Genetic Analysis and Cell Manipulation on Microfluidic Surfaces

Personalized cancer medicine is a cancer care paradigm in which diagnostic and therapeutic strategies are customized for individual patients. Microsystems that are created by Micro-Electro-Mechanical Systems (MEMS) technology and integrate various diagnostic and therapeutic methods on a single chip hold great potential to enable personalized cancer medicine. Toward ultimate realization of such microsystems, this thesis focuses on developing critical functional building blocks that perform genetic variation identification (single-nucleotide polymorphism (SNP) genotyping) and specific, efficient and flexible cell manipulation on microfluidic surfaces. For the identification of genetic variations, we first present a bead-based approach to detect single-base mutations by performing single-base extension (SBE) of SNP specific primers on solid surfaces. Successful genotyping of the SNP on exon 1 of HBB gene demonstrates the potential of the device for simple, rapid, and accurate detection of SNPs. In addition, a multi-step solution-based approach, which integrates SBE with mass-tagged dideoxynucleotides and solid-phase purification of extension products, is also presented. Rapid, accurate and simultaneous detection of 4 loci on a synthetic template demonstrates the capability of multiplex genotyping with reduced consumption of samples and reagents. For cell manipulation, we first present a microfluidic device for cell purification with surface-immobilized aptamers, exploiting the strong temperature dependence of the affinity binding between aptamers and cells. Further, we demonstrate the feasibility of using aptamers to specifically separate target cells from a heterogeneous solution and employing environmental changes to retrieve purified cells. Moreover, spatially specific capture and selective temperature-mediated release of cells on design-specified areas is presented, which demonstrates the ability to establish cell arrays on pre-defined regions and to collect only specifically selected cell groups for downstream analysis. We also investigate tunable microfluidic trapping of cells by exploiting the large compliance of elastomers to create an array of cell-trapping microstructures, whose dimensions can be mechanically modulated by inducing uniform strain via the application of external force. Cell trapping under different strain modulations has been studied, and capture of a predetermined number of cells, from single cells to multiple cells, has been achieved. In addition, to address the lack of aptamers for targets of interest, which is a major hindrance to aptamer-based cell manipulation, we present a microfluidic device for synthetically isolating cell-targeting aptamers from a randomized single-strand DNA (ssDNA) library, integrating cell culturing with affinity selection and amplification of cell-binding ssDNA. Multi-round aptamer isolation on a single chip has also been realized by using pressure-driven flow. Finally, some perspectives on future work are presented, and strategies and notable issues are discussed for further development of MEMS/microfluidics-based devices for personalized cancer medicine

Site-directed mutagenesis of a lytic polysaccharide monooxygenase from Micromonospora aurantiaca

