Pore forming toxins are known for their ability to efficiently form transmembrane pores which eventually leads to cell lysis. PFTs have potential applications in devel-oping novel drug and gene delivery strategies. Although structural aspects of many pore forming toxins have been studied, very little is known about the dynamics and subsequent rupture mechanisms. In the first part of the thesis, a combined experimental and modeling study to understand the lytic action of Cytolysin A (ClyA) toxins on red blood cells (RBCs) has been presented. Lysis experiments are carried out on a 1% suspension of RBCs for different initial toxin concentrations ranging from 100 – 500 ng/ml and the extent of lysis is monitored spectrophotometrically. Using a mean field approach, we propose a non – equilibrium adsorption-reaction model to quantify the rate of pore formation on the cell surface. By analysing the model in a pre-lysis regime, the number of pores per RBC to initiate rupture was found to lie between 400 and 800. The time constants for pore formation are estimated to lie between 1-25 s and monomer conformation time scales were found to be 2-4 times greater than the oligomerization times. Using this model, we are able to predict the extent of cell lysis as a function of the initial toxin concentration. Various kinetic models for oligomerization mechanism have been explored. Irreversible sequential kinetic model has the best agreement with the available experimental data. Subsequent to the mean field approach, a population balance model was also formulated. The mechanics of cell rupture due to pore formation is poorly understood. Efforts to address this issue are concerned with understanding the changes in the membrane mechanical properties such as the modulus and tension in the presence of pores. The second part of the thesis is concerned with using atomic force microscopy to measure the mechanical properties of cells. We explore the possibility of employing tapping mode AFM (TM-AFM) to obtain the elastic modulus of soft samples. The dynamics of TM-AFM is modelled to predict the elastic modulus of soft samples, and predict optimal cantilever stiffness for soft biological samples. From experiments using TM-AFM on Nylon-6,6 the elastic modulus is predicted to lie between 2 and 5 GPa. For materials having elastic moduli in the range of 1– 20 GPa, the cantilever stiffness from simulations is found to lie in the range of 1 – 50 N/m. For soft biological samples, whose elastic moduli are in the range of 10-1000 kPa, a narrower range of cantilever stiffness (0.1 – 0.6 N/m), should be used.

<p>Microbiological contamination from bacteria such as <em>Escherichia coli</em> and Salmonella is one of the main reasons for waterborne illness. Real time and accurate monitoring of water is needed in order to alleviate this human health concern. Performing multiple and parallel analysis of biomarkers such as DNA and mRNA that targets different regions of pathogen functionality provides a complete picture of its presence and viability in the shortest possible time. These biomarkers are present inside the cell and need to be extracted for analysis and detection. Hence, lysis of these pathogenic bacteria is an important part in the sample preparation for rapid detection. In addition, collecting a small amount of bacteria present in a large volume of sample and concentrating them before lysing is important as it facilitates the downstream assay. Various techniques, categorized as mechanical, chemical, thermal and electrical, have been used for lysing cells. In the electrical method, cells are lysed by exposure to an external electric field. The advantage of this method, in contrast to other methods, is that it allows lysis without the introduction of any chemical and biological reagents and permits rapid recovery of intercellular organelles. Despite the advantages, issues such as high voltage requirement, bubble generation and Joule heating are associated with the electrical method.</p> <p>To alleviate the issues associated with electrical lysis, a new design and associated fabrication process for a microfluidic cell lysis device is described in this thesis. The device consists of a nanoporous polycarbonate (PCTE) membrane sandwiched between two PDMS microchannels with electrodes embedded at the reservoirs of the microchannels. Microcontact printing is used to attach this PCTE membrane with PDMS.</p> <p>By using this PCTE membrane, it was possible to intensify the electric field at the interface of two channels while maintaining it low in the other sections of the device. Furthermore, the device also allowed electrophoretic trapping of cells before lysis at a lower applied potential. For instance, it could trap bacteria such as <em>E. coli</em> from a continuous flow into the intersection between two channels for lower electric field (308 V/cm) and lyse the cell when electric field was increased more than 1000 V/cm into that section.</p> <p>Application of lower DC voltage with pressure driven flow alleviated adverse effect from Joule heating. Moreover, gas evolution and bubble generation was not observed during the operation of this device.</p> <p>Accumulation and lysis of bacteria were studied under a fluorescence microscope and quantified by using intensity measurement. To observe the accumulation and lysis, LIVE/DEAD BacLight Bacterial Viability Kit consisting of two separate components of SYTO 9 and propidium iodide (PI) into the cell suspension in addition to GFP expressed <em>E. coli</em> were used. Finally, plate counting was done to determine the efficiency of the device and it was observed that the device could lyse 90% of bacteria for an operation voltage of 300V within 3 min.</p> <p>In conclusion, a robust, reliable and flexible microfluidic cell lysis device was proposed and analyzed which is useful for sample pretreatment in a Micro Total Analysis System.</p> / Master of Applied Science (MASc)

