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.

The Effects of Anesthetic Induced Loss of Consciousness on Quantitative Electroen Cephalogram, and Bispectral and Spectral Entropy Indices. Studies on Healthy Male

Changes in the electroencephalography (EEG) signal have been used to study the effects of anesthetic agents on the brain function. Several commercial EEG based anesthesia depth monitors have been developed to measure the level of the hypnotic component of anesthesia. Specific anesthetic related changes can be seen in the EEG, but still it remains difficult to determine whether the subject is consciousness or not during anesthesia. EEG reactivity to external stimuli may be seen in unconsciousness subjects, in anesthesia or even in coma. Changes in regional cerebral blood flow, which can be measured with positron emission tomography (PET), can be used as a surrogate for changes in neuronal activity. The aim of this study was to investigate the effects of dexmedetomidine, propofol, sevoflurane and xenon on the EEG and the behavior of two commercial anesthesia depth monitors, Bispectral Index (BIS) and Entropy. Slowly escalating drug concentrations were used with dexmedetomidine, propofol and sevoflurane. EEG reactivity at clinically determined similar level of consciousness was studied and the performance of BIS and Entropy in differentiating consciousness form unconsciousness was evaluated. Changes in brain activity during emergence from dexmedetomidine and propofol induced unconsciousness were studied using PET imaging. Additionally, the effects of normobaric hyperoxia, induced during denitrogenation prior to xenon anesthesia induction, on the EEG were studied. Dexmedetomidine and propofol caused increases in the low frequency, high amplitude (delta 0.5-4 Hz and theta 4.1-8 Hz) EEG activity during stepwise increased drug concentrations from the awake state to unconsciousness. With sevoflurane, an increase in delta activity was also seen, and an increase in alpha- slow beta (8.1-15 Hz) band power was seen in both propofol and sevoflurane. EEG reactivity to a verbal command in the unconsciousness state was best retained with propofol, and almost disappeared with sevoflurane. The ability of BIS and Entropy to differentiate consciousness from unconsciousness was poor. At the emergence from dexmedetomidine and propofol induced unconsciousness, activation was detected in deep brain structures, but not within the cortex. In xenon anesthesia, EEG band powers increased in delta, theta and alpha (8-12Hz) frequencies. In steady state xenon anesthesia, BIS and Entropy indices were low and these monitors seemed to work well in xenon anesthesia. Normobaric hyperoxia alone did not cause changes in the EEG. All of these results are based on studies in healthy volunteers and their application to clinical practice should be considered carefully.

Herpes simplex virus type 1 (HSV-1) pathogenesis and HSV gene therapy of experimental autoimmune encephalomyelitis

The aim of this thesis was to develop new herpes simplex virus (HSV) vectors for gene therapy of experimental autoimmune encephalomyelitis (EAE), the principal model of multiple sclerosis (MS), and to study the pathogenesis of wild-type HSV-1 and HSV-1 vectors in vivo. By introducing potential immunomodulatory factors into mice with EAE we strived to develop therapies and possibly find molecules improving recovery from EAE. We aimed at altering the immune response by inducing favorable Th2-type cytokines, thus shifting the immune response from a Th1- or a Th17-response. Our HSV vector expressing interleukin (IL)-5 modulated the cytokine responses, decreased inflammation and alleviated EAE. The use of a novel method, bacterial artificial chromosome (BAC), for engineering recombinant HSV facilitated the construction of a new vector expressing leukemia inhibitory factor (LIF). LIF is a neurotropic cytokine with broad functions in the central nervous system (CNS). LIF promotes oligodendrocyte maturation and decreases demyelination and oligodendrocyte loss. The BAC-derived HSV-LIF vector alleviated the clinical symptoms, induced a higher number of oligodendrocytes and modulated T cell responses. By administering HSV via different infection routes, e.g. peripherally via the nose or eye, or intracranially to the brain, the effect of the immune response on HSV spread at different points of the natural infection route was studied. The intranasal infection was an effective delivery route of HSV to the trigeminal ganglion and CNS, whereas corneal infection displayed limited spread. The corneal and intranasal infections induced different peripheral immune responses, which might explain the observed differences in viral spread.

