Optical Tweezers for Single Molecule Biology

Molecular machinery on the micro-scale, believed to be the fundamental building blocks of life, involve forces of 1-100 pN and movements of nanometers to micrometers. Micromechanical single-molecule experiments seek to understand the physics of nucleic acids, molecular motors, and other biological systems through direct measurement of forces and displacements. Optical tweezers are a popular choice among several complementary techniques for sensitive force-spectroscopy in the field of single molecule biology. The main objective of this thesis was to design and construct an optical tweezers instrument capable of investigating the physics of molecular motors and mechanisms of protein/nucleic-acid interactions on the single-molecule level. A double-trap optical tweezers instrument incorporating acousto-optic trap-steering, two independent detection channels, and a real-time digital controller was built. A numerical simulation and a theoretical study was performed to assess the signal-to-noise ratio in a constant-force molecular motor stepping experiment. Real-time feedback control of optical tweezers was explored in three studies. Position-clamping was implemented and compared to theoretical models using both proportional and predictive control. A force-clamp was implemented and tested with a DNA-tether in presence of the enzyme lambda exonuclease. The results of the study indicate that the presented models describing signal-to-noise ratio in constant-force experiments and feedback control experiments in optical tweezers agree well with experimental data. The effective trap stiffness can be increased by an order of magnitude using the presented position-clamping method. The force-clamp can be used for constant-force experiments, and the results from a proof-of-principle experiment, in which the enzyme lambda exonuclease converts double-stranded DNA to single-stranded DNA, agree with previous research. The main objective of the thesis was thus achieved. The developed instrument and presented results on feedback control serve as a stepping stone for future contributions to the growing field of single molecule biology.

Irradiation effects in graphene and related materials

Nanomaterials with a hexagonally ordered atomic structure, e.g., graphene, carbon and boron nitride nanotubes, and white graphene (a monolayer of hexagonal boron nitride) possess many impressive properties. For example, the mechanical stiffness and strength of these materials are unprecedented. Also, the extraordinary electronic properties of graphene and carbon nanotubes suggest that these materials may serve as building blocks of next generation electronics. However, the properties of pristine materials are not always what is needed in applications, but careful manipulation of their atomic structure, e.g., via particle irradiation can be used to tailor the properties. On the other hand, inadvertently introduced defects can deteriorate the useful properties of these materials in radiation hostile environments, such as outer space. In this thesis, defect production via energetic particle bombardment in the aforementioned materials is investigated. The effects of ion irradiation on multi-walled carbon and boron nitride nanotubes are studied experimentally by first conducting controlled irradiation treatments of the samples using an ion accelerator and subsequently characterizing the induced changes by transmission electron microscopy and Raman spectroscopy. The usefulness of the characterization methods is critically evaluated and a damage grading scale is proposed, based on transmission electron microscopy images. Theoretical predictions are made on defect production in graphene and white graphene under particle bombardment. A stochastic model based on first-principles molecular dynamics simulations is used together with electron irradiation experiments for understanding the formation of peculiar triangular defect structures in white graphene. An extensive set of classical molecular dynamics simulations is conducted, in order to study defect production under ion irradiation in graphene and white graphene. In the experimental studies the response of carbon and boron nitride multi-walled nanotubes to irradiation with a wide range of ion types, energies and fluences is explored. The stabilities of these structures under ion irradiation are investigated, as well as the issue of how the mechanism of energy transfer affects the irradiation-induced damage. An irradiation fluence of 5.5x10^15 ions/cm^2 with 40 keV Ar+ ions is established to be sufficient to amorphize a multi-walled nanotube. In the case of 350 keV He+ ion irradiation, where most of the energy transfer happens through inelastic collisions between the ion and the target electrons, an irradiation fluence of 1.4x10^17 ions/cm^2 heavily damages carbon nanotubes, whereas a larger irradiation fluence of 1.2x10^18 ions/cm^2 leaves a boron nitride nanotube in much better condition, indicating that carbon nanotubes might be more susceptible to damage via electronic excitations than their boron nitride counterparts. An elevated temperature was discovered to considerably reduce the accumulated damage created by energetic ions in both carbon and boron nitride nanotubes, attributed to enhanced defect mobility and efficient recombination at high temperatures. Additionally, cobalt nanorods encapsulated inside multi-walled carbon nanotubes were observed to transform into spherical nanoparticles after ion irradiation at an elevated temperature, which can be explained by the inverse Ostwald ripening effect. The simulation studies on ion irradiation of the hexagonal monolayers yielded quantitative estimates on types and abundances of defects produced within a large range of irradiation parameters. He, Ne, Ar, Kr, Xe, and Ga ions were considered in the simulations with kinetic energies ranging from 35 eV to 10 MeV, and the role of the angle of incidence of the ions was studied in detail. A stochastic model was developed for utilizing the large amount of data produced by the molecular dynamics simulations. It was discovered that a high degree of selectivity over the types and abundances of defects can be achieved by carefully selecting the irradiation parameters, which can be of great use when precise pattering of graphene or white graphene using focused ion beams is planned.