Cellulose and chitin are the two most abundant biopolymers in nature and they are valuable biomaterials that supplements fossil resources in the production of fuels, materials and chemicals. Due to the recalcitrant nature of these polysaccharides, efficient conversion into soluble sugars is of major importance for a sustainable bio-economy in the future. Traditionally, enzymatic degradation of cellulose and chitin were thought to rely on the synergistic action of hydrolytic enzymes, but the recent discovery of lytic polysaccharide monooxygenases (LPMOs) shows that these oxidative enzymes are important contributors to the depolymerization process. Cellulose-active AA10 LPMOs cleaves glycosidic bonds in the cellulose backbone by either specific C1 or C1/C4 oxidation. This oxidative regioselectivity is thought to be a result of different positioning of the LPMO on the cellulose, but the mechanisms behind the differences in regioselectivity is unknown. Understanding the mechanism of oxidative regioselectivity in LPMOs is important for both fundamental and applied reasons where optimization of enzyme cocktails is a major issue for biorefining of biomass. This study identifies a pair of amino acids in the sequence of LPMO10B from the bacterium Micromonospora aurantiaca that are likely to play an important role in the specificity of the protein’s oxidative regioselectivity. Highly conserved positions in the sequences of specific C1 oxidizing cellulose-active AA10 proteins were targeted for site-directed mutagenesis, and the C4 oxidizing activity of a MaLPMO10B variant, carrying two mutated residues, was almost completely lost. The finding suggests that both residues are highly important for the LPMO to carry out C4 oxidizing activity on cellulosic substrates. Characterization of two M. aurantiaca LPMOs were conducted: MaLPMO10B and MaLPMO10D. Both contain a CBM2 domain and are active on both cellulose (C1/C4 oxidizers) and chitin. The structure of MaLPMO10B was determined by X-ray crystallography to 1.08 Å. Structural comparison to other C1/C4 oxidizing cellulose-active AA10 proteins revealed a prolonged loop, comprising eight additional residues, near the substrate-binding surface of MaLPMO10B. From the research that has been conducted, it is possible to conclude that residues W82 and N85 play an important role in the determination of oxidative regioselectivity in MaLPMO10B. Further research will be necessary to identify additional residues to possibly eliminate the C4 oxidizing activity of the enzyme completely.Cellulose og kitin er de to mest forekommende biopolymerene i naturen, og de er verdifulle biomaterialer som supplerer fossile ressurser i produksjonen av drivstoff, materialer og kjemikaler. Effektiv konvertering av disse vanskelig nedbrytbare polysakkaridene til løselige sukkerarter er av stor viktighet for en fremtidig bærekraftig bioøkonomi. Det tradisjonelle synet på enzymatisk nedbryting av cellulose og kitin har innebåret synergistisk handling mellom hydrolytiske enzymer, men den nylige oppdagelsen av lytiske polysakkaridmonooksygenaser (LPMOer) viser at disse oksidative enzymene er viktige bidragsytere til depolymeriseringsprosessen. Cellulose-aktive AA10 LPMOer kløyver glykosidiske bånd i cellulose-kjedene ved å enten oksidere kun C1-karbonet eller både C1 og C4 karbonet. Denne oksidative regioselektiviteten mistenkes å være et resultat av ulik posisjonering av LPMOen på cellulose kjeden, men mekanismene bak dette er ukjent. Å forstå mekanismen bak oksidativ regioselektivitet i LPMOer er viktig for både fundamentale og anvendte årsaker der optimalisering av enzym-cocktails er av stor viktighet for bioraffinering av biomasse. Denne studien identifiserer et par aminosyrer i sekvensen til LPMO10B fra bakterien Micromonospora aurantiaca som er antatt å spille en viktig rolle i spesifiseringen av proteinets oksidative regioselektivitet. Høyt konserverte posisjoner i sekvensen til spesifikt C1-oksiderende cellulose-aktive AA10 proteiner var målrettet for seterettet mutagenese, og den C4-oksiderende aktiviteten til en MaLPMO10B-variant, som inneholdt to muterte residuer, var nesten helt fjernet. Dette funnet antyder at begge residuene er meget viktige for at LPMOen kan utføre C4-oksiderende aktivitet på cellulose-substrater. Karakterisering av to LPMOer fra M. aurantiaca ble utført: MaLPMO10B og MaLPMO10D. Begge inneholdt et CBM2-domene, og begge var aktive på både cellulose (C1/C4-oksiderende) og kitin. Strukturen til MaLPMO10B ble bestemt ved røntgen krystallografi til 1.08 Å. Strukturell sammenlikning med andre C1/C4-oksiderende cellulose-aktive AA10 proteiner avslører av MaLPMO10B inneholder en forlenget loop, bestående av åtte ekstra residuer, nær den substratbindende overflaten. Fra forskningen som har blitt utført i denne studien kan det trolig konkluderes at residuene W82 og N85 spiller en viktig rolle i bestemmelsen av oksidativ regioselektivitet hos MaLPMO10B. Videre forskning vil være nødvendig for å identifisere ytterligere residuer for å muligens eliminere den C4-oksiderende aktiviteten til enzymet fullstendig.M-BIOTE

Genome sequencing of Leptolyngbya Heron Island, 2Å crystal structure of phycoerythrin and spectroscopic investigation of chromatic acclimation

abstract: Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the L.HI genome - Since this is a newly discovered cyanobacterium, its genome was not previously elucidated. Illumina sequencing, a type of next generation sequencing (NGS) technology was employed to sequence the genome. Unfortunately, the natural isolate contained other contaminating and potentially symbiotic bacterial populations. A novel bioinformatics strategy for separating DNA from contaminating bacterial populations from that of L.HI was devised which involves a combination of tetranucleotide frequency, %(G+C), BLAST analysis and gene annotation. 2) Structural elucidation of phycoerythrin - Phycoerythrin is the most important protein in the PBS assembly because it is one of the few light harvesting proteins which absorbs green light. The protein was crystallized and its structure solved to a resolution of 2Å. This protein contains two chemically distinct types of chromophores: phycourobilin and phycoerythrobilin. Energy transfer calculations indicate that there is unidirectional flow of energy from phycourobilin to phycoerythrobilin. Energy transfer time constants using Forster energy transfer theory have been found to be consistent with experimental data available in literature. 3) Effect of chromatic acclimation on photosystems - Chromatic acclimation is a phenomenon in which an organism modulates the ratio of PE/PC with change in light conditions. Our investigation in case of L.HI has revealed that the PE is expressed more in green light than PC in red light. This leads to unequal harvesting of light in these two states. Therefore, photosystem II expression is increased in red-light acclimatized cells coupled with an increase in number of PBS.Dissertation/ThesisPh.D. Chemistry 201

Combining induced protease fragment assembly and microarray analysis to monitor signaling in living cells.

