Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Conventional and novel cardiovascular risk factors in young Finns and their associations with structural and functional vascular changes of subclinical atherosclerosis. The Cardiovascular Risk in Young Finns Study

Background: Atherosclerosis begins in early life progressing from asymptomatic to symptomatic as we age. Although substantial progress has been made in identifying the determinants of atherosclerosis in middle to older age adults at increased cardiovascular risk, there is lack of data examining determinants and prediction of atherosclerosis in young adults. Aims: The current study was designed to investigate levels of cardiovascular risk factors in young adults, subclinical measures of atherosclerosis, and prediction of subclinical arterial changes with conventional risk factor measures and novel metabolic profiling of serum samples. Subjects and Methods: This thesis utilised data from the follow-ups performed in 2001 and 2007 in the Cardiovascular Risk in Young Finns study, a Finnish population-based prospective cohort study that examined 2,204 subjects who were aged 30-45 years in 2007. Subclinical atherosclerosis was studied using noninvasive ultrasound measurements of carotid intima-media thickness (IMT), carotid arterial distensibility (CDist) and brachial flow-mediated dilation (FMD). Measurements included conventional risk factors and metabolic profiling using highthroughput nuclear magnetic resonance (NMR) methods that provided data on 42 lipid markers and 16 circulating metabolites. Results: Trends in lipids were favourable between 2001 and 2007, whereas waist circumference, fasting glucose, and blood pressure levels increased. To study the stability of noninvasive ultrasound markers, 6-year tracking (the likelihood to maintain the original fractile over time) in 6 years was examined. IMT tracked more strongly than CDist and FMD. Cardiovascular risk scores (Framingham, SCORE, Finrisk, Reynolds and PROCAM) predicted subclinical atherosclerosis equally. Lipoprotein subclass testing did not improve the prediction of subclinical atherosclerosis over and above conventional risk factors. However, circulating metabolites improved risk stratification. Tyrosine and docosahexaenoic acid were found to be novel biomarkers of high IMT. Conclusions: Prediction of cardiovascular risk in young Finnish adults can be performed with any of the existing risk scores. The addition of metabonomics to risk stratification improves prediction of subclinical changes and enables more accurate targeting of prevention at an early stage.

Novel force control methods in a teleoperation system of a hydraulic slider

The objective of the this research project is to develop a novel force control scheme for the teleoperation of a hydraulically driven manipulator, and to implement an ideal transparent mapping between human and machine interaction, and machine and task environment interaction. This master‘s thesis provides a preparatory study for the present research project. The research is limited into a single degree of freedom hydraulic slider with 6-DOF Phantom haptic device. The key contribution of the thesis is to set up the experimental rig including electromechanical haptic device, hydraulic servo and 6-DOF force sensor. The slider is firstly tested as a position servo by using previously developed intelligent switching control algorithm. Subsequently the teleoperated system is set up and the preliminary experiments are carried out. In addition to development of the single DOF experimental set up, methods such as passivity control in teleoperation are reviewed. The thesis also contains review of modeling of the servo slider in particular reference to the servo valve. Markov Chain Monte Carlo method is utilized in developing the robustness of the model in presence of noise.

Characterization of novel genes predominantly expressed in the epididymis – from genome analyses to genetically modified mice

In mammals, post-testicular sperm maturation taking place in the epididymis is required for the spermatozoa to acquire the abilities required to fertilize the egg in vivo. The epididymal epithelial cells secrete proteins and other small molecules into the lumen, where they interact with the spermatozoa and enable necessary maturational changes. In this study different in silico, in vitro and in vivo approaches were utilized in order to find novel genes responsible for the function of the epididymis and post-testicular sperm maturation in the mouse. Available online genomic databases were analyzed to identify genes potentially expressed in the epididymis, gene expression profiling was performed by studying their expression in different mouse tissues, and significance of certain genes to fertility was assessed by generating genetically modified mouse models. A recently discovered Pate (prostate and testis expression) gene family was found to be predominantly expressed in the epididymis. It represents one of the largest known gene families expressed in the epididymis, and the members code for proteins potentially involved in defense against microorganisms. Through genetically modified mouse models CRISP4 (cysteine-rich secretory protein 4) was identified to regulate sperm acrosome reaction, and BMYC to inhibit the expression of the Myc proto-oncogene in the developing testis. A mouse line expressing iCre recombinase specifically in the epididymis was also generated. This model can be used to generate conditional, epididymis-specific knock-out models, and will be a valuable tool in fertility studies.