Estimation of cutting volume with three inventory methods for harvest planning in Canadian boreal forests.

Two methods of pre-harvest inventory were designed and tested on three cutting sites containing a total of 197 500 m3 of wood. These sites were located on flat-ground boreal forests located in northwestern Quebec. Both methods studied involved scaling of trees harvested to clear the road path one year (or more) prior to harvest of adjacent cut-blocks. The first method (ROAD) considers the total road right-of-way volume divided by the total road area cleared. The resulting volume per hectare is then multiplied by the total cut-block area scheduled for harvest during the following year to obtain the total estimated cutting volume. The second method (STRATIFIED) also involves scaling of trees cleared from the road. However, in STRATIFIED, log scaling data are stratified by forest stand location. A volume per hectare is calculated for each stretch of road that crosses a single forest stand. This volume per hectare is then multiplied by the remaining area of the same forest stand scheduled for harvest one year later. The sum of all resulting estimated volumes per stand gives the total estimated cutting-volume for all cut-blocks adjacent to the studied road. A third method (MNR) was also used to estimate cut-volumes of the sites studied. This method represents the actual existing technique for estimating cutting volume in the province of Quebec. It involves summing the cut volume for all forest stands. The cut volume is estimated by multiplying the area of each stand by its estimated volume per hectare obtained from standard stock tables provided by the governement. The resulting total estimated volume per cut-block for all three methods was then compared with the actual measured cut-block volume (MEASURED). This analysis revealed a significant difference between MEASURED and MNR methods with the MNR volume estimate being 30 % higher than MEASURED. However, no significant difference from MEASURED was observed for volume estimates for the ROAD and STRATIFIED methods which respectively had estimated cutting volumes 19 % and 5 % lower than MEASURED. Thus the ROAD and STRATIFIED methods are good ways to estimate cut-block volumes after road right-of-way harvest for conditions similar to those examined in this study.

Effect of colostrum feeding method and presence of dam on the sleep, rest and sucking behaviour of newborn calves

"In rats, sucking milk reduces anxiety and promotes non-rapid eye movement (NREM) sleep, and in calves it induces resting but the effect on sleep is unknown. Here, we investigated how calves' sleep was affected by colostrum feeding methods. Forty-one calves were blocked by birth date and randomly allotted within blocks to the experimental treatments. Calves were housed for four days either with their dam (DAM) or individually with warm colostrum feeding (2 L four times a day) from either a teat bucket (TEAT) or an open bucket (BUCKET). DAM calves suckled their dam freely. Calves' sleeping and sucking behaviour was filmed continuously for 48 h at the ages of two and three days. Behavioural sleep (BS) was defined as calves resting at least 30 s with their head still and raised (non-rapid eye movement) or with their head against their body or the ground (rapid eye movement, REM). Latency from the end of colostrum feeding to the start of BS was recorded. We compared behaviour of TEAT calves with that of DAM and BUCKET calves using mixed models. Milk meal duration was significantly longer for TEAT calves than for BUCKET calves (mean +/- S.E.M.; 8.3 +/- 0.6 min vs. 5.2 +/- 0.6 min), but equal to that of DAM calves. We found no effect of feeding method on the duration of daily BS (12 h 59 min I h 38 min) but we found a tendency for the daily amount of NREM sleep; BUCKET calves had less NREM sleep per day than TEAT calves (6 h 18 min vs. 7 h 48 min, S.E.M. = 45 min) and also longer latencies from milk ingestion to BS (21.9 +/- 2.0 min vs. 16.2 +/- 2.0 min). DAM calves slept longer bouts than TEAT calves (10.8 +/- 1.0 min vs. 8.3 +/- 1.0 min) and less often (78 +/- 4 vs. 92 +/- 4). Sucking colostrum from a teat bucket compared with drinking from an open"