Identification of Some Additional Loss Components in High-Power Low-Voltage Permanent Magnet Generators

Permanent magnet generators (PMG) represent the cutting edge technology in modern wind mills. The efficiency remains high (over 90%) at partial loads. To improve the machine efficiency even further, every aspect of machine losses has to be analyzed. Additional losses are often given as a certain percentage without providing any detailed information about the actual calculation process; meanwhile, there are many design-dependent losses that have an effect on the total amount of additional losses and that have to be taken into consideration. Additional losses are most often eddy current losses in different parts of the machine. These losses are usually difficult to calculate in the design process. In this doctoral thesis, some additional losses are identified and modeled. Further, suggestions on how to minimize the losses are given. Iron losses can differ significantly between the measured no-load values and the loss values under load. In addition, with embedded magnet rotors, the quadrature-axis armature reaction adds losses to the stator iron by manipulating the harmonic content of the flux. It was, therefore, re-evaluated that in salient pole machines, to minimize the losses and the loss difference between the no-load and load operation, the flux density has to be kept below 1.5 T in the stator yoke, which is the traditional guideline for machine designers. Eddy current losses may occur in the end-winding area and in the support structure of the machine, that is, in the finger plate and the clamping ring. With construction steel, these losses account for 0.08% of the input power of the machine. These losses can be reduced almost to zero by using nonmagnetic stainless steel. In addition, the machine housing may be subjected to eddy current losses if the flux density exceeds 1.5 T in the stator yoke. Winding losses can rise rapidly when high frequencies and 10–15 mm high conductors are used. In general, minimizing the winding losses is simple. For example, it can be done by dividing the conductor into transposed subconductors. However, this comes with the expense of an increase in the DC resistance. In the doctoral thesis, a new method is presented to minimize the winding losses by applying a litz wire with noninsulated strands. The construction is the same as in a normal litz wire but the insulation between the subconductors has been left out. The idea is that the connection is kept weak to prevent harmful eddy currents from flowing. Moreover, the analytical solution for calculating the AC resistance factor of the litz-wire is supplemented by including an end-winding resistance in the analytical solution. A simple measurement device is developed to measure the AC resistance in the windings. In the case of a litz-wire with originally noninsulated strands, vacuum pressure impregnation (VPI) is used to insulate the subconductors. In one of the two cases studied, the VPI affected the AC resistance factor, but in the other case, it did not have any effect. However, more research is needed to determine the effect of the VPI on litz-wire with noninsulated strands. An empirical model is developed to calculate the AC resistance factor of a single-layer formwound winding. The model includes the end-winding length and the number of strands and turns. The end winding includes the circulating current (eddy currents that are traveling through the whole winding between parallel strands) and the main current. The end-winding length also affects the total AC resistance factor.

Molecular Mechanisms of Sexual Dimorphism in Threespine Stickleback

Sexual dimorphism is commonly understood as differences in external features, such as morphological features or coloration. However, it can more broadly encompass behavior and physiology and at the core of these differences is the genetic mechanism – mRNA and protein expression. How, and which, molecular mechanisms influence sexually dimorphic features is not well understood thus far. DNA, RNA and proteins are the template required to create the phenotype of an individual, and they are connected to each other via processes of transcription and translation. As the genome of males and females are almost identical with the exception of the few genes on the sex chromosome or the sex-determining alleles (in the case of organisms without sex chromosomes), it is likely that many of the downstream processes resulting in sexual dimorphism are produced by changes in gene regulation and result from a regulatory cascade and not from a vastly different gene composition. Thus, in this thesis a systems biology approach is used to understand sexual dimorphism at all molecular levels and how different genomic features, e.g. sex chromosome evolution, can affect the interplay of these molecules. The threespine stickleback, Gasterosteus aculeatus, is used as the model to investigate molecular mechanisms of sexual dimorphism. It has well-characterized ecology and behavior, especially in the breeding season when sexual dimorphism is high. Moreover, threespine stickleback has a recently evolved Y chromosome in the early stages of sex chromosome evolution, characterized by a lack of recombination leading to degeneration (i.e. gene loss). The aim of my thesis is to investigate how the genotype links to the molecular phenotype and relates to differences in molecular expression between males and females. Based on previous research on sex differences in mRNA expression, I investigated sex-biased protein expression in adult fish outside the breeding season to see if differences persisted after translation. As sex-biased expression also prevailed in the proteome and previous transcription expression seemed to be related to the sex chromosomes, I investigated the genome level with a particular focus on the sex-chromosomes. I characterized the status of Y chromosome degeneration in the threespine stickleback and its effects on gene function. Furthermore, since the degeneration process leaves genes in a single copy in males, I examined whether the resulting dosage difference of messenger RNA for hemizygous genes is compensated as it is in other organisms. In addition, threespine sticklebacks have wellcharacterized behavioral differences related to the male’s social status during the breeding season. To understand the connection between the genotype and behavior, I examined gene expression patterns related to breeding behavior using dominant and subordinate males as well as female