High-Throughput Screening for Novel Prostate Cancer Drug Targets –Getting Personal

Prostate cancers form a heterogeneous group of diseases and there is a need for novel biomarkers, and for more efficient and targeted methods of treatment. In this thesis, the potential of microarray data, RNA interference (RNAi) and compound screens were utilized in order to identify novel biomarkers, drug targets and drugs for future personalized prostate cancer therapeutics. First, a bioinformatic mRNA expression analysis covering 9873 human tissue and cell samples, including 349 prostate cancer and 147 normal prostate samples, was used to distinguish in silico prevalidated putative prostate cancer biomarkers and drug targets. Second, RNAi based high-throughput (HT) functional profiling of 295 prostate and prostate cancer tissue specific genes was performed in cultured prostate cancer cells. Third, a HT compound screen approach using a library of 4910 drugs and drug-like molecules was exploited to identify potential drugs inhibiting prostate cancer cell growth. Nine candidate drug targets, with biomarker potential, and one cancer selective compound were validated in vitro and in vivo. In addition to androgen receptor (AR) signaling, endoplasmic reticulum (ER) function, arachidonic acid (AA) pathway, redox homeostasis and mitosis were identified as vital processes in prostate cancer cells. ERG oncogene positive cancer cells exhibited sensitivity to induction of oxidative and ER stress, whereas advanced and castrate-resistant prostate cancer (CRPC) could be potentially targeted through AR signaling and mitosis. In conclusion, this thesis illustrates the power of systems biological data analysis in the discovery of potential vulnerabilities present in prostate cancer cells, as well as novel options for personalized cancer management.

Novel CO2 Regulated Proteins in Synechocystis PCC 6803

The large biodiversity of cyanobacteria together with the increasing genomics and proteomics metadata provide novel information for finding new commercially valuable metabolites. With the advent of global warming, there is growing interest in the processes that results in efficient CO2 capture through the use of photosynthetic microorganisms such as cyanobacteria. This requires a detailed knowledge of how cyanobacteria respond to the ambient CO2. My study was aimed at understanding the changes in the protein profile of the model organism, Synechocystis PCC 6803 towards the varying CO₂ level. In order to achieve this goal I have employed modern proteomics tools such as iTRAQ and DIGE, recombinant DNA techniques to construct different mutants in cyanobacteria and biophysical methods to study the photosynthetic properties. The proteomics study revealed several novel proteins, apart from the well characterized proteins involved in carbon concentrating mechanisms (CCMs), that were upregulated upon shift of the cells from high CO₂ concentration (3%) to that in air level (0.039%). The unknown proteins, Slr0006 and flavodiiron proteins (FDPs) Sll0217-Flv4 and Sll0219-Flv2, were selected for further characterization. Although slr0006 was substantially upregulated under C_i limiting conditions, inactivation of the gene did not result in any visual phenotype under various environmental conditions indicating that this protein is not essential for cell survival. However, quantitative proteomics showed the induction of novel plasmid and chromosome encoded proteins in deltaslr0006 under air level CO₂ conditions. The expression of the slr0006 gene was found to be strictly dependent on active photosynthetic electron transfer. Slr0006 contains conserved dsRNA binding domain that belongs to the Sua5/YrdC/YciO protein family. Structural modelling of Slr0006 showed an alpha/beta twisted open-sheet structure and a positively charged cavity, indicating a possible binding site for RNA. The 3D model and the co-localization of Slr0006 with ribosomal subunits suggest that it might play a role in translation or ribosome biogenesis. On the other hand, deletions in the sll0217-sll218- sll0219 operon resulted in enhanced photodamage of PSII and distorted energy transfer from phycobilisome (PBS) to PSII, suggesting a dynamic photoprotection role of the operon. Constructed homology models also suggest efficient electron transfer in heterodimeric Flv2/Flv4, apparently involved in PSII photoprotection. Both Slr0006 and FDPs exhibited several common features, including negative regulation by NdhR and ambiguous cellular localization when subjected to different concentrations of divalent ions. This strong association with the membranes remained undisturbed even in the presence of detergent or high salt. My finding brings ample information on three novel proteins and their functions towards carbon limitation. Nevertheless, many pathways and related proteins remain unexplored. The comprehensive understanding of the acclimation processes in cyanobacteria towards varying environmental CO₂ levels will help to uncover adaptive mechanisms in other organisms, including higher plants.

Techno-Economic Feasibility of Novel On-Line Condition Monitoring Methods in Low Voltage Distribution Networks

The focus in this thesis is to study both technical and economical possibilities of novel on-line condition monitoring techniques in underground low voltage distribution cable networks. This thesis consists of literature study about fault progression mechanisms in modern low voltage cables, laboratory measurements to determine the base and restrictions of novel on-line condition monitoring methods, and economic evaluation, based on fault statistics and information gathered from Finnish distribution system operators. This thesis is closely related to master’s thesis “Channel Estimation and On-line Diagnosis of LV Distribution Cabling”, which focuses more on the actual condition monitoring methods and signal theory behind them.

