Mitotic regulation by Polo-like kinase 1 and the Chromosomal Passenger Complex

During mitosis, the duplicated genome must be accurately divided between two daughter cells. Polo-like kinase 1 (Plk1) and Aurora B kinase, together with its binding partners Incenp, Survivin and Borealin (chromosomal passenger complex, CPC), have key roles in coordinating mitotic events. The accuracy of cell division is safeguarded by a signaling cascade termed the mitotic spindle checkpoint (SC), which ensures that chromosomes are not physically separated before correct bipolar attachments have been formed between kinetochores and spindle microtubules (MT). An inhibitory “wait anaphase” signal, which delays chromosome separation (anaphase onset), is created at individual kinetochores and broadcasted throughout the cell in response to lack of kinetochore-microtubule (kMT) attachment or proper interkinetochore tension. It is believed that the fast turnover of SC molecules at kinetochores contributes to the cell’s ability to produce this signal and enables rapid responses to changing cellular conditions. Kinetochores that lack MT attachment and tension express a certain phosphoepitope called the 3F3/2 phosphoepitope, which has been linked to SC signaling. In the experimental part, we investigated the regulation of the 3F3/2 phosphoepitope, analyzed whether CPC molecules turn over at centromeres, and dissected the mitotic roles of the CPC using a microinjection technique that allowed precise temporal control over its function. We found that the kinetochore 3F3/2 phosphoepitope is created by Plk1, and that CPC proteins exhibit constant exchange at centromeres. Moreover, we found that CPC function is necessary in the regulation of chromatid movements and spindle morphology in anaphase. In summary, we identified new functions of key mitotic regulators Plk1 and CPC, and provided insighs into the coordination of mitotic events.

Skill Development in Music Reading. The Eye-Movement Approach

This dissertation examined skill development in music reading by focusing on the visual processing of music notation in different music-reading tasks. Each of the three experiments of this dissertation addressed one of the three types of music reading: (i) sight-reading, i.e. reading and performing completely unknown music, (ii) rehearsed reading, during which the performer is already familiar with the music being played, and (iii) silent reading with no performance requirements. The use of the eye-tracking methodology allowed the recording of the readers’ eye movements from the time of music reading with extreme precision. Due to the lack of coherence in the smallish amount of prior studies on eye movements in music reading, the dissertation also had a heavy methodological emphasis. The present dissertation thus aimed to promote two major issues: (1) it investigated the eye-movement indicators of skill and skill development in sight-reading, rehearsed reading and silent reading, and (2) developed and tested suitable methods that can be used by future studies on the topic. Experiment I focused on the eye-movement behaviour of adults during their first steps of learning to read music notation. The longitudinal experiment spanned a nine-month long music-training period, during which 49 participants (university students taking part in a compulsory music course) sight-read and performed a series of simple melodies in three measurement sessions. Participants with no musical background were entitled as “novices”, whereas “amateurs” had had musical training prior to the experiment. The main issue of interest was the changes in the novices’ eye movements and performances across the measurements while the amateurs offered a point of reference for the assessment of the novices’ development. The experiment showed that the novices tended to sight-read in a more stepwise fashion than the amateurs, the latter group manifesting more back-and-forth eye movements. The novices’ skill development was reflected by the faster identification of note symbols involved in larger melodic intervals. Across the measurements, the novices also began to show sensitivity to the melodies’ metrical structure, which the amateurs demonstrated from the very beginning. The stimulus melodies consisted of quarter notes, making the effects of meter and larger melodic intervals distinguishable from effects caused by, say, different rhythmic patterns. Experiment II explored the eye movements of 40 experienced musicians (music education students and music performance students) during temporally controlled rehearsed reading. This cross-sectional experiment focused on the eye-movement effects of one-bar-long melodic alterations placed within a familiar melody. The synchronizing of the performance and eye-movement recordings enabled the investigation of the eye-hand span, i.e., the temporal gap between a performed note and the point of gaze. The eye-hand span was typically found to remain around one second. Music performance students demonstrated increased professing efficiency by their shorter average fixation durations as well as in the two examined eye-hand span measures: these participants used larger eye-hand spans more frequently and inspected more of the musical score during the performance of one metrical beat than students of music education. Although all participants produced performances almost indistinguishable in terms of their auditory characteristics, the altered bars indeed affected the reading of the score: the general effects of expertise in terms of the two eye- hand span measures, demonstrated by the music performance students, disappeared in the face of the melodic alterations. Experiment III was a longitudinal experiment designed to examine the differences between adult novice and amateur musicians’ silent reading of music notation, as well as the changes the 49 participants manifested during a nine-month long music course. From a methodological perspective, an opening to research on eye movements in music reading was the inclusion of a verbal protocol in the research design: after viewing the musical image, the readers were asked to describe what they had seen. A two-way categorization for verbal descriptions was developed in order to assess the quality of extracted musical information. More extensive musical background was related to shorter average fixation duration, more linear scanning of the musical image, and more sophisticated verbal descriptions of the music in question. No apparent effects of skill development were observed for the novice music readers alone, but all participants improved their verbal descriptions towards the last measurement. Apart from the background-related differences between groups of participants, combining verbal and eye-movement data in a cluster analysis identified three styles of silent reading. The finding demonstrated individual differences in how the freely defined silent-reading task was approached. This dissertation is among the first presentations of a series of experiments systematically addressing the visual processing of music notation in various types of music-reading tasks and focusing especially on the eye-movement indicators of developing music-reading skill. Overall, the experiments demonstrate that the music-reading processes are affected not only by “top-down” factors, such as musical background, but also by the “bottom-up” effects of specific features of music notation, such as pitch heights, metrical division, rhythmic patterns and unexpected melodic events. From a methodological perspective, the experiments emphasize the importance of systematic stimulus design, temporal control during performance tasks, and the development of complementary methods, for easing the interpretation of the eye-movement data. To conclude, this dissertation suggests that advances in comprehending the cognitive aspects of music reading, the nature of expertise in this musical task, and the development of educational tools can be attained through the systematic application of the eye-tracking methodology also in this specific domain.

