Merging microfluidics and micro-array concepts: from molecular to nematode-based bioassays

Essential in biomedical research is the necessity of gathering statistically relevant data about large populations of specific biological entities, e.g. organisms, cells or molecules, while preserving detailed information about each single entity under investigation. This thesis deals with this need and proposes the combination of microfluidics and micro-arraying techniques in developing technological tools to conceive bio-assays at single molecule/cell/organism resolution. First, we propose an on-chip immunoassay technique, through which we demonstrated detection of the biomarker tumor necrosis factor alpha in serum down to concentrations in the attomolar range (10-18 M). In particular, we provide a comprehensive predictive model of the assay, which employs micro-arrays of superparamagnetic beads. We introduce the concept of magnetic particle-scanning, as a method for building immunoassays with extremely low limit of detection, down to the single-molecule level. Afterwards, we modified our bead micro-arraying technique, to make it suitable for the immobilization of particles and cells of various sizes and properties. Specifically, we present a method for the electrostatic self-assembly of dielectric microspheres in well templates, as a technique for fast and versatile fabrication of microlens arrays. By combining these arrays with microfluidics, we created a new tool for single-nanoparticle detection in flowing media, able to detect moving objects of sub-diffraction size through conventional low-magnification microscopes. An analogous micro-arraying method was then developed to seed large populations of non-adherent cells in isolated micro-compartments. In combination with an electrowetting-on-dielectric microfluidic platform, this technique allows implementing high-throughput cytotoxicity assays on yeast cells, at single-cell resolution. Subsequently, we conceived technological solutions for the automated analysis of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. First, we developed a microfluidic platform for on-chip nematode culture and creation of synchronized C. elegans embryo micro-arrays. Long-term multi-dimensional imaging in our device allows systematic phenotyping studies at single-embryo resolution. We could discriminate embryonic development variations with unprecedented accuracy and we successfully analyzed the impact of perturbations of the mitochondrial functions on the embryogenesis. A second generation prototype of the device is then presented, enabling long-term automated studies on C. elegans at single-nematode resolution and over the whole organism development, from early embryogenesis to adulthood. Finally, we introduce a third generation prototype, which features: (i) a new microfluidic design tailored for the isolation of larvae at a desired developmental stage and for their successive culture and treatment; (ii) a method for reversible immobilization of nematodes, enabling long-term high-resolution imaging. We successfully employed this platform to analyze protein aggregation in a C. elegans model for human amyotrophic lateral sclerosis (ALS). The device allows precisely localizing protein aggregates within the nematode tissues, as well as monitoring the evolution of single aggregates over consecutive days at sub-cellular level

Social relationships in captive Bornean orangutans (Pongo pygmaeus)

This study investigates the social relationships in captive groups of Bornean orangutans (Pongo pygmaeus) concerning their social system and sociality. Wild orangutans are supposed to live in fission-fusion societies with a high flexibility in spatial and social structures. Zoo-living orangutans however are usually housed in stable groups. It is unclear whether a permanent group-living under the spatial restrictions of captivity over longer periods of time corresponds to the orangutans` social potential. The orangutans’ social system is still not well understood. Proximate data referring to mechanisms regulating the social relationships, including a sufficient sample size of individuals, are largely missing so far. This study examines the structure of social relationships, the persistence and form of fission-fusion tendencies, and how the individuals deal with the group-living conditions. Three sets of orangutans were comparatively studied. Two groups were housed under constant group-keeping conditions; one group was kept under more flexible conditions. The analyses focused on the patterns of the interactive and spatial behaviour. Additionally, long-term observations were carried out on one of the groups, and a second sampling method was implemented allowing a sequence analysis of behaviours. The results of this study support the assumption that orangutans have the potential to engage in a higher amount of social activities compared to the wild without many conflicts. To evaluate this social potential under natural conditions is hardly possible. The structure of social relationships between the studied animals was largely compatible to what is described for free-ranging orangutans, though the relationships tended to be generally friendlier. The different keeping conditions seem to have little influence on the social patterns. The socio-spatial patterns were characterized by frequent encounters and leavings; interactions were mainly sociopositive but brief. On the structural level, this pattern is in accordance with the fission-fusion sociality described for wild orangutans. Orangutans may possibly have an intrinsic motivation to merge and split regularly and to establish only weak social bonds. Future work should refer to this, both under the conditions in the wild and under human care. The findings of this study can contribute to the further development of new concepts on fission-fusion dynamics in primates. This work points out that a detailed analysis of the interactive and spatial-temporal patterns under the conditions in captivity provides important and supplemental clues about the sociality of orangutans. This may account also for the management of this endangered species in zoos and fragmented areas in the wild

Application of microfabrication in electrochemical sensing.

