The use of ultrasonic standing waves for contactless manipulation of microparticles in microfluidic systems is a field with potential to become a new standard tool in lab-on-chip systems. Compared to other contactless manipulation methods ultrasonic standing wave manipulation shows promises of gentle cell handling, low cost, and precise temperature control. The technology can be used both for batch handling, such as sorting and aggregation, and handling of single particles. This doctoral Thesis presents multi-dimensional ultrasonic manipulation, i.e., manipulation in both two and three spatial dimensions as well as time-dependent manipulation of living cells and microbeads in microfluidic systems. The lab-on-chip structures used allow for high-quality optical microscopy, which is central to many bio-applications. It is demonstrated how the ultrasonic force fields can be spatially confined to predefined regions in the system, enabling sequential manipulation functions. Furthermore, it is shown how frequency-modulated signals can be used both for spatial stabilization of the force fields as well as for flow-free transport of particles in a microchannel. Design parameters of the chip-transducer systems employed are investigated experimentally as well as by numerical simulations. It is shown that three-dimensional resonances in the solid structure of the chip strongly influences the resonance shaping in the channel. / QC 20100730

Cellular homeostasis is the effort of all living cells to maintain their intracellular content when facing physiological change(s) in the extracellular environment. To date, cellular homeostasis is known to be regulated mainly by time-consuming active mechanisms and via multiple signaling pathways within the cells. The aim of this thesis is to show that time-efficient passive (physical) mechanisms also, under the control and regulation of bio-physical factors such as cell morphology and distribution and co-localization of transport proteins in the cell membrane, can regulate cellular homeostasis. This thesis has been developed in an interface between physics and biology and focuses on critical cases in which cells face physiologically unstable environments at their steady state and therefore may need a constituent effort to maintain their homeostasis. The main hypothesis here is that the cell geometry is oriented in such a way that cellular homeostasis is preserved in a given environment. For exploring these cases, comparative spatial models have been developed that combine transporting function of membrane proteins with simple versus complex geometries of cells. Models confirm the hypothesis and show that cell morphology, size of extracellular space and intercellular distances are important for a dynamic regulation of water and ion homeostasis at steady state. The main clue is the existence of diffusion limited space (DLS) in the bulk extracellular space (ECS). DLS can, despite being ECS, maintain its ionic content and water balance due a controlled function of transport proteins in the membrane facing part of DLS. This can significantly regulate cellular water and ion homeostasis and play an important role in cell physiology. In paper I, the role of DLS is explored in the kidney whereas paper II addresses the brain. The response of cells to change in osmolarity is of critical importance for water homeostasis. Cells primarily respond to osmotic challenge by transport of water via their membranes. As water moves into or out of cells, the volumes of intra- and extracellular compartments consequently change. Water transport across the cell membrane is enhanced by a family of water channel proteins (aquaporins) which play important roles in regulation of both cell and the extracellular space dimensions. Paper III explores a role for aquaporins in renal K+ transport. Experimentally this role is suggested to be different from bulk water transport. In a geometrical model of a kidney principal cell with several DLS in the basolateral membrane, a biophysical role for DLS-aquaporins is suggested that also provides physiological relevance for this study. The biophysical function of water channels is then extensively explored in paper IV where the main focus has been the dynamics of the brain extracellular space following water transport. Both modeling and experimental data in this paper confirmed the importance of aquaporin-4 expressed in astrocytes for potassium kinetics in the brain extracellular space. Finally, geometrically controlled transport mechanisms are studied on a molecular level, using silicon particles as a simplified model system for cell studies (paper V and VI). In paper V the role of electrostatic forces (around the nano-pores and in between the loaded material and the silicon surface) is studied with regard to transport processes.  In paper VI the roles of pore size and molecular weight of loaded material are studied. All together this thesis presents various modeling approaches that employ biophysical aspects of transport mechanisms combined with cell geometry to explain cell homeostasis and address cell physiology-based questions. / QC20100727