Analytic evaluation of three-phase short circuit demagnetization and hysteresis loss risk in rotor-surface-magnet Permanent-Magnet Synchronous Machines

Permanent magnet synchronous machines (PMSM) have become widely used in applications because of high efficiency compared to synchronous machines with exciting winding or to induction motors. This feature of PMSM is achieved through the using the permanent magnets (PM) as the main excitation source. The magnetic properties of the PM have significant influence on all the PMSM characteristics. Recent observations of the PM material properties when used in rotating machines revealed that in all PMSMs the magnets do not necessarily operate in the second quadrant of the demagnetization curve which makes the magnets prone to hysteresis losses. Moreover, still no good analytical approach has not been derived for the magnetic flux density distribution along the PM during the different short circuits faults. The main task of this thesis is to derive simple analytical tool which can predict magnetic flux density distribution along the rotor-surface mounted PM in two cases: during normal operating mode and in the worst moment of time from the PM’s point of view of the three phase symmetrical short circuit. The surface mounted PMSMs were selected because of their prevalence and relatively simple construction. The proposed model is based on the combination of two theories: the theory of the magnetic circuit and space vector theory. The comparison of the results in case of the normal operating mode obtained from finite element software with the results calculated with the proposed model shows good accuracy of model in the parts of the PM which are most of all prone to hysteresis losses. The comparison of the results for three phase symmetrical short circuit revealed significant inaccuracy of the proposed model compared with results from finite element software. The analysis of the inaccuracy reasons was provided. The impact on the model of the Carter factor theory and assumption that air have permeability of the PM were analyzed. The propositions for the further model development are presented.

Effects of Obesity and Weight Loss Following Bariatric Surgery on Brain Function, Structural Integrity and Metabolism

Obesity is one of the key challenges to health care system worldwide and its prevalence is estimated to rise to pandemic proportions. Numerous adverse health effects follow with increasing body weight, including increased risk of hypertension, diabetes, hypercholesterolemia, musculoskeletal pain and cancer. Current evidence suggests that obesity is associated with altered cerebral reward circuit functioning and decreased inhibitory control over appetitive food cues. Furthermore, obesity causes adverse shifts in metabolism and loss of structural integrity within the brain. Prior cross-sectional studies do not allow delineating which of these cerebral changes are recoverable after weight loss. We compared morbidly obese subjects with healthy controls to unravel brain changes associated with obesity. Bariatric surgery was used as an intervention to study which cerebral changes are recoverable after weight loss. In Study I we employed functional magnetic resonance imaging (fMRI) to detect the brain basis of volitional appetite control and its alterations in obesity. In Studies II-III we used diffusion tensor imaging (DTI) and voxel-based morphometry (VBM) to quantify the effects of obesity and the effects of weight loss on structural integrity of the brain. In study IV we used positron emission tomography (PET) with [18F]-FDG in fasting state and during euglycemic hyperinsulinemia to quantify effects of obesity and weight loss on brain glucose uptake. The fMRI experiment revealed that a fronto-parietal network is involved in volitional appetite control. Obese subjects had lower medial frontal and dorsal striatal brain activity during cognitive appetite control and increased functional connectivity within the appetite control circuit. Obese subjects had initially lower grey matter and white matter densities than healthy controls in VBM analysis and loss of integrity in white matter tracts as measured by DTI. They also had initially elevated glucose metabolism under insulin stimulation but not in fasting state. After the weight loss following bariatric surgery, obese individuals’ brain volumes recovered and the insulin-induced increase in glucose metabolism was attenuated. In conclusion, obesity is associated with altered brain function, coupled with loss of structural integrity and elevated glucose metabolism, which are likely signs of adverse health effects to the brain. These changes are reversed by weight loss after bariatric surgery, implicating that weight loss has a causal role on these adverse cerebral changes. Altogether these findings suggest that weight loss also promotes brain health.Key words: brain, obesity, bariatric surgery, appetite control, structural magnetic resonance