The human body is made of billions of cells and nearly all have the same genome. However, there is a high diversity of cells, resulted from what part of the genome the cells use, i.e. which RNA molecules are expressed. Rapid advances within the field of sequencing allow us to determine the RNA molecules expressed in a specific cell at a certain time. The use of the new technologies has expanded our view of the human transcriptome and increased our understanding of when, where, and how each RNA molecule is expressed. The work presented in this thesis focuses on analysis of the human transcriptome. In Paper I, we describe an automated approach for sample preparation. This protocol was compared with the standard manual protocol, and we demonstrated that the automated version outperformed the manual process in terms of sample throughput while maintaining high reproducibility. Paper II addresses the impact of nuclear transcripts on gene expression. We compared total RNA from whole cells and from cytoplasm, showing that transcripts with long, structured 3’- and 5’-untranslated regions and transcripts with long protein coding sequences tended to be retained in the nucleus. This resulted in increased complexity of the total RNA fraction and fewer reads per unique transcript. Papers III and IV describe dynamics of the human muscle transcriptome. For Paper III, we systematically investigated the transcriptome and found remarkably high tissue homogeneity, however a large number of genes and isoforms were differentially expressed between genders. Paper IV describes transcriptome differences in response to repeated training. No transcriptome-based memory was observed, however a large number of isoforms and genes were affected by training. Paper V describes a transcript profiling protocol based on the method Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification. We designed the method for a few selected transcripts whose expression patterns are important for detecting breast cancer cells, and optimized the method for single cell analysis. We successfully detected cells in human blood samples and applied the method to single cells, confirming the heterogeneity of a cell population. / Människokroppen är uppbyggd av miljarder celler och nästan alla innehåller samma arvsmassa. Trots detta finns det många olika celler med olika funktioner vilket är en följd av vilken del av arvsmassan som cellerna använder, dvs vilka RNA-molekyler som finns i varje cell. Den snabba utvecklingen av sekvenseringstekniker har gjort det möjligt att studera när, var och hur varje RNA-molekyl är uttryckt och att få en djupare förståelse för hur människans celler fungerar. Arbetet som presenteras i denna avhandling fokuserar på analys av RNA-molekyler i människans celler. I artikel I beskriver vi en automatiserad metod för att förbereda cellprov för RNA-sekvensering. Det automatiserade protokollet jämfördes med det manuella protokollet, och vi visade att det automatiserade protokollet överträffade det manuella när det gällde provkapacitet samtidigt som en höga reproducerbarheten behölls. I artikel II undersökte vi effekterna som RNA-molekyler från en del av cellen (cellkärnan) har på den totala mängden uttryckta RNA-molekyler. Vi jämförde RNA från hela cellen och från en del av cellen (cytoplasman) och visade att RNA-molekyler med långa och strukturerade 3'- och 5'-otranslaterade regioner och RNA-molekyler med långa proteinkodande sekvenser tenderade att hållas kvar i cellkärnan till en högre grad. Detta resulterade i en ökad komplexitet av RNA-molekylerna i hela cellen, medan vi i cytoplasma-fraktionen lättare kunde hitta de korta och svagt uttryckta RNA-molekyler. I Artikel III och IV studerar vi RNA-molekyler i människans skelettmuskler. I artikel III visar vi att andelen RNA-molekyler uttryckta i skelettmuskler är väldigt lika mellan muskler och mellan olika personer, men att ett stort antal RNA-molekyler var uttryckta i olika nivåer hos kvinnor och män. Artikel IV beskriver RNA-nivåer som svar på upprepade perioder av uthållighetsträning. Artikel V beskriver en metod för att studera ett fåtal utvalda RNA-molekyler. Vi valde RNA-molekyler vars uttryck är viktigt vid analys av bröstcancerceller, och optimerade metoden för analys av enskilda celler. Vi analyserade cancerceller från blodprov och använde metoden för att titta på RNA-nivåer i enskilda celler från en grupp av celler och visade på skillnader i RNA-nivåer inom gruppen. / <p>QC 20150115</p>

In 2008 Ontario implemented a screening program for colorectal cancer, which drew attention to the increasing demand for colonoscopies in the province. This trend and the forecasted demand of the new screening program created a need to increase capacity in hospital endoscopy suites. This thesis addresses this need by investigating throughput gains from scheduling according to physician specific procedure durations in endoscopy suites. This is accomplished through the development of a scheduler and a generic discrete event simulation. Case study results show that physician specific scheduling can increase throughput in the endoscopy suite while reducing undertime and only slightly increasing overtime. They further indicate the trade off between a 1:2 and 1:1 physician to room ratio, finding that while a 1:1 ratio increases throughput by 33% over a 1:2 ratio, physicians are 1.5 times more productive under a 1:2 ratio.