<p>The recent decades of research in molecular biology have resulted in break-throughs concerning our knowledge of the genetic code, protein structures and functions of the different cellular components. With this new information follows an increased interest in constructing computational models of the biological systems. A computational model can range from a description of one specific protein to a complete cell or organism. The aim of a computational model is often to complement the experimental studies and help identify essential mechanisms of a system.</p><p>All processes taking place in our cells, from general metabolic processes to cell specific actions, originates from information encoded in our DNA. The first step in transferring the genetic information to a functional protein or RNA, is through the transcription of a gene. The transcription process is controlled by cellular proteins binding to DNA regions called promoters. The term "genetic switch", used in the title of this thesis, refers to a specific change in transcription activity, where one or several promoters get activated or silenced.</p><p>In this thesis, I present studies of the regulation mechanisms in two different genetic switches. The first is a switch between two central promoters in the Epstein- Barr virus. This human virus is mostly known for causing the ’kissing disease’, but is also coupled to several cancer types. Infected cells can change between a resting and a proliferating phenotype, depending on which viral promoter is active. In order to understand what causes uncontrolled proliferation in tumors, it is important to understand the regulation of these viral promoters. The other switch is present in the phage λ, a bacterial virus. This virus has one specific promoter region, controlling expression of two proteins that determine if the phage will remain silent (lysogenic) in the host cell, or start producing new viral particles (go lytic). For the Epstein- Barr virus we tested, and confirmed, the hypothesis that the regulation of the two central promoters can be obtained by only one viral and one human protein. Further, we studied the cooperative effects on one of the promoters, showing that steric hindrance at the promoter region results in a more effective switching than with only cooperative binding present. For the bacteriophage λ we studied the genetically altered λ- Lac mutants, presented by Little & Atsumi in 2006. We demonstrate that the experimental results cannot, in terms of its equilibria, be explained by the mechanisms generally believed to be in control of the lysogenic/ lytic switch.</p>

Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process. Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory. Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.

<p>Polymeric nanoparticles are potential candidates as carriers for pharmaceutical agents. Development of such nanoparticles generally requires molecules immobilized on the particle surfaces to ensure biocompatibility and/or targeting abilities. Following particle preparation and surface modification, excess reagents must be removed. Ultracentrifugation, which is the most widely used purification technique as per today, is not feasible in industrial applications. In this diploma work, tangential flow filtration is studied as an alternative purification method which is better suited for implementation in a large-scale process.</p><p>Comparison of ultracentrifugation and tangential flow filtration in diafiltration mode for purification of nanoparticles, indicate that they are comparable with respect to particle stability and the removal of the surfactant SDS from methacrylic anhydride nanoparticles. The purification efficiency of tangential flow filtration is superior to that of ultracentrifugation. Conductivity measurements of filtrates and supernatant liquids show that a stable conductivity value can be reached 6 times faster in filtration than in centrifugation with equipment and settings used. This conductivity arises from several types of molecules, and the contribution from surfactant molecules alone is not known. However, protein adsorption on the particles indicates successful removal of surfactant. Conductivity and tensiometry were evaluated as potential methods to quantify surfactant in solutions, but both proved unsatisfactory.</p><p>Using bovine serum albumin as a model protein, the extent of immobilization to nanoparticles is evaluated at different pH. A maximum amount of 6,8 mg/m2 is immobilized, whereof an unknown part is covalently bound. This coverage is achieved at pH 4,0 and is probably partly due to low electrostatic repulsion between particle and protein. An estimation of 2,0 µmol covalently bound BSA per gram of nanoparticles corresponds to 5,3 mg/m2 and a surface coverage of 76%. Removal of excess reagents after surface modification is done with ultracentrifugation instead of filtration, as particle aggregates present after the immobilization reaction might foul the membrane.</p>