Novel Regression Methods For Spectral Data

Singular Value Decomposition (SVD), Principal Component Analysis (PCA) and Multiple Linear Regression (MLR) are some of the mathematical pre- liminaries that are discussed prior to explaining PLS and PCR models. Both PLS and PCR are applied to real spectral data and their di erences and similarities are discussed in this thesis. The challenge lies in establishing the optimum number of components to be included in either of the models but this has been overcome by using various diagnostic tools suggested in this thesis. Correspondence analysis (CA) and PLS were applied to ecological data. The idea of CA was to correlate the macrophytes species and lakes. The di erences between PLS model for ecological data and PLS for spectral data are noted and explained in this thesis. i

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.

Identification and characterization of CIP2A as a novel oncogenic inhibitor of PP2A

Inhibition of the tumor suppressor protein phosphatase 2A (PP2A) activity has been identified as one of the five key alterations required for human cell transformation. Regardless of this crucial role in human cancer development, the detailed mechanisms by which PP2A inhibition occurs in human cancers remain largely uncharacterized. PP2A regulates a plethora of cellular signaling cascades. One of the targets of PP2A is Myc oncoprotein, which is destabilized and degraded in response to PP2A-mediated dephosphorylation of Myc serine 62. In this study we identify Cancerous Inhibitor of PP2A (CIP2A) as a previously uncharacterized endogenous inhibitor of PP2A in human cancer cells. CIP2A inhibits PP2A activity leading to subsequent stabilization of the Myc protein. CIP2A promotes malignant growth of cancer cells in vitro and xenograft tumor formation in vivo and is overexpressed in cancer. Moreover, we explored the effect of CIP2A on global transcriptional profiles and validated a CIP2A-dependent transcriptional signature. Analysis of the CIP2A signature revealed both Myc-dependent and -independent functions for CIP2A. Importantly, we demonstrate that the CIP2A signature has clinical relevance in human breast cancer subtypes. Finally, we identify the genes potentially mediating the long-term growth suppression in CIP2A depleted cancer cells. Taken together, this work identifies CIP2A as a novel human oncoprotein and describes its function in cancer cells. These results may open novel possibilities for patient stratification and therapeutic intervention of cancer.

Detection of the Vulnerable Atherosclerotic Plaque. Pre-Clinical Evaluation of Novel PET Imaging Probes With Experimental Models

Atherosclerosis is a life-long vascular inflammatory disease and the leading cause of death in Finland and in other western societies. The development of atherosclerotic plaques is progressive and they form when lipids begin to accumulate in the vessel wall. This accumulation triggers the migration of inflammatory cells that is a hallmark of vascular inflammation. Often, this plaque will become unstable and form vulnerable plaque which may rupture causing thrombosis and in the worst case, causing myocardial infarction or stroke. Identification of these vulnerable plaques before they rupture could save lives. At present, in the clinic, there exists no appropriated, non-invasive method for their identification. The aim of this thesis was to evaluate novel positron emission tomography (PET) probes for the detection of vulnerable atherosclerotic plaques and to characterize, two mouse models of atherosclerosis. These studies were performed by using ex vivo and in vivo imaging modalities. The vulnerability of atherosclerotic plaques was evaluated as expression of active inflammatory cells, namely macrophages. Age and the duration of high-fat diet had a drastic impact on the development of atherosclerotic plaques in mice. In imaging of atherosclerosis, 6-month-old mice, kept on high-fat diet for 4 months, showed matured, metabolically active, atherosclerotic plaques. [18F]FDG and 68Ga were accumulated in the areas representative of vulnerable plaques. However, the slow clearance of 68Ga limits its use for the plaque imaging. The novel synthesized [68Ga]DOTA-RGD and [18F]EF5 tracers demonstrated efficient uptake in plaques as compared to the healthy vessel wall, but the pharmacokinetic properties of these tracers were not optimal in used models. In conclusion, these studies resulted in the identification of new strategies for the assessment of plaque stability and mouse models of atherosclerosis which could be used for plaque imaging. In the used probe panel, [18F]FDG was the best tracer for plaque imaging. However, further studies are warranted to clarify the applicability of [18F]EF5 and [68Ga]DOTA-RGD for imaging of atherosclerosis with other experimental models.