This thesis describes the development of microfluidic (MF) platforms for the study of biological and chemical processes. In particular the thesis is divided into two distinct parts: (i) development of a MF methodology to generate tunable cell-laden microenvironments for detailed studies of cell behavior, and (ii) the design and fabrication of MF reactors for studies of chemical reactions. First, this thesis presented the generation of biopolymer microenvironments for cell studies. In the first project we demonstrated a high-throughput MF system for generating cell-laden agarose microgels with a controllable ratio of two different types of cells. The MF co-encapsulation system was shown to be a robust method for identifying autocrine and/or paracrine dependence of specific cell subpopulations. In the second project we studied the effect of the mechanical properties on the behavior of acute myeloid leukemia (AML2) cancer cells. Cell-laden macroscopic agarose gels were prepared at varying agarose concentrations. A modest range of the elastic modulus of the agarose gels were achieved, ranging from 0.62 kPa to 20.21 kPa at room temperature. We observed a pronounced decrease in cell proliferation in stiffer gels when compared to the gels with lower elastic moduli. The second part of the thesis focuses on the development of MF platforms for studying chemical reactions. In the third project presented in this thesis, we exploited the temperature dependent solubility of CO2 in order to: (i) study the temperature mediated CO2 transfer between the gas and the various liquid phases on short time scales, and (ii) to generate bubbles with a dense layer of colloid particles (armoured bubbles). The fourth project involved the fabrication of a multi-modal MF device with integrated analytical probes. The MF device comprised a pH, temperature, and ATR-FTIR probes for in-situ analysis of chemical reactions in real-time. Furthermore, the MF reactor featured a temperature controlled feedback system capable of maintaining on-chip temperatures at flow rates up to 50 mL/hr.

In 2008 Ontario implemented a screening program for colorectal cancer, which drew attention to the increasing demand for colonoscopies in the province. This trend and the forecasted demand of the new screening program created a need to increase capacity in hospital endoscopy suites. This thesis addresses this need by investigating throughput gains from scheduling according to physician specific procedure durations in endoscopy suites. This is accomplished through the development of a scheduler and a generic discrete event simulation. Case study results show that physician specific scheduling can increase throughput in the endoscopy suite while reducing undertime and only slightly increasing overtime. They further indicate the trade off between a 1:2 and 1:1 physician to room ratio, finding that while a 1:1 ratio increases throughput by 33% over a 1:2 ratio, physicians are 1.5 times more productive under a 1:2 ratio.

This research investigates cross-layer design in multi-hop wireless networks with random access. Due to the complexity of the problem, we study cross-layer design with a simple slotted ALOHA medium access control (MAC) protocol without considering any network dynamics. Firstly, we study the optimal joint configuration of routing and MAC parameters in slotted ALOHA based wireless networks under a signal to interference plus noise ratio based physical interference model. We formulate a joint routing and MAC (JRM) optimization problem under a saturation assumption to determine the optimal max-min throughput of the flows and the optimal configuration of routing and MAC parameters. The JRM optimization problem is a complex non-convex problem. We solve it by an iterated optimal search (IOS) technique and validate our model via simulation. Via numerical and simulation results, we show that JRM design provides a significant throughput gain over a default configuration in a slotted ALOHA based wireless network. Next, we study the optimal joint configuration of routing, MAC, and network coding in wireless mesh networks using an XOR-like network coding without opportunistic listening. We reformulate the JRM optimization problem to include the simple network coding and obtain a more complex non-convex problem. Similar to the JRM problem, we solve it by the IOS technique and validate our model via simulation. Numerical and simulation results for different networks illustrate that (i) the jointly optimized configuration provides a remarkable throughput gain with respect to a default configuration in a slotted ALOHA system with network coding and (ii) the throughput gain obtained by the simple network coding is significant, especially at low transmission power, i.e., the gain obtained by jointly optimizing routing, MAC, and network coding is significant even when compared to an optimized network without network coding. We then show that, in a mesh network, a significant fraction of the throughput gain for network coding can be obtained by limiting network coding to nodes directly adjacent to the gateway. Next, we propose simple heuristics to configure slotted ALOHA based wireless networks without and with network coding. These heuristics are extensively evaluated via simulation and found to be very efficient. We also formulate problems to jointly configure not only the routing and MAC parameters but also the transmission rate parameters in multi-rate slotted ALOHA systems without and with network coding. We compare the performance of multi-rate and single rate systems via numerical results. We model the energy consumption in terms of slotted ALOHA system parameters. We found out that the energy consumption for various cross-layer systems, i.e., single rate and multi-rate slotted ALOHA systems without and with network coding, are very close.