<p>Gene expression is regulated in response to metabolic necessities and environmental changes throughout the life of a cell.</p><p>A major part of this regulation is governed at the level of transcription, deciding whether messengers to specific genes are produced or not.</p><p>This decision is triggered by the action of transcription factors, proteins which interact with specific sites on DNA and thus influence the rate of transcription of proximal genes.</p><p>Mapping the organisation of these transcription factor binding sites sheds light on potential causal relations between genes and is the key to establishing networks of genetic interactions, which determine how the cell adapts to external changes.</p><p>In this work I review briefly the basics of genetics and summarise popular approaches to describe transcription factor binding sites, from the most straight forward to finally discuss a biophysically motivated representation based on the estimation of free energies of molecular interactions.</p><p>Two articles on transcription factors are contained in this thesis, one published (Aurell, Fouquier d'Hérouël, Malmnäs and Vergassola, 2007) and one submitted (Fouquier d'Hérouël, 2008).</p><p>Both rely strongly on the representation of binding sites by matrices accounting for the affinity of the proteins to specific nucleotides at the different positions of the binding sites.</p><p>The importance of non-specific binding of transcription factors to DNA is briefly addressed in the text and extensively discussed in the first appended article:</p><p>In a study on the affinity of yeast transcription factors for their binding sites, we conclude that measured in vivo protein concentrations are marginally sufficient to guarantee the occupation of functional sites, as opposed to unspecific emplacements on the genomic sequence.</p><p>A common task being the inference of binding site motifs, the most common statistical method is reviewed in detail, upon which I constructed an alternative biophysically motivated approach, exemplified in the second appended article.</p>

Genetics is among the most rewarding fields of biology for the theoretically inclined, offering both room and need for modeling approaches in the light of an abundance of experimental data of different kinds. Many aspects of the field are today understood in terms of physical and chemical models, joined by information theoretical descriptions. This thesis discusses different mechanisms and phenomena related to genetics, employing tools from statistical physics along with experimental biomolecular methods. Five articles support this work. Two articles deal with interactions between proteins and DNA. The first one reports on the properties of non-specific binding of transcription factors proteins in the yeast Saccharomyces cerevisiae, due to an effective background free energy which describes the affinity of a single protein for random locations on DNA. We argue that a background pool of non-specific binding sites is filled up before specific binding sites can be occupied with high probability, thus presenting a natural filter for genetic responses to spurious transcription factor productions. The second article describes an algorithm for the inference of transcription factor binding sites for proteins using a realistic physical model. The functionality of the method is verified on a set of known binding sequences for Escherichia coli transcription factors. The third article describes a possible genetic feedback mechanism between human cells and the ubiquitous Epstein-Barr virus (EBV). 40 binding regions for the major EBV transcription factor EBNA1 are identified in human DNA. Several of these are located nearby genes of particular relevance in the context of EBV infection and the most interesting ones are discussed. The fourth article describes results obtained from a positional autocorrelation analysis of the human genome, a simple technique to visualize and classify sequence repeats, constituting large parts of eukaryotic genomes. Applying this analysis to genome sequences in which previously known repeats have been removed gives rise to signals corroborating the existence of yet unclassified repeats of surprisingly long periods. The fifth article combines computational predictions with a novel molecular biological method based on the rapid amplification of cDNA ends (RACE), coined 5’tagRACE. The first search for non-coding RNAs encoded in the genome of the opportunistic bacterium Enterococcus faecalis is performed here. Applying 5’tagRACE allows us to discover and map 29 novel ncRNAs, 10 putative novelm RNAs and 16 antisense transcriptional organizations. Further studies, which are not included as articles, on the monitoring of secondary structure formation of nucleic acids during thermal renaturation and the inference of genetic couplings of various kinds from massive gene expression data and computational predictions, are outlined in the central chapters. / QC 20110316

