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.

Consumer neuroscience: usability of brain imaging methods in marketing research

Consumer neuroscience (neuromarketing) is an emerging field of marketing research which uses brain imaging techniques to study neural conditions and processes that underlie consumption. The purpose of this study was to map this fairly new and growing field in Finland by studying the opinions of both Finnish consumers and marketing professionals towards it and comparing the opinions to the current consumer neuroscience literature, and based on that evaluate the usability of brain imaging techniques as a marketing research method. Mixed methods research design was chosen for this study. Quantitative data was collected from 232 consumers and 28 marketing professionals by means of online surveys. Both respondent groups had either neutral opinions or lacked knowledge about the four themes chosen for this study: benefits, limitations and challenges, ethical issues and future prospects of consumer neuroscience. Qualitative interview data was collected from 2 individuals from Finnish neuromarketing companies to deepen insights gained from quantitative research. The four interview themes were the same as in the surveys and the interviewees’ answers were mostly in line with the current literature, although more optimistic about the future of the field. The interviews also exposed a gap between academic consumer neuroscience research and practical level applications. The results of this study suggest that there are still many unresolved challenges and relevant populations either have neutral opinions or lack information about consumer neuroscience. The practical level applications are, however, already being successfully used and this new field of marketing research is growing both globally and in Finland.

Consumer neuroscience: usability of brain imaging methods in marketing research

Consumer neuroscience (neuromarketing) is an emerging field of marketing research which uses brain imaging techniques to study neural conditions and processes that underlie consumption. The purpose of this study was to map this fairly new and growing field in Finland by studying the opinions of both Finnish consumers and marketing professionals towards it and comparing the opinions to the current consumer neuroscience literature, and based on that evaluate the usability of brain imaging techniques as a marketing research method. Mixed methods research design was chosen for this study. Quantitative data was collected from 232 consumers and 28 marketing professionals by means of online surveys. Both respondent groups had either neutral opinions or lacked knowledge about the four themes chosen for this study: benefits, limitations and challenges, ethical issues and future prospects of consumer neuroscience. Qualitative interview data was collected from 2 individuals from Finnish neuromarketing companies to deepen insights gained from quantitative research. The four interview themes were the same as in the surveys and the interviewees’ answers were mostly in line with the current literature, although more optimistic about the future of the field. The interviews also exposed a gap between academic consumer neuroscience research and practical level applications. The results of this study suggest that there are still many unresolved challenges and relevant populations either have neutral opinions or lack information about consumer neuroscience. The practical level applications are, however, already being successfully used and this new field of marketing research is growing both globally and in Finland.

Towards desired crystalline product properties: In-situ monitoring of batch crystallization

The objective of industrial crystallization is to obtain a crystalline product which has the desired crystal size distribution, mean crystal size, crystal shape, purity, polymorphic and pseudopolymorphic form. Effective control of the product quality requires an understanding of the thermodynamics of the crystallizing system and the effects of operation parameters on the crystalline product properties. Therefore, obtaining reliable in-line information about crystal properties and supersaturation, which is the driving force of crystallization, would be very advantageous. Advanced techniques, such asRaman spectroscopy, attenuated total reflection Fourier transform infrared (ATR FTIR) spectroscopy, and in-line imaging techniques, offer great potential for obtaining reliable information during crystallization, and thus giving a better understanding of the fundamental mechanisms (nucleation and crystal growth) involved. In the present work, the relative stability of anhydrate and dihydrate carbamazepine in mixed solvents containing water and ethanol were investigated. The kinetics of the solvent mediated phase transformation of the anhydrate to hydrate in the mixed solvents was studied using an in-line Raman immersion probe. The effects of the operation parameters in terms of solvent composition, temperature and the use of certain additives on the phase transformation kineticswere explored. Comparison of the off-line measured solute concentration and the solid-phase composition measured by in-line Raman spectroscopy allowedthe identification of the fundamental processes during the phase transformation. The effects of thermodynamic and kinetic factors on the anhydrate/hydrate phase of carbamazepine crystals during cooling crystallization were also investigated. The effect of certain additives on the batch cooling crystallization of potassium dihydrogen phosphate (KDP) wasinvestigated. The crystal growth rate of a certain crystal face was determined from images taken with an in-line video microscope. An in-line image processing method was developed to characterize the size and shape of thecrystals. An ATR FTIR and a laser reflection particle size analyzer were used to study the effects of cooling modes and seeding parameters onthe final crystal size distribution of an organic compound C15. Based on the obtained results, an operation condition was proposed which gives improved product property in terms of increased mean crystal size and narrowersize distribution.