To many people love is special, sacred even. Love plays a countless number of roles for a countless number of people. Contemporary ideas about love, however, are more in alignment with the philosophies of Aristotle, and not of Plato. Aristotle held that love could exist as many people see it today – wishing well for others purely for their own sake. But Plato disagreed. Plato claimed that love was a way by which one could better themselves and become wiser. In this thesis, I explain Plato’s theory of love put forth in the Symposium. I also explore the textual evidence for the selfish nature of Plato’s love.

Cette thèse est consacrée à l’étude d’une technique innovante de lyse de microorganismes, fondée sur l’utilisation d’impulsions haute tension en milieu aqueux. Cette technique se distingue de l’électroporation qui exploite le champ électrique produit pour dégrader la membrane cellulaire ; dans notre étude les impulsions haute tension permettent la formation d’arcs électriques produisant de multiples phénomènes physico-chimiques qui peuvent entraîner la lyse des microorganismes.L’efficacité du procédé a été évalué sur les microorganismes suivants : des microalgues productrices d’huile (Nannochloropsis gaditana et Phaeodactylum tricornutum) et des bactéries couramment utilisées comme modèles de laboratoire (Escherishia coli et Bacillus subtilis). Dans ces travaux, nous avons montré que les ondes de pression produites sont principalement responsables de la lyse.En fin d’étude, des perspectives sont explorées en vue du développement de systèmes autonomes soit dans le cadre de la bioproduction, soit dans le cadre de l’analyse cellulaire. / This thesis focuses on the study of an innovative technology for microorganisms lysis, based on high voltage pulses generated in an aqueous medium. This technology is different from electroporation which operates thanks to the electric field for damaging cell membranes ; in our study high voltage pulses generate an electric arc leading to various physicochemical phenomena supposed to lyse microorganisms.The technology efficiency is evaluated with the following microorganims : some lipid producting microalgae (Nannochloropsis gaditana and Phaeodactylum tricornutum) and classical laboratory model bacteria (Escherishia coli and Bacillus subtilis). In this work, we found that generated shock waves are mainly responsible of the cells lysis.At the end, the development of self-functioning devices is investigated either for bioproduction or for cell analysis.

