where titanium metal was used as a reducing agent. Tungsten nitride was deposited with a well-known process from tungsten hexafluoride and ammonia, but tungsten nitride carbide as a new material required a new process chemistry. In addition to material properties, the process integration for the copper metallization was studied making compatibility experiments on different surface materials. Based on these studies, titanium nitride and tungsten nitride processes were found to be incompatible with copper metal. However, tungsten nitride carbide film was compatible with copper and exhibited the most promising properties to be integrated for the copper metallization scheme. The process scale-up on 300 mm wafer comprised extensive film uniformity studies, which improved understanding of non-uniformity sources of the ALD growth and the process-specific requirements for the ALD reactor design. Based on these studies, it was discovered that the TiN process from titanium tetrachloride and ammonia required the reactor design of perpendicular flow for successful scale-up. The copper metallization scheme also includes process steps of the copper oxide reduction prior to the barrier deposition and the copper seed deposition prior to the copper metal deposition. Easy and simple copper oxide reduction process was developed, where the substrate was exposed gaseous reducing agent under vacuum and at elevated temperature. Because the reduction was observed efficient enough to reduce thick copper oxide film, the process was considered also as an alternative method to make the copper seed film via copper oxide reduction.

Studies on diamond-like carbon and novel diamond-like carbon polymer hybrid coatings deposited with filtered pulsed arc discharge method

The main obstacle for the application of high quality diamond-like carbon (DLC) coatings has been the lack of adhesion to the substrate as the coating thickness is increased. The aim of this study was to improve the filtered pulsed arc discharge (FPAD) method. With this method it is possible to achieve high DLC coating thicknesses necessary for practical applications. The energy of the carbon ions was measured with an optoelectronic time-of-flight method. An in situ cathode polishing system used for stabilizing the process yield and the carbon ion energies is presented. Simultaneously the quality of the coatings can be controlled. To optimise the quality of the deposition process a simple, fast and inexpensive method using silicon wafers as test substrates was developed. This method was used for evaluating the suitability of a simplified arc-discharge set-up for the deposition of the adhesion layer of DLC coatings. A whole new group of materials discovered by our research group, the diamond-like carbon polymer hybrid (DLC-p-h) coatings, is also presented. The parent polymers used in these novel coatings were polydimethylsiloxane (PDMS) and polytetrafluoroethylene (PTFE). The energy of the plasma ions was found to increase when the anode-cathode distance and the arc voltage were increased. A constant deposition rate for continuous coating runs was obtained with an in situ cathode polishing system. The novel DLC-p-h coatings were found to be water and oil repellent and harder than any polymers. The lowest sliding angle ever measured from a solid surface, 0.15 ± 0.03°, was measured on a DLC-PDMS-h coating. In the FPAD system carbon ions can be accelerated to high energies (≈ 1 keV) necessary for the optimal adhesion (the substrate is broken in the adhesion and quality test) of ultra thick (up to 200 µm) DLC coatings by increasing the anode-cathode distance and using high voltages (up to 4 kV). An excellent adhesion can also be obtained with the simplified arc-discharge device. To maintain high process yield (5µm/h over a surface area of 150 cm2) and to stabilize the carbon ion energies and the high quality (sp3 fraction up to 85%) of the resulting coating, an in situ cathode polishing system must be used. DLC-PDMS-h coating is the superior candidate coating material for anti-soiling applications where also hardness is required.

Progression of diffuse gliomas - from the first diagnosis to recurrence

Gliomas are the most frequent primary brain tumours. The cardinal features of gliomas are infiltrative growth pattern and progression from low-grade tumours to a more malignant phenotype. These features of gliomas generally prevent their complete surgical excision and cause their inherent tendency to recur after initial treatment and lead to poor long-term prognosis. Increasing knowledge about the molecular biology of gliomas has produced new markers that supplement histopathological diagnostics. Molecular markers are also used to evaluate the prognosis and predict therapeutic response. The purpose of this thesis is to study molecular events involved in the malignant progression of gliomas. Gliomas are highly vascularised tumours. Contrast enhancement in magnetic resonance imaging (MRI) reflects a disrupted blood-brain barrier and is often seen in malignant gliomas. In this thesis, 62 astrocytomas, oligodendrogliomas and oligoastrocytomas were studied by MRI and immunohistochemistry. Contrast enhancement in preoperative MRI was associated with angiogenesis, tumour cell proliferation and histological grade of gliomas. Activation of oncogenes by gene amplification is a common genetic aberration in gliomas. EGFR amplification on chromosome 7p12 occurs in 30-40% of glioblastomas. PDGFRA, KIT and VEGFR2 are receptor tyrosine kinase genes located on chromosome 4q12. Amplification of these genes was studied using in situ hybridisation in the primary and recurrent astrocytomas, oligodendrogliomas and oligoastrocytomas of 87 patients. PDGFRA, KIT or VEGFR2 amplification was found in 22% of primary tumours and 36% of recurrent tumours including low-grade and malignant gliomas. The most frequent aberration was KIT amplification, which occurred in 10% of primary tumours and in 27% of recurrent tumours. The expression of ezrin, cyclooxygenase 2 (COX-2) and HuR was studied immunohistochemically in a series of primary and recurrent gliomas of 113 patients. Ezrin is a cell membrane-cytoskeleton linking-protein involved in the migration of glioma cells. The COX-2 enzyme is implicated in the carcinogenesis of epithelial neoplasms and is overexpressed in gliomas. HuR is an RNA-stabilising protein, which regulates the expression of several proteins including COX-2. Ezrin, COX-2 and HuR were associated with histological grade and the overall survival of glioma patients. However, in multivariate analysis they were not independent prognostic factors. In conclusion, these results suggest that contrast enhancement in MRI can be used as a surrogate marker for the proliferative and angiogenic potential of gliomas. Aberrations of PDGFRA, KIT and VEGFR2 genes, as well as the dysregulated expression of ezrin, COX-2 and HuR proteins, are linked to the progression of gliomas.