In this work, micro fabrication techniques are explored not only to simplify the production of complex lab on-a-chip devices (LOC), but also micro fabrication will be utilized to create intelligent design features that will enhance an electrochemical sensor\u27s capabilities. First, a low temperature adhesive bonding procedure for LOC glass devices was evaluated for capillary electrophoresis (CE) applications. This low temperature method utilizes UV adhesive to bond the glass microchips under the assistance of a mask aligner. The bonding process was carried out at room temperature in \u3c 30 minutes, and provided a near 100% success rate. Microchips exhibited similar electroosmotic flow, separation characteristics, stable long-term performance, excellent chip-to-chip reproducibility, as their thermally bonded counter parts. This bonding approach required new but easily implemented structural features. In addition to cost effective and reliable fabrication techniques, microchips designed for long-term unattended electrochemical sensing have been evaluated. Specific advantages of the micro fabrication approach include the capability to create an intelligent design containing features such as redundant sensing electrodes, on-chip reference and auxiliary electrodes, and in situ electrode regeneration/calibration. One system targeted involves continuous pH monitoring in drinking water at solid-state iridium oxide electrodes. Microchips utilized consist of a flow-through silicon platform containing patterned gold electrodes onto which iridium oxide was deposited electrochemically. To simulate drinking water detection scenarios, sensors were integrated into a flow system. Elven equivalent pH electrodes where evaluated for electrode-to-electrode reproducibility, long-term drift, and response to expected interfering agents. With on-chip voltage treatment, absolute potentials measured for an electrode array are within ± 4 mY, with identical (±1 mY/pH unit) calibration slopes. This performance level is sustainable over weeks. Sensors for exhaustive coulometry were designed, fabricated and evaluated. Microchips contained thin-film gold working and Ag/AgCI pseudo-reference electrodes. A custom flow cell containing a counter electrode chamber was constructed to integrate the sensor and to create an electrolysis chamber with a fixed volume. Different chip designs were evaluated for reproducibility and longevity using Fe(CN)63-/4- as model analytes. The relative standard deviation (RSD) for a chip (over 42 days) was 5.5% whereas the sensor-to-sensor reproducibility was within 6.3%. A more practical application for utilizing exhaustive coulometry by the determination of free chlorine in drinking water is briefly evaluated. Initially studies will outline the challenges involved by analyzing hypochlorite

Ageing with Smartphones in Urban China: From the cultural to the digital revolution in Shanghai

If we want to understand contemporary China, the key is through understanding the older generation. This is the generation in China whose life courses almost perfectly synchronised with the emergence and growth of the ‘New China’ under the rule of the Communist Party (1949). People in their 70s and 80s have double the life expectancy of their parents’ generation. The current oldest generation in Shanghai was born in a time when the average household could not afford electric lights, but today they can turn their lights off via their smartphone apps. Based on 16-month ethnographic fieldwork in Shanghai, Ageing with Smartphones in Urban China tackles the intersection between the ‘two revolutions’ experienced by the older generation in Shanghai: the contemporary smartphone-based digital revolution and the earlier communist revolutions. We find that we can only explain the smartphone revolution if we first appreciate the long-term consequences of these people’s experiences during the communist revolutions. The context of this book is a wide range of dramatic social transformations in China, from the Cultural Revolution to the individualism and Confucianism in Digital China. Supported by detailed ethnographic material, the observations and analyses provide a panoramic view of the social landscape of contemporary China, including topics such as the digital and everyday life, ageing and healthcare, intergenerational relations and family development, community building and grassroots organizations, collective memories and political attitudes among ordinary Chinese people