<p>Fotosyntesen kallas den process som omvandlar ljusenergi till kemisk energi. Fotosyntesen sker i tylakoidmembranet och drivs av två stora proteinkomplex, fotosystem II (PSII) och fotosystem I (PSI) då de tillförs energi i form av fotoner. PSI-N är ett mindre protein på ca 10 kDa som ingår i PSI. På något sätt, som ännu inte är klarlagt, samverkar PSI-N med PSI-F och plastocyanin när det dockar till PSI. Det är därför av viktigt att rena fram större mängder av PSI-N för att få djupare kunskaper om proteinet samt dess struktur och funktioner. Tidigare undersökningar har utförts i ämnet och ett fusionsprotein innehållande PSI-N har uttryckts i <em>Escherichia coli</em> (<em>E.coli</em>). Problem har dock uppstått efter lysis av cellerna då det har visat sig att fusionsproteinet har degraderats. Vårt examensarbete strävar efter att rena fram intakt fusionsprotein med hjälp av, framför allt, mekanisk lysis och proteasinhibitorer.</p> / <p>The process where light is converted into chemical energy is called photosyntesis. The reaction takes place in the thylakoid membrane and is driven by two major protein complexes, photosystem II (PSII) and photosystem I (PSI) when energy in form of photons are received. PSI-N, a subunit in PSI, is a smaller protein with a mass of approximately 10 kDa. In some way, which is not yet clarified, PSI-N collaborates with PSI-F and plastocyanin when plastocyanin is docking to PSI. It is therefore important to purify larger amounts of the protein to acquire deeper knowledge of its structure and function. In earlier research the PSI-N protein has been expressed in <em>Escherichia coli</em> (<em>E.coli</em>). The problem has been degradation of the fusion protein after lysis. Our goal with this project is to obtain the purified protein intact using mechanic lysis and protease inhibitors.</p>

Development of a simple, specific, sensitive and rapid RNA microchip for detection of Head and Neck Cancer (HNC) mRNA, pathogenic bacteria and dengue virus (DENV) RNA is reported. By use of nucleases and polymerases specific RNAs are selectively labeled and detected without separation, reverse transcription and or polymerase chain reaction. This is accomplished by designing specific Hybrid probes consisting of DNA-2’-O-Me-RNA-DNA regions to target the RNA of interest. Upon hybridization with the target RNA, RNase H digestion is used to remove the 3’- RNA sequences which exposes the template for Klenow extension with reporter molecules such as hapten or fluorophore labels. This novel RNA microchip is fast (ca. 1 h detection time), selective as individual RNAs are detected in a synthetic mixture and total RNA mixtures, specific for single nucleotide polymorphisms (SNPs) discrimination and sensitive up to attomole level for chemiluminescence detection and lower femtomole for gold nanoparticles (AuNPs) and silver staining method. Using chemiluminescence, HNC biomarkers, VCAM1 and IL8 are specifically labeled and detected in the presence of thousands of other mRNAs in cancer cell lines and human colon cancer total RNA without interference. Furthermore, the method is highly specific as shown with DENV SNPs discrimination. Moreover, we report rapid (ca 1hour), selective, specific multi-marker detection of pathogenic mRNAs and HNC mRNAs using AuNPs-silver staining on the RNA microchip. Streptavidin gold nanoparticles technology has a potential in the analysis of specific mRNAs in a wide array of field including infectious diseases diagnosis, viral infections, food safety, gene expression profiling and cancer detection. A simple and rapid NaOH RNA extraction procedure was developed for E. coli total RNA extraction with specific results on the RNA microchip using both chemiluminescence and AuNPs silver staining. This extraction avoids the use of commercial RNA purification kits thus reducing the cost. Furthermore, visual detection on the RNA microchip is simple, does not require electricity or special equipment, and therefore is a good candidate for field diagnostics with minimum resources.