<p>The fabrication of zone plate lenses that are used for focusing X-rays relies on nanofabrication techniques such as e-beam lithography, reactive ion etching, and electroplating. The circular grating-like zone plate pattern can have a smallest half-period, a so-called zone width, of down to 20 nm while it also needs to have a height that is 5 to 10 times the zone width to have good diffraction efficiency. This high aspect ratio structuring is a very challenging field of nanofabrication.</p><p>This diploma project has focused on improving the process step of fabricating the electroplating mold by cryo-cooling the polymer during the reactive ion etching with O₂. The low temperature causes passivation of the sidewalls of the mold during etching which results in a more ideal rectangular profile of the high aspect ratio plating mold.</p><p>By etching at -100 °C, structures with highly vertical sidewalls and no undercut were realized. The experiments showed that there is a tradeoff between the anisotropy of the zone profile and the formation rate of polymer residue, so-called RIE grass. Through a proper choice of process parameters the grass could be completely removed without introducing any undercut.</p> / QC 20100414

As a new pediatric dummy family, the Q-family, is suggested for the European childsafety regulations (R44) and the updated EuroNCAP, it needed to be tested andcompared to the older pediatric dummy family, Hybrid III, used in testing at Autolivtoday.In this study, tests were performed with the Q6 and the Hybrid III 6-year-old. Bothdummies were subjected to eight sled tests using a EuroNCAP acceleration pulse. The sled represented the interior of a Volvo V70, with integrated booster cushions mounted onto the car body through a rigid fixture. Standard belt were used for all tests, except one where pretensioning was used. Static tests investigated how the chest deflection on Q6 was affected by the shoulder belt geometry. Large difference in belt interaction was observed between the dummies. The beltslipped off the Hybrid III’s shoulder for all tests except one, while the belt on the Qdummy’swas hard to provoke off the shoulder. The overall kinematic behavior, beforethe belt slipped off the Hybrid III’s shoulder, were similar for both dummies. Differences in chest deflection on the Q6, depending on the belt geometry, were observed in both the dynamic and the static tests; a shoulder belt geometry closer to theneck resulted in minor displacement than a mid-shoulder belt geometry. After testing, five different damages were observed on the Q6. / Då en ny familj av barnkrockdockor, Q-familjen, är föreslagen för det europeiskalagkravet som reglerar barnsäkerhet (R44), uppstod ett behov av att testa och jämföradessa mot den äldre familjen av barnkrockdockor som används vid testning på Autoliv idag, Hybrid III. I den här studien utfördes tester på Q6 och Hybrid III 6 år. Båda dockorna utsattes föråtta stycken slädtest i en accelerationspuls enligt EuroNCAP. Släden representerade enVolvo V70 med integrerade barnkuddar som monterats i riggen via en stel fixtur. I alla test utom ett användes standardbälten (i undantagsfallet användes försträckare). Statiska tester gjordes för att undersöka hur Q6 påverkades av olika geometrier på axelbältet. Stora skillnader observerades mellan dockornas bältesinteraktion. Bältet gled av HybridIII:s axel i alla test förutom ett, medan det istället var svårt att provocera av bältet från Q-dockans axel. Innan Hybrid III gled ur bältet var dockornas kinematik liknande. I både statiska och dynamiska tester observerades skillnader i bröstintryckning på Q6, beroende på bältesgeometrin; en geometri där axelbältet var placerat nära nackenresulterade i en mindre intryckning än då axelbältet var placerat mitt på axeln. Efte ravslutad testning upptäcktes fem skador på Q6.

First principle electronic structure theory is used to describe the effect of crystal binding on radiation detectors, electron transport properties, and structural damage induced by intense irradiation. A large database containing general electronic structure results to which data mining algorithms can be applied in the search for new functional materials, a case study is presented for scintillator detector materials. Inelastic cross sections for the generation of secondary electron cascades through impact ionization are derived from the dielectric response of an electron gas and evolved in time with Molecular Dynamics (MD). Qualitative and quantitive estimates are presented for the excitation and relaxation of a sample irradiated with Free Electron Laser pulses. A study is presented in where the structural damage on covalent bonded crystals following intense irradiation is derived from a Tight Binding approach and evolved in time with MD in where the evolution of the sample is derived from GW theory for the quasiparticle spectra and a dedicated Boltzmann transport equation for the impact ionization.