INVESTIGATION OF CHEMOMETRICS METHODS FOR CHARACTERISING DRIFT PHENOMENA IN iCK-AES

The objective of this study was to fully characterise drift phenomena in inductively coupled plasma atomic emission spectroscopy (ICP-AES) in order to develop novel correction procedures to aid routine analysis. Long-term drift of the analytical signal continues to be a potential disadvantage when using ICP-AES and often necessitates regular recalibration. The long-term stability of three commercially available Instruments was studied using In each case a range of analyte and intrinsic plasma emission lines. Long-term fluctuations were observed which generated drift bias of up to 20% on the initial values. The drift pafterns were characterised and found to be qualitatively reproducible. In most cases, similar long-term fluctuations were observed independent of the analyte or nature of the emission line. In addition, high inter-element correlation was observed on the long-term fluctuations even when sequential acquisition was employed. In order to study the fundamental causes of drift, the effect of two key instrumental parameters, i.e. the RF power and the nebutiser gas flow rate were studied with respect to the stability of the signal. Different drift patterns were found depending on the working conditions. Classical statistical methods and a multi-way approach. PARAFAC. were then employed to describe the system. The use of intemal standards to correct for drift has also been investigated, but found to be of benefit only under certain defined conditions (i.e. robust conditions, high RF power and low nebuliserflow rate). At soft conditions, low RF power and medium to high nebuliser flow rate, the system Is very unstable and intemal standardisation is not fully effective as a correction method. For such conditions, a novel correction procedure has been developed, which employs the drift pattem of one intrinsic plasma line (i.e. an argon line) and a correction factor which is specific for each emission line. The drift values were reduced from around 20% before correction to better than ±2% following the described protocol. Finally, the effects of chemical matrices on the long-term stability of the emission signals have been evaluated. Three synthetic matrices were prepared simulating nitric, soil and water matrices. The stability of the instrument when working wrth these matrices at both robust and soft conditions was found to be poor, especially when the solution was matched with the soil matrix. The use of more robust conditions did not improve the long-temi stability of the emission signals. The outcome of this study proved to be a better understanding of drift phenomena and a novel method for drift correction.BRITISH GEOLOGICAL SURVE

Long-term ecological research program: shortgrass steppe

Proposal submitted to National Science Foundation Ecosystems Studies Program; Principal investigators: W. K. Lauenroth, I. C. Burke, J. Van Schilfgaarde, J. R. Forwood.Includes bibliographical references (pages 132-140).The SGS-LTER research site was established in 1980 by researchers at Colorado State University as part of a network of long-term research sites within the US LTER Network, supported by the National Science Foundation. Scientists within the Natural Resource Ecology Lab, Department of Forest and Rangeland Stewardship, Department of Soil and Crop Sciences, and Biology Department at CSU, California State Fullerton, USDA Agricultural Research Service, University of Northern Colorado, and the University of Wyoming, among others, have contributed to our understanding of the structure and functions of the shortgrass steppe and other diverse ecosystems across the network while maintaining a common mission and sharing expertise, data and infrastructure.We propose to continue the long-term ecological research project in the shortgrass steppe, at the Central Plains Experimental Range in north central Colorado. The objective is to improve our understanding of the long-term processes responsible for the origin and sustainability of shortgrass steppe ecosystems. Our concept for long-term study of the shortgrass steppe is that the major controls over ecosystem structure and function are climate, geomorphology, and landuse management. Each of these controls has important spatial and temporal heterogeneity across a range of scales, and exerts its influence at a particular combination of levels. We hypothesize that the most important controls at the CPER are soil texture and landuse. We propose to continue current work and initiate new studies to further examine the influence of soil texture and landuse over shortgrass steppe ecosystem structure and function using field experimental research, simulation, and regional analysis

"Walking the fine line?" : Young people, sporting risk, health and embodied identities

sociological literature suggests that adult sports participation is occurring in a 'culture of risk' which glorifies pain, rationalises risk and promotes the practice of playing hurt (Messner, 1990; Nixon, 1992; Curry 1993; Pike, 2000; Roderick et aI, 2000, Safai, 2003; Howe, 2004; Young, 2004a; Liston et aI. 2006). Using this corpus of knowledge as a point of departure, this study directs attention towards young people's sporting risk encounters within the specific context of school sport. Guided by a process-sociological framework (Elias, 1978, 1991,2000 [1939]), it offers an insight into the ways in which young people interpret, experience and manage sporting risk and episodes of sporting pain and injury whilst at school. The research draws on data generated by 1,651 young people aged between ten and sixteen years old using a three-phase data collection programme. The programme incorporated self-report questionnaires, semi-structured interviews and group-based creative tasks and was conducted in six secondary schools located in "Churchill", a major English conurbation. The findings suggest that school sport worlds (re )produce two entwined, yet competing sets of beliefs, attitudes and practices related to sporting pain and injury and are best described as webs of risks and precaution and protectionism. Rather than adopting a more cautious approach to pain and injury the data indicates that this cluster of young people frequently play hurt, normalise injury and engage in forms of 'injury talk' that discredit episodes of sporting pain. In so doing, they may be placing their short and long-term physical, psychological, social and moral health in jeopardy. However, it is argued that this collection of sporting practices are highly valued by young people and are integral to the ways in which they assign and perform a range of dissecting and fluid embodied identities. Notwithstanding the potential for sporting risk encounters to engender damaging, disrupting and debilitating outcomes, the data also emphasises the potential for these experiences to act as important spaces in which young people are able to probe their bodily limits, develop corporeal knowledge and experience pleasurable emotions (Maguire, 199Ia). This thesis draws attention to the duality of sport and calls for a more reality-congruent approach to the sport-health-risk-youth nexus in the development of future (school) sport worlds