The recent breakthroughs in genomics and molecular diagnostics will not be reflected in health-care systems unless the biogenetic or other nucleic acid-based tests are transferred from the laboratory to clinical market. Developments in microfabrication techniques brought lab-on-a-chip (LOC) into being the best candidate for conducting sample preparation for such clinical devices, or point-of-care testing set-ups. Sample preparation procedure consists of several stages including cell transportation, separation, cell lysis and nucleic acid purification and detection. LOC, as a subset of Microelectromechanical systems (MEMS), refers to a tiny, compact, portable, automated and easy-to-use microchip capable of performing the sample-preparation stages together. Complexity in micro-fabrications and inconsistency of the stages oppose integration of them into one chip. Among the variety of mechanisms utilized in LOC for cell lysis, electrical methods have the highest potential to be integrated with other microchip-based mechanisms. There are, however, major limitations in electrical cell lysis methods: the difficulty and high-cost fabrication of microfluidic chips and the high voltage requirements for cell lysis. Addressing these limitations, the focus of this thesis is on realization of cell lysis microchips suitable for LOC applications. We have developed a new methodology of fabricating microfluidic chips with electrical functionality. Traditional lithography of microchannel with electrode, needed for making electro-microfluidic chips, is considerably complicated. We have combined several easy-to-implement techniques to realize electro-microchannel with laser-ablated polyimide. The current techniques for etching polyimide are by excimer lasers in bulky set-ups and with involvement of toxic gas. We present a method of ablating microfluidic channels in polyimide using a 30W CO2 laser. Although this technique has poorer resolution, this approach is more cost effective, safer and easier to handle. We have verified the performance of the fabricated electro-microfluidic chips on electroporation of mammalian cells. Electrical cell lysis mechanisms need an operational voltage that is relatively high compared to other cell manipulation techniques, especially for lysing bacteria. Microelectro-devices have dealt with this limitation mostly by reducing the inter-distance of electrodes. The technique has been realized in tiny flow-through microchips with built-in electrodes in a distance of a few micrometers which is in the scale of cell size. In addition to the low throughput of such devices, high probability of blocking cells in such tiny channels is a serious challenge. We have developed a cell lysis device featured with aligned carbon nanotube (CNT) to reduce the high voltage requirement and to improve the throughput. The vertically aligned CNT on an electrode inside a MEMS device provides highly strengthened electric field near the tip. The concept of strengthened electric field by means of CNT has been applied in field electron emission but not in cell lysis. The results show that the incorporation of CNT in lysing bacteria reduces the required operational voltage and improves throughput. This achievement is a significant progress toward integration of cell lysis in a low-voltage, high-throughput LOC. We further developed the proposed fabrication methodology of micro-electro-fluidic chips, described earlier, to perform electroporation of single mammalian cell. We have advanced the method of embedding CNT in microchannel so that on-chip fluorescent microscopy is also feasible. The results verify the enhancement of electroporation by incorporating CNT into electrical cell lysis. In addition, a novel methodology of making CNT-embedded microfluidic devices has been presented. The embedding methodology is an opening toward fabrication of a CNT-featured LOC for other applications.

This study proposes a navel method for continuously particle sorting utilizing cascade squeeze jumping effect under microfluidic configuration. Microparticles with different sizes can be successfully separated at different stages of squeezing sheath flow. The method is based on that particles can not flow stably within a flow stream with the smaller stream width than their sizes. Big particles will jump from their original flow stream into the wider neighboring sheath flow. In this study, we have successfully designed and fabricated two kinds of particles/cells sorters using MEMS (Micro-electro-mechanical Systems) technology. The proposed microchip device includes a multi-stage sheath flow particles/cells sorter and an improved design of a cascade squeezed flow scheme. In the study, theoretical formulations, computer simulations and experimental operations are used to analyze the flow field in the microchip and evaluate the sorting performance of the devices. Results show the good sorting performance with cell recovery rate of 87.7% and yield rate of 94.1% can be obtained using the proposed micro particles/cells sorter. Furthermore, it is also important to continiously prepare reagents for in-column bio-chemical reactions. Therefore, this study presents a sheath-flow based microfluidic device for concentration fraction delivery of liquid samples. The simple and novel structure proposed in this study is able to prepare reagent with different concentration and is also easy to be integrated with other multifunctional microfluidic device. In order to demonstrate the feasibility and performance of the proposed concentration fraction delivery device, this study designs an integrated microchip device for in-line preparation of lysin reagent for cell lysis and an integrated T-form microfluidic mixer for demonstration of RBC lysis in the same microchip. Reagents for cell lysis are firstly prepared by the concentration faction delivery part of the chip. The prepared reagent is mixed with RBC sample downstream in the reaction channel using the T-form mixer. Results show a high RBC lysing rate of upto 100% in 10 mm downstream the T-junction can be achieved utilizing the proposed chip. In this study, we have successfully demonstrated three kinds of microfluidic device including a micro particles/cells sorter, a concentration fraction delivery device and a cell lysis reactor. Numerical analysis and experimental investigation confirm the proposed concepts and performance of the microfluidic devices. The contributions of the study are highly potential for developing a low-cost bioreactor system in the