Domestic labour relations in India : Vulnerability and gendered life courses in Jaipur

This study explores labour relations between domestic workers and employers in India. It is based on interviews with both employers and workers, and ethnographically oriented field work in Jaipur, carried out in 2004-2007. Combining development studies with gender studies, labour studies, and childhood studies, it asks how labour relations between domestic workers and employers are formed in Jaipur, and how female domestic workers trajectories are created. Focusing on female part-time maids and live-in work arrangements, the study analyses children s work in the context of overall work force, not in isolation from it. Drawing on feminist Marxism, domestic labour relations are seen as an arena of struggle. The study takes an empirical approach, showing class through empiria and shows how paid domestic work is structured and stratified through intersecting hierarchies of class, caste, gender, age, ethnicity and religion. The importance of class in domestic labour relations is reiterated, but that of caste, so often downplayed by employers, is also emphasized. Domestic workers are crucial to the functioning of middle and upper middle class households, but their function is not just utilitarian. Through them working women and housewives are able to maintain purity and reproduce class disctinctions, both between poor and middle classes and lower and upper middle classes. Despite commodification of work relations, traditional elements of service relationships have been retained, particularly through maternalist practices such as gift giving, creating a peculiar blend of traditional and market practices. Whilst employers of part-time workers purchase services in a segmented market from a range of workers for specific, traditional live-in workers are also hired to serve employers round the clock. Employers and workers grudgingly acknowledged their dependence on one another, employers seeking various strategies to manage fear of servant crime, such as the hiring of children or not employing live-in workers in dual-earning households. Paid domestic work carries a heavy stigma and provide no entry to other jobs. It is transmitted from mothers to daughters and working girls were often the main income providers in their families. The diversity of working conditions is analysed through a continuum of vulnerability, generic live-in workers, particularly children and unmarried young women with no close family in Jaipur, being the most vulnerable and experienced part-time workers the least vulnerable. Whilst terms of employment are negotiated informally and individually, some informal standards regarding salary and days off existed for maids. However, employers maintain that workings conditions are a matter of individual, moral choice. Their reluctance to view their role as that of employers and the workers as their employees is one of the main stumbling blocks in the way of improved working conditions. Key words: paid domestic work, India, children s work, class, caste, gender, life course

Vieraan aksentin arviointi ja mittaaminen suomessa

In the field of second language (L2) acquisition, the term `foreign accent´ is often used to refer to speech characteristics that differ from the pronunciation of native speakers. Foreign accent may affect the intelligibility and perceived comprehensibility of speech and it is also sometimes associated with negative attitudes. The degree of L2 learners foreign accent and the speech characteristics that account for it have previously been studied through speech perception experiments and acoustic measurements. Perception experiments have shown that native listeners are easily able to identify foreign accent in speech. However to date, no studies have been done on the assessment of foreign accent in the speech of non-native speakers of Finnish. The aim of this study is to examine how native speakers of Finnish rate the degree of foreign accentedness in the speech of Russian L2 learners of Finnish. Furthermore, phonetic analysis is used to study the characteristics of speech that affect the perceived strength of foreign accent. Altogether 96 native speakers of Finnish listened to excerpts of read-aloud and spontaneous Finnish speech from ten Russian and six Finnish female speakers. The Russian speakers were intermediate and advanced learners of Finnish and had all immigrated to Finland as adults. Among the listeners, was a group of teachers of Finnish as an L2, and it was presumed that these teachers had been exposed to foreign accent in Finnish and were used to hearing it. The temporal aspects and segmental properties of speech were phonetically analysed in the speech of the Russian speakers in order to measure their effect on the perceived degree of accent. Although wide differences were observed in the use of the rating scale among the listeners, they were still quite unanimous on which speakers had the strongest foreign accent and which had the mildest. The listeners background factors had little effect on their ratings, and the ratings of the teachers of Finnish as an L2 did not differ from those of the other listeners. However, a clear difference was noted in the ratings of the two types of stimuli used in the perception experiment: the read-aloud speech was rated as more strongly accented than the spontaneous speech. It is important to note that the assessment of foreign accent is affected by many factors and their complex interactions in the experimental setting. Futher the study found that, both the temporal aspects of speech, often associated with fluency, and the number of single deviant phonetic segments contributed to the perceived degree of accentedness in the speech of the native Russian speakers.