Märkäosan lisäaineet ja kuituflokit konesihdin ympäristössä

Painelajittelu on yksi yleisimmistä yksikköprosesseista paperin ja sellun valmistuksessa. Suurelta osin lajittimet toimivat niille asetettujen vaatimusten mukaisesti, mutta joissakin tapauksissa lajittimissa saattaa esiintyä ei-toivottavaa kuitujen kasautumista sekä kehräymän muodostusta. Niiden seurauksena lajittimien kapasiteetti alenee ja lajittelutulos heikkenee. Tämän työn tarkoituksena on uutta kuvantamistekniikkaa hyödyntäen selvittää miten kehräymät ja kuitukasaumat syntyvät painelajittimen sihtipinnalla ja miten retentioaineen syöttö sihdin ympäristössä vaikuttaa niiden syntyyn. Työn kirjallisuusosassa tarkastellaan painelajittimen toimintaa, rakennetta sekä lyhyen kierron konesihdin erityispiirteitä. Lisäksi tarkastellaan retentiokemikaalien käyttäytymistä leikkausvoimien alaisuudessa ja kuitukehräymien syntyä painelajittimissa. Kokeellisessa osassa on raportoitu kuvantamisjärjestelmällä saatuja tuloksia sekä esitetään havaintoja kehräymien ja kuitukasaumien synnystä ja niiden vaikutuksista painelajittimen toimintaan. Kuvausten perusteella voidaan sanoa, että kehräymän syntyminen sihdissä vaatii aina jonkinlaisen kuitukasauman olemassaoloa. Tällaista alkukasaumaa tarvitaan, jotta kuidut voivat ankkuroitua siihen kiinni ja johon kiinnittyneenä kuidut alkavat pyöriä virtauksessa muodostaen kehräymää. Kuitukasauman muodostuminen painelajittimessa johtuu pääosin sihdissä olevasta epäjatkuvuuskohdasta, massassa olevista epäpuhtauksista ja kuituflokeista jotka jäävät kiinni sihtipinnan aukkoihin tai lajittimen kapasiteetin ylittymisestä. Kehräymän syntyä kasauman jäljessä voidaan pitää enemmän sääntönä kuin poikkeuksena, mutta kehräytyminen on vähäisempää reikäsihdillä kuin rakosihdillä. Silloituspolymeerillä flokattu massa ei muodosta herkemmin kuitukasaumia sihtipintaan verrattuna flokkaamattomaan massaan. Lajiteltavan massan sakeuden nosto vähentää kuitukasaumien ja kehräymien syntyä. Kuitukasaumien ja kehräymien välttämiseksi on tärkeää, että sihtiä ei ajeta suunniteltua mitoitusaluetta suuremmilla tuotannoilla tai virtauksilla.

Flexible multibody approach in bone strain estimation during physical activity:

Bone strain plays a major role as the activation signal for the bone (re)modeling process, which is vital for keeping bones healthy. Maintaining high bone mineral density reduces the chances of fracture in the event of an accident. Numerous studies have shown that bones can be strengthened with physical exercise. Several hypotheses have asserted that a stronger osteogenic (bone producing) effect results from dynamic exercise than from static exercise. These previous studies are based on short-term empirical research, which provide the motivation for justifying the experimental results with a solid mathematical background. The computer simulation techniques utilized in this work allow for non-invasive bone strain estimation during physical activity at any bone site within the human skeleton. All models presented in the study are threedimensional and actuated by muscle models to replicate the real conditions accurately. The objective of this work is to determine and present loading-induced bone strain values resulting from physical activity. It includes a comparison of strain resulting from four different gym exercises (knee flexion, knee extension, leg press, and squat) and walking, with the results reported for walking and jogging obtained from in-vivo measurements described in the literature. The objective is realized primarily by carrying out flexible multibody dynamics computer simulations. The dissertation combines the knowledge of finite element analysis and multibody simulations with experimental data and information available from medical field literature. Measured subject-specific motion data was coupled with forward dynamics simulation to provide natural skeletal movement. Bone geometries were defined using a reverse engineering approach based on medical imaging techniques. Both computed tomography and magnetic resonance imaging were utilized to explore modeling differences. The predicted tibia bone strains during walking show good agreement with invivo studies found in the literature. Strain measurements were not available for gym exercises; therefore, the strain results could not be validated. However, the values seem reasonable when compared to available walking and running invivo strain measurements. The results can be used for exercise equipment design aimed at strengthening the bones as well as the muscles during workout. Clinical applications in post fracture recovery exercising programs could also be the target. In addition, the methodology introduced in this study, can be applied to investigate the effect of weightlessness on astronauts, who often suffer bone loss after long time spent in the outer space.

Machine vision methods for process measurements in pulping

The papermaking industry has been continuously developing intelligent solutions to characterize the raw materials it uses, to control the manufacturing process in a robust way, and to guarantee the desired quality of the end product. Based on the much improved imaging techniques and image-based analysis methods, it has become possible to look inside the manufacturing pipeline and propose more effective alternatives to human expertise. This study is focused on the development of image analyses methods for the pulping process of papermaking. Pulping starts with wood disintegration and forming the fiber suspension that is subsequently bleached, mixed with additives and chemicals, and finally dried and shipped to the papermaking mills. At each stage of the process it is important to analyze the properties of the raw material to guarantee the product quality. In order to evaluate properties of fibers, the main component of the pulp suspension, a framework for fiber characterization based on microscopic images is proposed in this thesis as the first contribution. The framework allows computation of fiber length and curl index correlating well with the ground truth values. The bubble detection method, the second contribution, was developed in order to estimate the gas volume at the delignification stage of the pulping process based on high-resolution in-line imaging. The gas volume was estimated accurately and the solution enabled just-in-time process termination whereas the accurate estimation of bubble size categories still remained challenging. As the third contribution of the study, optical flow computation was studied and the methods were successfully applied to pulp flow velocity estimation based on double-exposed images. Finally, a framework for classifying dirt particles in dried pulp sheets, including the semisynthetic ground truth generation, feature selection, and performance comparison of the state-of-the-art classification techniques, was proposed as the fourth contribution. The framework was successfully tested on the semisynthetic and real-world pulp sheet images. These four contributions assist in developing an integrated factory-level vision-based process control.

Computational Techniques for Spectral Image Analysis

Multispectral images are becoming more common in the field of remote sensing, computer vision, and industrial applications. Due to the high accuracy of the multispectral information, it can be used as an important quality factor in the inspection of industrial products. Recently, the development on multispectral imaging systems and the computational analysis on the multispectral images have been the focus of a growing interest. In this thesis, three areas of multispectral image analysis are considered. First, a method for analyzing multispectral textured images was developed. The method is based on a spectral cooccurrence matrix, which contains information of the joint distribution of spectral classes in a spectral domain. Next, a procedure for estimating the illumination spectrum of the color images was developed. Proposed method can be used, for example, in color constancy, color correction, and in the content based search from color image databases. Finally, color filters for the optical pattern recognition were designed, and a prototype of a spectral vision system was constructed. The spectral vision system can be used to acquire a low dimensional component image set for the two dimensional spectral image reconstruction. The data obtained by the spectral vision system is small and therefore convenient for storing and transmitting a spectral image.