In order to meet the increasing demand for higher data rates, next generation wireless networks must incorporate additional functionalities to enhance network throughput. Multihop networks are considered as a promising alternative due to their ability to exploit spatial reuse and to extend coverage. Recently, industry has shown increased interest in multihop networks as they do not require additional infrastructure and have relatively low deployment costs. Many advances in physical and network layer techniques have been proposed in the recent past and they have been studied mostly in single-hop networks. Very few studies, if any, have tried to quantify the gains that these techniques could provide in multihop networks. We investigate the impact of simple network coding, advanced physical layer and cooperative techniques on the maximum achievable throughput of wireless multihop networks of practical size. We consider the following advanced physical layer techniques: successive interference cancellation, superposition coding, dirty-paper coding, and some of their combinations. We achieve this by formulating several cross-layer frameworks when these techniques are jointly optimized with routing and scheduling. We also formulate power allocation subproblems for the cases of continuous power control and superposition coding. We also provide numerous engineering insights by solving these problems to optimality.

The initial research of this thesis involves the evaluation of different strategies for developing diverse chemistries of highly stable coatings for the automated 96-blade (thin-film) solid phase microextraction (SPME) system. Thin-film geometry increases the volume of extractive phase, and consequently improves the sensitivity of the analysis. Sol-gel technology was used for the preparation of octadecyl (C18)-silica gel thin-film coating. The evaluation of the C18-silica gel SPME extractive phase resulted in stable physical and chemical characteristics and long-term reusability with a high degree of reproducibility. Biocompatible polyacrylonitrile (PAN) polymer was used for the preparation of particle-based extractive phases in order to improve the biocompatible characteristics of SPME coatings for the extraction from biological samples. Three different immobilization strategies were evaluated for developing highly stable coatings for the automated 96-blade SPME system. The spraying was found to be the optimal method in terms of stability and reusability for long-term use. The optimized C18-PAN coating demonstrated improved biocompatibility, stability, and reusability for the extraction of benzodiazepines from human plasma in comparison with those of C18-silica gel coating. To improve the biocompatible properties of the C18-PAN SPME coating for long-term direct analysis from whole blood, different modification strategies were studied and evaluated. The modification of the coating with an extra layer of biocompatible polyacrylonitrile resulted in significant improvement in the blood compatibility in long-term use. ‘Extracted blood spot’ (EBS) sampling was introduced as a novel approach to overcome the limitations of dried blood spot sampling. EBS includes the application of a biocompatible SPME coating for spot sampling of blood or other biofluids. The compatibility of EBS sampling with different analytical methods was demonstrated. The utilization of EBS as a fast sampling and sample preparation method resulted in a significant reduction of matrix effects through efficient sample clean-up. Modified polystyrene-divinylbenzene (PS-DVB)-PAN and phenylboronic acid (PBA)-PAN 96-blade SPME coatings were developed and evaluated for the extraction of analytes in a wide range of polarity. These coatings demonstrated efficient extraction recovery for both polar and non-polar groups of compounds, and presented chemical and mechanical stabilities and reproducible extraction efficiencies for more than 100 usages in biological sample.

Breast cancers with overexpression or amplification of the HER2 tyrosine kinase receptor are more aggressive, resistant to chemotherapy, and associated with a worse prognosis. Currently, these breast cancers are treated with the monoclonal antibody trastuzumab (Herceptin®). Unfortunately, not all patients respond to trastuzumab drug therapy; some patients show de novo resistance, while others acquire resistance during treatment. This thesis describes our RNAi studies to identify novel regulators of the HER2 signaling pathway in breast cancer. Three kinome-wide siRNA screens were performed on five HER2 amplified and seven HER2 non-amplified breast cancer cell lines, two normal breast cell lines, as well as two HER2-positive breast cancer cell lines with acquired trastuzumab resistance and their isogenic trastuzumab-sensitive controls. To understand the main kinase drivers of HER2 signaling, we performed a comprehensive screen that selected against growth inhibitors of the non-HER2 amplified breast cancer cell lines. This screen identified the loss of the HER2/HER3 heterodimer as the most prominent selective inhibitor of HER2-amplified breast cancers. In a trastuzumab sensitization screen on five trastuzumab-treated breast cancer cell lines, we identified several siRNA against the PI3K pathway as well as various other signaling pathways that inhibited proliferation. Finally, in a screen for acquired trastuzumab resistance, PKCη and its downstream targets were identified. Loss of PKCη resulted in a decrease in G1/S transition and upregulation of the cyclin dependent kinase inhibitor p27. Initial data suggest that PKCη promotes p27 ubiquitination and degradation. Taken together, these studies provide novel insight into the complex signaling of HER2-positive breast cancers and the mechanisms of resistance to trastuzumab therapy. This work describes how various kinases can modulate cell proliferation, and points to possible novel drug targets for the treatment of HER2-positive breast cancers.

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