Cells switch phenotypes or behaviors to adapt to various environmental stimuli. Often there are multiple alternative phenotypes, hence a cell chooses one phenotype among them, a process which we term a ``decision switch'. At the cellular level, decision switches are governed by gene regulation, hence they are intrinsically stochastic. Here we investigate two aspects of decision switches: how copy number of genetic components facilitates multiple phenotypes and how temporal dynamics of gene regulation with stochastic fluctuations affect switching a cell fate. First, we demonstrate that gene expression can be sensitive to changes in the copy number of genes and promoters, and alternative phenotypes may arise due to bistability within gene regulatory networks. Our analysis in phage-lambda-infected E. coli cells exhibit drastic change in gene expression by changing the copy number of viral genes, suggesting phages can determine their fates collectively via sharing gene products. Second, we examine decision switches mediated by temporal dynamics of gene regulation. We consider a case when temporal gene expression triggers a corresponding cell fate, and apply it to the lysis-lysogeny decision switch by phage lambda. Our analysis recapitulates the systematic bias between lysis and lysogeny by the viral gene copy number. We also present a quantitative measure of cell fate predictability based on temporal gene expression. Analyses using our framework suggest that the future fate of a cell can be highly correlated with temporal gene expression, and predicted if the current gene expression is known.

Microfluidics promises to re-shape the current health-care system by transferring diagnostic tools from central laboratories to close vicinity of the patient (point-of-care). One of the most important operational steps in any diagnostic platform is sample preparation, which is the main subject in this thesis. The goal of sample preparation is to isolate targets of interest from their surroundings. The work in this thesis is based on three ways to isolate bacteria:  immune-based isolation, selective cell lysis, size-based separation. The first sample-preparation approach uses antibodies against lipopolysaccharides (LPS), which are surface molecules found on all gram-negative bacteria. There are two characteristics that make this surface molecule interesting. First, it is highly abundant: one bacterium has approximately a million LPS molecules on its cell-wall. Second, the molecule has a conserved region within all gram-negative bacteria, so using one affinity molecule to isolate disease-causing gram-negative bacteria is an attractive option, particularly from the point of view of sample preparation. The main challenge, however, is antigen accessibility. To address this, we have developed a treatment protocol that improves the capturing efficiency. The strategy behind selective cell lysis takes advantage of the differences between the blood-cell membrane and the bacterial cell-wall. These fundamental differences make it possible to lyse (destroy) blood-cells selectively while keeping the target of interest, here the bacteria, intact and, what is more important alive. Viability plays an important role in determining antibiotic susceptibility. Difference in size is another well-used characteristic for sample- separation. Inertial microfluidics can focus size-dependent particle at high flow-rates. Thus, particles of 10 µm diameter were positioned in precise streamlines within a curved channel.  The focused particles can then be collected at defined outlets.  This approach was then used to isolate white blood cells, which account for approximately 1% of the whole blood.  In such a device particles of 2µm diameter (size of bacteria) would not be focused and thereby present at every outlet. To separate bacteria from blood elasto-inertial microfluidics was used. Here, e blood components are diverted to center of the channels while smaller bacteria remain in the side streams and can subsequently be separated. / <p>QC 20141212</p>