CT and PET/CT Hybrid Imaging of Coronary Artery Disease

Coronary artery disease (CAD) is a chronic process that evolves over decades and may culminate in myocardial infarction (MI). While invasive coronary angiography (ICA) is still considered the gold standard of imaging CAD, non-invasive assessment of both the vascular anatomy and myocardial perfusion has become an intriguing alternative. In particular, computed tomography (CT) and positron emission tomography (PET) form an attractive combination for such studies. Increased radiation dose is, however, a concern. Our aim in the current thesis was to test novel CT and PET techniques alone and in hybrid setting in the detection and assessment of CAD in clinical patients. Along with diagnostic accuracy, methods for the reduction of the radiation dose was an important target. The study investigating the coronary arteries of patients with atrial fibrillation (AF) showed that CAD may be an important etiology of AF because a high prevalence of CAD was demonstrated within AF patients. In patients with suspected CAD, we demonstrated that a sequential, prospectively ECG-triggered CT technique was applicable to nearly 9/10 clinical patients and the radiation dose was over 60% lower than with spiral CT. To detect the functional significance of obstructive CAD, a novel software for perfusion quantification, CarimasTM, showed high reproducibility with 15O-labelled water in PET, supporting feasibility and good clinical accuracy. In a larger cohort of 107 patients with moderate 30-70% pre-test probability of CAD, hybrid PET/CT was shown to be a powerful diagnostic method in the assessment of CAD with diagnostic accuracy comparable to that of invasive angiography and fractional flow reserve (FFR) measurements. A hybrid study may be performed with a reasonable radiation dose in a vast majority of the cases, improving the performance of stand-alone PET and CT angiography, particularly when the absolute quantification of the perfusion is employed. These results can be applied into clinical practice and will be useful for daily clinical diagnosis of CAD.

The Possibilities and Dosimetric Limitations of MLC-Based Intensity-Modulated Radiotherapy Delivery and Optimization Techniques

The use of intensity-modulated radiotherapy (IMRT) has increased extensively in the modern radiotherapy (RT) treatments over the past two decades. Radiation dose distributions can be delivered with higher conformality with IMRT when compared to the conventional 3D-conformal radiotherapy (3D-CRT). Higher conformality and target coverage increases the probability of tumour control and decreases the normal tissue complications. The primary goal of this work is to improve and evaluate the accuracy, efficiency and delivery techniques of RT treatments by using IMRT. This study evaluated the dosimetric limitations and possibilities of IMRT in small (treatments of head-and-neck, prostate and lung cancer) and large volumes (primitive neuroectodermal tumours). The dose coverage of target volumes and the sparing of critical organs were increased with IMRT when compared to 3D-CRT. The developed split field IMRT technique was found to be safe and accurate method in craniospinal irradiations. By using IMRT in simultaneous integrated boosting of biologically defined target volumes of localized prostate cancer high doses were achievable with only small increase in the treatment complexity. Biological plan optimization increased the probability of uncomplicated control on average by 28% when compared to standard IMRT delivery. Unfortunately IMRT carries also some drawbacks. In IMRT the beam modulation is realized by splitting a large radiation field to small apertures. The smaller the beam apertures are the larger the rebuild-up and rebuild-down effects are at the tissue interfaces. The limitations to use IMRT with small apertures in the treatments of small lung tumours were investigated with dosimetric film measurements. The results confirmed that the peripheral doses of the small lung tumours were decreased as the effective field size was decreased. The studied calculation algorithms were not able to model the dose deficiency of the tumours accurately. The use of small sliding window apertures of 2 mm and 4 mm decreased the tumour peripheral dose by 6% when compared to 3D-CRT treatment plan. A direct aperture based optimization (DABO) technique was examined as a solution to decrease the treatment complexity. The DABO IMRT technique was able to achieve treatment plans equivalent with the conventional IMRT fluence based optimization techniques in the concave head-and-neck target volumes. With DABO the effective field sizes were increased and the number of MUs was reduced with a factor of two. The optimality of a treatment plan and the therapeutic ratio can be further enhanced by using dose painting based on regional radiosensitivities imaged with functional imaging methods.