De productie en moleculaire verschijningsvorm van radiotoxisch Po-210 in kernfusie- en splijtingsreactoren = The Production and Molecular Occurrence of Radiotoxic Po-210 in Nuclear Fusion and Fission Reactors

In order to counter the climate change, there is a need for a radical revolution in the global energy supply. One of the most realistic scenarios is a substantial shift towards renewable energy sources, supported by a flexible baseload from carbon-free nuclear energy. The main requirements for this new generation of nuclear reactors are intrinsic safety, high fuel efficiency and a minimum amount of (long-lived) radioactive waste. A promising candidate for the near future is the lead (alloy) cooled fast fission reactor. An ideal long-term solution are nuclear fusion reactors, which produce only a small amount of short-lived radioactive waste. A problem that occurs in both these reactor types is the undesired production of the highly radiotoxic polonium-210 isotope. In this PhD thesis, the total production of this isotope in a nuclear fusion reactor is determined using neutron transport and inventory calculations. Next, quantum chemical calculations are performed to predict the molecular form in which Po-210 will occur in both discussed reactor types. Combining both aspects allows to estimate the risk associated with the presence of Po-210 in a nuclear reactor and can help to design efficient Po-210 extraction systems

Long-term genetic modification of the ocular outflow tract and the retinal pigment epithelium with feline immunodeficiency viral vectors

The central goal to engineer feline immunodeficiency viral (FIV) vectors into a more modular platform for long-term ocular gene therapy was accomplished collaboratively by separating envelope, packaging and transfer function, the latter of which I optimized. FIV mediated gene transfer to the aqueous humor outflow tract and to the retina was established and long-term vector function and biocompatibility were confirmed. Transduction of the outflow tract was highly efficient and resulted in genetic modification of nearly the entire trabecular meshwork. For this purpose, I developed methods for administration and sensitive monitoring of FIV vectors in the anterior chamber and a novel subretinal injection technique. Bicistronic FIV vectors generated high levels of two different transgenes eGFP and β-galactosidase, which allowed live in vivo tracking and sensitive yet specific cell labeling in tissue specimen, respectively. An integrase mutant control vector that differed in only one amino acid resulted in no significant transduction while preserving all other biochemical properties. I developed a novel protocol for scaled-up production FIV vectors using cell factories and large-volume concentration that proved useful in the animal studies of this thesis. Especially the results achieved in the larger animal model, the domestic cat, validated the protocol for large-scale production. Transient transfection of 10 times lessDNA into 293T cells within high surface area cell factories and high volume, fixed-angle ultracentrifugation resulted in high titer vectors

Multilingual computer-mediated communication practice and the development of symbolic competence:insider research from a Japanese university

Amidst a rapid global proliferation of digital social media, Japanese youth have been adopting, adapting, and developing a diversity of multilingual computer mediated communication (CMC) practices on a variety of digital platforms. Previous research has pointed to commonalities in these practices with the practices of youth in other locales, but also to unique localized ways that Japanese youth communicate across digital media. This quasi-ethnographic case study was designed to explore English language learners’ multilingual CMC practices and perceptions from within a mediumsized private regional university in central Japan, and to point to pedagogical affordances that might lead to the development of symbolic competence. The project operates within a social constructivist paradigm and employs a theoretical approach largely consistent with cultural-historical activity theory (CHAT), but also drawing upon other theoretical traditions. Data were collected using narrative frames and field notes gathered during long-term participant observation that constituted insider research at the research site. The rationale for this study comes from the researcher’s interest in exploring ways that new technological affordances can best be used to enhance educational outcomes in a Japanese higher education context. Beyond the design affordances and constraints of various CMC technologies, myriad local factors and cultural assumptions were seen to contribute to the ‘cultures of use’ that inform students’ multilingual and multimodal interactions through various media. This research revealed that students rapidly develop new multilingual CMC practices through their interactions with their peers after entering university and returning from study abroad. A significant lack of broader media literacy, however, was seen to impede the development of symbolic competence. Pedagogical recommendations are offered to overcome this and other barriers to the development of symbolic competence