Pico Cell Integration to Operator’s Incident Management Process

Mobile network coverage is traditionally provided by outdoor macro base stations, which have a long range and serve several of customers. Due to modern passive houses and tightening construction legislation, mobile network service is deteriorated in many indoor locations. Typically, solutions for indoor coverage problem are expensive and demand actions from the mobile operator. Due to these, superior solutions are constantly researched. The solution presented in this thesis is based on Small Cell technology. Small Cells are low power access nodes designed to provide voice and data services.. This thesis concentrates on a specific Small Cell solution, which is called a Pico Cell. The problem regarding Pico Cells and Small Cells in general is that they are a new technological solution for the mobile operator, and the possible problem sources and incidents are not properly mapped. The purpose of this thesis is to figure out the possible problems in the Pico Cell deployment and how they could be solved within the operator’s incident management process. The research in the thesis is carried out with a literature research and a case study. The possible problems are investigated through lab testing. Pico Cell automated deployment process was tested in the lab environment and its proper functionality is confirmed. The related network elements were also tested and examined, and the emerged problems are resolvable. Operators existing incident management process can be used for Pico Cell troubleshooting with minor updates. Certain pre-requirements have to be met before Pico Cell deployment can be considered. The main contribution of this thesis is the Pico Cell integrated incident management process. The presented solution works in theory and solves the problems found during the lab testing. The limitations in the customer service level were solved by adding the necessary tools and by designing a working question pattern. Process structures for automated network discovery and pico specific radio parameter planning were also added for the mobile network management layer..

Imaging techniques applied for detection of secretion, transgene delivery and G proteincoupled receptors in reproductive and endocrine cells in vivo and in vitro

Post-testicular sperm maturation occurs in the epididymis. The ion concentration and proteins secreted into the epididymal lumen, together with testicular factors, are believed to be responsible for the maturation of spermatozoa. Disruption of the maturation of spermatozoa in the epididymis provides a promising strategy for generating a male contraceptive. However, little is known about the proteins involved. For drug development, it is also essential to have tools to study the function of these proteins in vitro. One approach for screening novel targets is to study the secretory products of the epididymis or the G protein-coupled receptors (GPCRs) that are involved in the maturation process of the spermatozoa. The modified Ca2+ imaging technique to monitor release from PC12 pheochromocytoma cells can also be applied to monitor secretory products involved in the maturational processes of spermatozoa. PC12 pheochromocytoma cells were chosen for evaluation of this technique as they release catecholamines from their cell body, thus behaving like endocrine secretory cells. The results of the study demonstrate that depolarisation of nerve growth factor -differentiated PC12 cells releases factors which activate nearby randomly distributed HEL erythroleukemia cells. Thus, during the release process, the ligands reach concentrations high enough to activate receptors even in cells some distance from the release site. This suggests that communication between randomly dispersed cells is possible even if the actual quantities of transmitter released are extremely small. The development of a novel method to analyse GPCR-dependent Ca2+ signalling in living slices of mouse caput epididymis is an additional tool for screening for drug targets. By this technique it was possible to analyse functional GPCRs in the epithelial cells of the ductus epididymis. The results revealed that, both P2X- and P2Y-type purinergic receptors are responsible for the rapid and transient Ca2+ signal detected in the epithelial cells of caput epididymides. Immunohistochemical and reverse transcriptase-polymerase chain reaction (RTPCR) analyses showed the expression of at least P2X1, P2X2, P2X4 and P2X7, and P2Y1 and P2Y2 receptors in the epididymis. Searching for epididymis-specific promoters for transgene delivery into the epididymis is of key importance for the development of specific models for drug development. We used EGFP as the reporter gene to identify proper promoters to deliver transgenes into the epithelial cells of the mouse epididymis in vivo. Our results revealed that the 5.0 kb murine Glutathione peroxidase 5 (GPX5) promoter can be used to target transgene expression into the epididymis while the 3.8 kb Cysteine-rich secretory protein-1 (CRISP-1) promoter can be used to target transgene expression into the testis. Although the visualisation of EGFP in living cells in culture usually poses few problems, the detection of EGFP in tissue sections can be more difficult because soluble EGFP molecules can be lost if the cell membrane is damaged by freezing, sectioning, or permeabilisation. Furthermore, the fluorescence of EGFP is dependent on its conformation. Therefore, fixation protocols that immobilise EGFP may also destroy its usefulness as a fluorescent reporter. We therefore developed a novel tissue preparation and preservation techniques for EGFP. In addition, fluorescence spectrophotometry with epididymal epithelial cells in suspension revealed the expression of functional purinergic, adrenergic, cholinergic and bradykinin receptors in these cell lines (mE-Cap27 and mE-Cap28). In conclusion, we developed new tools for studying the role of the epididymis in sperm maturation. We developed a new technique to analyse GPCR dependent Ca2+ signalling in living slices of mouse caput epididymis. In addition, we improved the method of detecting reporter gene expression. Furthermore, we characterised two epididymis-specific gene promoters, analysed the expression of GPCRs in epididymal epithelial cells and developed a novel technique for measurement of secretion from cells.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

To Divide or Not to Divide; MicroRNAs and Small Compounds as Modulators of Mitosis

Mitosis is under the stringent quality control of the spindle assembly checkpoint (SAC). However, in cancer cells this control can fail, leading to excessive cellular proliferation and ultimately to the formation of a tumor. Novel cancer cell selective therapies are needed to stop the uncontrolled cell proliferation and tumor growth. The aim of the research presented in this thesis was to identify microRNAs (miRNAs) that could play a role in cancer cell proliferation as well as low molecular weight (LMW) compounds that could interfere with cell division. The findings could be used to develop better cancer diagnostics and therapies in the future. First, a high-throughput screen (HTS) was performed to identify LMW compounds that possess a similar chemical interaction field as rigosertib, an anti-cancer compound undergoing clinical trials. A compound termed Centmitor-1 was discovered that phenocopied the cellular impact of rigosertib by affecting the microtubule dynamics. Next, another HTS aimed at identifying compounds that would target the Hec1 protein, which mediates the interaction between spindle microtubules and chromosomes. Perturbation of this connection should prevent cell division and induce cell death. A compound termed VTT-006 was discovered that abrogated mitosis in several cell line models and exhibited binding to Hec1 in vitro. Lastly, using a cell-based HTS two miRNAs were identified that affected cancer cell proliferation via Aurora B kinase, which is an important mitotic regulator. MiR-378a-5p was found to indirectly suppress the production of the kinase whereas let-7b showed direct binding to the 3’UTR of Aurora B mRNA and repressed its translation. The miRNA-mediated perturbation of Aurora B induced defects in mitosis leading to abnormal chromosome segregation and induction of aneuploidy. The results of this thesis provide new information on miRNA signaling in cancer, which could be utilized for diagnostic purposes. Moreover, the thesis introduces two small compounds that may benefit future drug research.