The Design and Implementation of Loudspeaker Control Network Protocols

The networking and digitalization of audio equipment has created a need for control protocols. These protocols offer new services to customers and ensure that the equipment operates correctly. The control protocols used in the computer networks are not directly applicable since embedded systems have resource and cost limitations. In this master's thesis the design and implementation of new loudspeaker control network protocols are presented. The protocol stack was required to be reliable, have short response times, configure the network automatically and support the dynamic addition and removal of loudspeakers. The implemented protocol stack was also required to be as efficient and lightweight as possible because the network nodes are fairly simple and lack processing power. The protocol stack was thoroughly tested, validated and verified. The protocols were formally described using LOTOS (Language of Temporal Ordering Specifications) and verified using reachability analysis. A prototype of the loudspeaker network was built and used for testing the operation and the performance of the control protocols. The implemented control protocol stack met the design specifications and proved to be highly reliable and efficient.

Venous thromboembolism and obstruction after pacemaker implantation. - A Prospective Study of 150 Consecutive Cardiac Pacing Device Implantations

Background: Pacemaker implantation (PMI) may predispose to venous thromboembolism (VTE) and obstruction (VO). This prospective study aimed at quantifying changes in venous calibers, and at determining the incidence of symptomatic and asymptomatic VTE/VO after PMI. Further goals included an assessment of the role of transesophageal echocardiography (TEE) in the diagnosis of lead-related central venous thrombi (CVT), and determination of predictors for VTE/VO. Methods: 150 (mean age 67; 61% male) consecutive patients with first PMI were enrolled and followed for 6 months. Contrast venography was performed at baseline and 6 months after PMI to measure venous diameters, and to detect stenosis, total occlusions and thrombi. TEE was conducted in 66 patients. Based on clinical suspicion, work-up for pulmonary embolism (PE) or acute deep vein thrombosis (DVT) were performed as needed. A total of 50 cases underwent longer-term (mean 2.4 years) follow-up venography. All cases with VTE/VO during the initial 6 months, and their matched controls, were selected for a case-control study focused on possible predictive role of laboratory and patient-related factors for the development of VTE/VO. Results: 10 (7 %) patients were found to have baseline venous abnormalities (e.g. 8 obstructions). Mean venous diameters diminished significantly during the first 6 months, but no further reduction occurred in late follow-up. New VO was discovered in 19 patients (14 %; 14 stenosis, 5 total occlusions; all asymptomatic). Small non-obstructive thrombi were found in 20/140 (14 %) 6-month venograms. TEE at 6 months disclosed CVT in 6 (9 %) patients. One (0.7 %) patient had acute symptomatic upper-extremity DVT, and PE was discovered in 5/150 (3.3 %) patients during the first 6 months with no further cases thereafter. At 6 months, the total number of cases with VTE/VO amounted to 47 (31.3 %). Additionally, the later 2-year venograms (n=50) disclosed 4 (8 %) total occlusions and 1 (2 %) stenosis. In the case-control study, no parameter was predictive of venous end-points as a single variable, but there appeared to be significant clustering of traditional VTE risk-factors among the cases. Laboratory parameters showed a definite acute hypercoagulative state induced by PMI, but its degree did not predict subsequent development of VTE/VO. Conclusions: This study shows that VTE/VO is relatively common after PMI with an overall incidence of at least 30 %. Although the majority of the lesions are asymptomatic and clinically benign, cases of PE were also encountered, and totally occluded veins may hamper future upgrading or replacement of pacing system. Venous complications seem difficult to prognosticate as firm predictors were not identified from a wide range of parameters analyzed in this study, although clustering of classic VTE risk factors may be a predisposing factor. Parameters related to implantation procedure or pacing systems and the severity of implantation-induced trauma did not emerge as predictors.