Impaired spermatogenesis in Finnish boars and bulls

Maintenance of breeding efficiency and high semen quality is essential for reproductive success in farm animals. Early recognition of possible inheritable factors causing infertility requires constant attention. This thesis focuses on describing different manifestations of impaired spermatogenesis, their impact on fertility and partly also their incidence in populations. The reasons for spermatogenic failure are various. An interruption of germ cell differentiation, spermatogenic arrest, can lead to infertility. The incidence of azoospermia was investigated in the 1996 2005 survey of Finnish AI and farm breeding boars. We focused on the diagnosis, testicular morphometry and the possible reasons for the condition. The incidence of azoospermia was significantly higher in Yorkshire boars than in the Landrace breed. The most common diagnosis in Yorkshire boars was germ cell arrest at the primary spermatocyte level. The second most frequent diagnosis in Yorkshire boars was segmental aplasia of the Wolffian ducts with idiopathic epididymal obstruction. Other reasons for azoospermia were infrequent. In the second study we investigated the incidence of two relatively well-defined specific sperm defects in Finnish Yorkshire and Landrace boars during the same survey, the immotile short-tail sperm (ISTS) defect and the knobbed acrosome (KA) defect. In the Finnish Yorkshire boars the inherited ISTS defect, and the probably inherited KA defect, were important causes of infertility during 1996 2005. The ISTS defect was found in 7.6% and the KA defect in 0.8% of the Yorkshire boars. No Landrace boars were diagnosed with either of these two defects. In the third study we described a new sterilizing sperm defect in an oligoasthenoterazoospermic bull. Because of its morphological characteristics this defect was termed the multinuclear-multiflagellar sperm (MNMFS) defect. The number of Sertoli cells in the seminiferous tubuli was highly increased in the MNMFS bull compared with the number in normal bulls. In the following two studies we used a combined approach of fluorescence in situ hybridization (FISH), flow cytometry and morphometric studies to provide information on the cytogenetic background of macrocephalic bull spermatozoa. We described cellular features of diploid spermatozoa and compared the failures in the first and second meiotic divisions. In the last study we describe how the transplantation of testicular cells was used to determine whether spermatogonia derived from donor animals are able to colonize and produce motile spermatozoa in immune-competent unrelated boars suffering the ISTS defect. Transplantation resulted in complete focal spermatogenesis, indicated by the appearance of motile spermatozoa and confirmed by genotyping.

Asuntopolitiikka ja vieraskielisen väestön alueellinen keskittyminen Helsingissä vuosina 1992-2005