Identification of novel regulators for tumor progression in breast cancer

Breast cancer is the most frequent solid tumor among women and the leading cause of cancer related death in women worldwide. The prognosis of breast cancer patients is tightly correlated with the degree of spread beyond the primary tumor. In this thesis, the aim was to identify novel regulators of tumor progression in breast cancer as well as to get insights into the molecular mechanisms of breast cancer progression and metastasis. First, the role of phospholipid remodeling genes and enzymes important for breast cancer progression was studied in breast cancer samples as well as in cultured breast cancer cells. Tumor samples displayed increased de novo synthesized fatty acids especially in aggressive breast cancer. Furthermore, RNAi mediated cell based assays implicated several target genes critical for breast cancer cell proliferation and survival. Second, the role of arachidonic acid pathway members 15-hydroxyprostaglandin dehydrogenase (HPGD) and phospholipase A2 group VII (PLA2G7) in tumorigenesis associated processes was explored in metastatic breast cancer cells. Both targets were found to contribute to epithelial-mesenchymal transition related processes. Third, a high-throughput RNAi lysate microarray screen was utilized to identify novel vimentin expression regulating genes. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was found to promote cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion. Taken together, this study identified several putative targets for breast cancer therapy. In addition, these results provide novel information about the mechanisms and factors underlying breast cancer progression.

Identification and characterisation of a novel adhesin of Streptococcus suis and its use as a target of adhesion inhibition and bacterial detection

Streptococcus suis is an important pig pathogen but it is also zoonotic, i.e. capable of causing diseases in humans. Human S. suis infections are quite uncommon but potentially life-threatening and the pathogen is an emerging public health concern. This Gram-positive bacterium possesses a galabiose-specific (Galalpha1−4Gal) adhesion activity, which has been studied for over 20 years. P-fimbriated Escherichia coli−bacteria also possess a similar adhesin activity targeting the same disaccharide. The galabiose-specific adhesin of S. suis was identified by an affinity proteomics method. No function of the protein identified was formerly known and it was designated streptococcal adhesin P (SadP). The peptide sequence of SadP contains an LPXTG-motif and the protein was proven to be cell wall−anchored. SadP may be multimeric since in SDS-PAGE gel it formed a protein ladder starting from about 200 kDa. The identification was confirmed by producing knockout strains lacking functional adhesin, which had lost their ability to bind to galabiose. The adhesin gene was cloned in a bacterial expression host and properties of the recombinant adhesin were studied. The galabiose-binding properties of the recombinant protein were found to be consistent with previous results obtained studying whole bacterial cells. A live-bacteria application of surface plasmon resonance was set up, and various carbohydrate inhibitors of the galabiose-specific adhesins were studied with this assay. The potencies of the inhibitors were highly dependent on multivalency. Compared with P-fimbriated E. coli, lower concentrations of galabiose derivatives were needed to inhibit the adhesion of S. suis. Multivalent inhibitors of S. suis adhesion were found to be effective at low nanomolar concentrations. To specifically detect galabiose adhesin−expressing S. suis bacteria, a technique utilising magnetic glycoparticles and an ATP bioluminescence bacterial detection system was also developed. The identification and characterisation of the SadP adhesin give valuable information on the adhesion mechanisms of S. suis, and the results of this study may be helpful for the development of novel inhibitors and specific detection methods of this pathogen.

Administration of Bifidobacterium animalis subsp. lactis BB-12 and xylitol with a novel pacifier in early childhood

Probiotic bifidobacteria are used in the prevention and treatment of childhood diseases. On the other hand, these bacteria are also connected to dental caries. The purpose of the present work was to test a food supplement containing Bifidobacterium animalis subsp. lactis BB-12 (B. lactis BB-12) and xylitol, and to investigate its health effects, properties and safety when used in a novel pacifier in early childhood. In a double-blind, placebo-controlled trial, newborn infants (n=163) were assigned randomly to receive B. lactis BB-12, xylitol, or sorbitol from the age of 1– 2 monthsto 2 years with a pacifier or a spoon. Children were followed up to four years of age. A part of the parents participating in the clinical trial evaluated the feasibility of the novel administration method. The pattern of tablet release from the pouch of the pacifier was tested in adults. The food supplement tablet containing B. lactis BB-12 and xylitol could be delivered in a safe and controlled way with the novel pacifier. The early administration of B. lactis BB-12 did not result in permanent oral colonization of this probiotic or affect the colonization of mutans streptococci in early childhood. Moreover, B. lactis BB-12 did not increase the occurrence of caries. Controlled administration of B. lactis BB-12 significantly reduced the incidence of respiratory infections during the first eight months of life in a Finnish population with breastfed infants. To conclude, administration of B. lactis BB-12 in early childhood is safe with regard to the future dental health of the child. In addition, B. lactis BB-12 may add to the protection against respiratory infections provided by human breast milk in infancy.