Detection of Pathologic Changes Following Traumatic Brain Injury Using Magnetic Resonance Imaging

Background: Approximately two percent of Finns have sequels after traumatic brain injury (TBI), and many TBI patients are young or middle-aged. The high rate of unemployment after TBI has major economic consequences for society, and traumatic brain injury often has remarkable personal consequences, as well. Structural imaging is often needed to support the clinical TBI diagnosis. Accurate early diagnosis is essential for successful rehabilition and, thus, may also influence the patient’s outcome. Traumatic axonal injury and cortical contusions constitute the majority of traumatic brain lesions. Several studies have shown magnetic resonance imaging (MRI) to be superior to computed tomography (CT) in the detection of these lesions. However, traumatic brain injury often leads to persistent symptoms even in cases with few or no findings in conventional MRI. Aims and methods: The aim of this prospective study was to clarify the role of conventional MRI in the imaging of traumatic brain injury, and to investigate how to improve the radiologic diagnostics of TBI by using more modern diffusion-weighted imaging (DWI) and diffusion tensor imaging (DTI) techniques. We estimated, in a longitudinal study, the visibility of the contusions and other intraparenchymal lesions in conventional MRI at one week and one year after TBI. We used DWI-based measurements to look for changes in the diffusivity of the normal-appearing brain in a case-control study. DTI-based tractography was used in a case-control study to evaluate changes in the volume, diffusivity, and anisotropy of the long association tracts in symptomatic TBI patients with no visible signs of intracranial or intraparenchymal abnormalities on routine MRI. We further studied the reproducibility of different tools to identify and measure white-matter tracts by using a DTI sequence suitable for clinical protocols. Results: Both the number and extent of visible traumatic lesions on conventional MRI diminished significantly with time. Slightly increased diffusion in the normal-appearing brain was a common finding at one week after TBI, but it was not significantly associated with the injury severity. Fractional anisotropy values, that represent the integrity of the white-matter tracts, were significantly diminished in several tracts in TBI patients compared to the control subjects. Compared to the cross-sectional ROI method, the tract-based analyses had better reproducibility to identify and measure white-matter tracts of interest by means of DTI tractography. Conclusions: As conventional MRI is still applied in clinical practice, it should be carried out soon after the injury, at least in symptomatic patients with negative CT scan. DWI-related brain diffusivity measurements may be used to improve the documenting of TBI. DTI tractography can be used to improve radiologic diagnostics in a symptomatic TBI sub-population with no findings on conventional MRI. Reproducibility of different tools to quantify fibre tracts vary considerably, which should be taken into consideration in the clinical DTI applications.

Developing correlative Optical-Photoacustic microscopy for high-resolution in vivo imaging

PhotoAcoustic Imaging (PAI) is a branch in clinical and pre-clinical imaging, that refers to the techniques mapping acoustic signals caused by the absorption of the short laser pulse. This conversion of electromagnetic energy of the light to the mechanical (acoustic) energy is usually called photoacoustic effect. PAI, by combining optical excitation with acoustical detection, is able to preserve the diffraction limited spatial resolution. At the same time, the penetration depth is extended beyond the diffusive limit. The Laser-Scanning PhotoAcoustic Microscope system (LS-PAM) has been developed, that offers the axial resolution of 7.75 µm with the lateral resolution better than 10 µm. The first in vivo imaging experiments were carried out. Thus, in vivo label-free imaging of the mouse ear was performed. The principle possibility to image vessels located in deep layers of the mouse skin was shown. As well as that, a gold printing sample, vasculature of the Chick Chorioallantoic Membrane Assay, Drosophila larvae were imaged by PAI. During the experimental work, a totally new application of PAM was found, in which the acoustic waves, generated by incident light can be used for further imaging of another sample. In order to enhance the performance of the presented system two main recommendation can be offered. First, the current system should be transformed into reflection-mode setup system. Second, a more powerful source of light with the sufficient repetition rate should be introduced into the system.