The aim of the thesis was to study the extent of spatial concentration of immigrant population in Helsinki and to analyse the impact of housing policy on ethnic residential segregation in 1992-2005. For the purpose of the study, immigrant population was defined based on the language spoken at home. The theory of residential segregation by Andersson and Molina formed the main theoretical framework for the study. According to Andersson and Molina ethnic residential segregation results from different dynamic intra-urban migration processes. Institutionally generated migration, i.e. migration patterns generated by various housing and immigrant policies and procedures, is one of the central factors in the development of ethnic segregation. The data of the study consisted of population and housing statistics and housing and immigrant policy documents of Helsinki municipality. Spatial concentration of immigrant population was studied both at district and building levels using GIS-methods and statistical methods. The housing policy of Helsinki municipality was analysed using a method created by Musterd et al. Musterd et al. categorise two types of policy approaches to residential segregation: spatial dispersion policy and compensating policy. The housing policy of Helsinki has a strong focus on social mixing and spatial dispersion of housing stock. Ethnic segregation is regarded as a threat. The importance of ethnic communities and networks is, however, acknowledged and small-scale concentration is therefore not considered harmful. Despite the spatial dispersion policy, the immigrant population is concentrated in the eastern, north-eastern and north-western suburbs of Helsinki. The spatial pattern of concentration was formed already at the beginning of the 1990's when immigration to Finland suddenly peaked. New immigrant groups were housed in the neighbourhoods where public housing was available at the time. Housing policy, namely the location of new residential areas and public housing blocks and the policies of public housing allocation were key factors influencing the residential patterns of immigrant population in the 1990's. The immigration and refugee policies of the state have also had an impact on the development. The concentration of immigrant population has continued in the same areas in the beginning of the 2000's. Dispersion to new areas has mainly taken place within the eastern and north-eastern parts of the city or in the adjacent areas. The migration patterns of native population and the reasonably rapid changes in the housing market have emerged as new factors generating and influencing the ethnic residential segregation in Helsinki in the 2000's. Due to social mixing and spatial dispersion policies, ethnic segregation in Helsinki has so far been fairly small-scale, concentrated in particular housing blocks. The number of residential buildings with a high share of immigrant population is very modest. However, the number of such buildings has doubled between 1996-2002. The concentration of immigrant population concerns mainly the public housing sector. The difference in the level of concentration between the public housing sector and privately owned housing companies is remarkable.

Helsingin kävelykeskustan suunnittelu ydinkeskustan kadunvarsiliikeyrittäjien näkökulmasta

Aiempien tutkimusten mukaan keskustayrittäjien kielteinen asenne on olennaisesti vaikeuttanut kävelykatujen toteuttamista kaupunkikeskustoissa. Yrittäjät pelkäävät ostokykyisten asiakkaiden kaikkoavan ja liikevaihtonsa pienenevän kävelykeskustauudistusten myötä. Yrittäjien kielteiset asenteet ovat usein myös painottuneet suuriin kaupunkeihin. Etenkin ydinkeskustassa sijaitsevat kadunvarsiliikeyrittäjät ovat kokeneet kävelykadut ongelmiksi. Tämän tutkimuksen tavoitteena on selvittää, mikä on ydinkeskustan kadunvarsiliikeyrittäjien näkemys Helsingin kävelykeskustan suunnittelusta, millaisena ydinkeskustan kadunvarsiliikeyrittäjät näkevät sijaintikatunsa kävelykeskustan suunnittelupäämäärien kautta tarkasteltuna, ja onko kävelykatuyrittäjien ja kävelykatujen ulkopuolella sijaitsevien yrittäjien näkemysten välillä eroja. Taustana tälle tarkastellaan Helsingin kävelykeskustaa pohjoismaisessa kontekstissa, ja käydään läpi Helsingin kävelykeskustan suunnittelun ja rakentumisen historiaa ja päämääriä. Tutkimuksen aineisto koostuu Helsingin ydinkeskustan kadunvarsiliikeyrittäjille tehdystä kyselystä, kävelykeskustoihin liittyvistä tutkimuksista ja selvityksistä, kaupunkisuunnitteluun ja -tutkimukseen liittyvästä tutkimuskirjallisuudesta, sanomalehtiartikkeleista, ydinkeskustassa tehdystä empiirisestä havainnoinnista ja kahdesta asiantuntijahaastattelusta. Kyselyaineistoa analysoidaan tutkimuksessa tilastollisten menetelmien avulla. Helsingin kävelykeskustan suunnittelu ja rakentuminen on ollut pitkällinen prosessi. Vuoden 1 989 kävelykeskustan periaatesuunnitelmasta on konkretisoitunut Kluuvikatu ja Mikonkatu. Keskustatunnelihanke on vaakalaudalla, minkä vuoksi kävelykeskustan uuden periaatesuunnitelman toteutuminen on epävarmaa. Kävelykeskustan rakentuminen kuitenkin etenee. Keskuskatu ja Kalevankadun itäpää muutetaan kävelykaduiksi ja ydinkeskustan jalankulkuympäristöä parannetaan ja kehitetään jatkuvasti. Tällä hetkellä kävelykeskustan suunnittelun painopiste on Aleksanterinkadun kortteleiden ympäristössä, ja suunnittelun tärkeimpiä päämääriä ovat viihtyisyyden, turvallisuuden, kaupallisen vetovoimaisuuden ja saavutettavuuden parantaminen. Kyselyyn vastanneiden yrittäjien mielestä kävelykeskustaa tulisi laajentaa, sillä laajemman kävelykeskustan nähdään kasvattavan liikevaihtoa ja lisäävän yleisesti ydinkeskustan vetovoimaa. Kävelykatuyrittäjien näkemykset kävelykeskustan suunnittelusta ja kehittämisestä olivat kävelykatujen ulkopuolisia yrittäjiä kielteisempiä. Em. yrittäjien asenteisiin vaikuttavat oletettavasti tutkituilla kävelykaduilla ilmenneet ongelmat. Kyse voi myös olla siitä, että uusien kävelykatujen rakentaminen haittaa kävelykatujen saavutettavuutta. Kadunvarsiliikeyrittäjien yleinen suhtautuminen kävelykeskustan kehittämiseen ja suunnittelupäämääriin on kuitenkin pääosin positiivista. Jatkotutkimuksen kannalta olisi kiinnostavaa selvittää, onko muiden ydinkeskustassa toimivien yritysten, kuten esimerkiksi tavaratalojen ja kauppakeskusten johdon ja kiinteistösijoittajien ja -omistajien suhtautuminen kävelykeskustan kehittämiseen myös positiivista. Tämän lisäksi olisi mielenkiintoista selvittää keskustan käyttäjien ja keskustassa asuvien asenteita kävelykeskustan suunnitteluun, ja verrata tuloksia tässä tutkimuksessa selvitettyihin kadunvarsiliikeyrittäjien näkemyksiin.

Elämää keskustassa ja kaupunkiseudun reunoilla. Urbaani ja jälkiesikaupungillinen elämäntyyli asumisen valinnoissa ja arkiliikkumisessa Helsingin seudulla.

The lifestyles of people living in single-family housing areas on the outskirts of the Greater Helsinki Region (GHR) are different from those living in inner city area. The urban structure of the GHR is concentrated in the capital on the one hand, and spread out across the outskirts on the other. Socioeconomic spatial divisions are evident as well-paid and educated residents move to the inner city or the single-family house dominated suburban neighbourhoods depending on their housing preferences and life situations. The following thesis explores how these lifestyles have emerged through the housing choices and daily mobility of the residents living in the new single-family housing areas on the outskirts of the GHR and the inner city. The study shows that, when it comes to lifestyles, residents on the outskirts of the region have different housing preferences and daily mobility patterns when compared with their inner city counterparts. Based on five different case study areas my results show that these differences are related to residents values, preferences and attitudes towards the neighbourhood, on the one hand, and limited by urban structure on the other. This also confirms earlier theoretical analyses and findings from the GHR. Residents who moved to the outskirts of Greater Helsinki Region and the apartment buildings of the inner city were similar in the basic elements of their housing preferences: they sought a safe and peaceful neighbourhood close to the natural environment. However, where housing choices, daily mobility and activities vary different lifestyles develop in both the outskirts and the inner city. More specifically, lifestyles in the city apartment blocks were inherently urban. Liveliness and highest order facilities were appreciated and daily mobility patterns were supported by diverse modes of transportation for the purposes of work, shopping and leisure time. On the outskirts, by contrast, lifestyles were largely post-suburban and child-friendliness appreciated. Due to the heterachical urban structure, daily mobility was more car-dependent since work, shopping and free time activities of the residents are more spread around the region. The urban structure frames the daily mobility on the outskirts of the region, but this is not to say that short local trips replace longer regional ones. This comparative case study was carried out in the single-family housing areas of Sundsberg in Kirkkonummi, Landbo in Helsinki and Ylästö in Vantaa, as well as in the inner city apartment building areas of Punavuori and Katajanokka in Helsinki. The data is comprised of residential surveys, interviews, and statistics and GIS data sets that illustrate regional daily mobility, socio-economic structure and vis-à-vis housing stock.