Die Fähigkeit Signalkaskaden zu vermessen ist für das Verständnis komplexer biologischer Prozesse essentiell. Bis jetzt versorgt uns die DNA Microarray Technologie mit umfassenden Daten, deren Auflösung jedoch auf der Ebene der Genexpression endet. Diese Informationen reichen nicht aus um die vorgeschalteten regulatorischen Mechanismen der Genexpression zu verstehen. Die meisten proteomischen Technologien hängen von in vitro synthetisierten Peptiden ab oder benötigen weitere biochemische Manipulationen. Für die Charakterisierung und Beobachtung einzelner Bestandteile von Signalkaskaden in lebenden Zellen sind Hochdurchsatz-Verfahren notwendig. In der vorliegenden Arbeit wird ein experimentelles Verfahren namens EXTassay beschrieben, dass eine quantitative und parallele Messung multipler Signal-Ereignisse ermöglicht, die der mRNA Expression vorgelagert sind. EXTassays vereinen verschiedene zelluläre Assays, die an die Reporter Gen Expression gekoppelt sind. Um Multiplexing zu erreichen wurde eine komplexe und optimierte Bibliotek kurzer expressed oligonucleotide tags (EXTs) generiert. Jedes einzelne EXT ersetzt hierbei ein klassisches Reportergen und dient als eindeutiger Identifikator für einen definierten zellulären Assay. Es können verschiedene EXTs, die entweder in einer Zelle oder in einer Zellpopulation exprimiert sein können, über Microarray Hybridisierung analysiert werden. In dieser Arbeit wurden Protokolle für das verlässliche Auslesen von Microarrays für EXTs optimiert. Weiterhin wurden EXT-basierte Assays verwendet, um die durch Neuregulin-1 induzierte Dimerisierung und Aktivierung von Rezeptortyrosinkinasen der ErbB Familie zu untersuchen. Für die quantitative Messung von Rezeptordimeriserung und phosphorylationsabhängige Kopplung an Interaktionspartner wurden Protein-Komplementations-Assays der TEV Protease, split-TEV Assays, verwendet. Hierzu wurde jeder Assay an eindeutige EXT-Reporter gekoppelt. Zusätzlich wurde die Aktivierung von 30 verschiedenen EXT-gekoppelten cis-regulatorischen Elementen erfaßt, um so einen Einblick in die nachfolgende Aspekte der Signalverarbeitung zu erhalten. Alle Assays wurden mit eindeutigen EXTs durchgeführt und mittels Microarray analysiert. Die simultane Analyse dreier verschiedener und regulierter Rezeptor Komplexe (ErbB2/2, 2/3, 2/4) zeigte, dass EXT-basierte Assays geeignet sind rezeptor-spezifische Signalereignisse zu unterscheiden. EXTassays sind daher geeignet quantitative Profile aktivierter Signalkaskaden in Zellen erstellen zu können.The ability to monitor multiple signaling events simultaneously in living cells is essential to better understand complex biological processes. So far, DNA-microarray technologies provide global scale data mainly restricted to the level of gene expression. This information is not sufficient to understand the upstream regulatory mechanisms that lead to gene expression changes. Most proteomic technologies also provide large scale measurement but usually depend on in vitro synthesized peptides or require biochemical manipulations. High throughput technologies are required for functional characterization and monitoring of signaling components in living cells. Here, an experimental approach is presented termed EXTassay that enables quantitative and parallel measurements of various signaling events upstream of mRNA expression. EXTassay incorporates various cellular assays that are coupled to reporter gene expression. To achieve multiplexing, we have generated a complex and optimized library of short expressed oligonucleotide tags (EXTs). Each unique EXT can replace a classical reporter gene and serves as a unique identifier for tracking and quantification of a defined cellular assay. Multiple EXT-reporters expressed in the same cell or cell population can be isolated and analyzed by custom microarray hybridization. We have established protocols and optimized the microarray readout for reliable EXT quantification. We applied the EXTassay to analyze the neuregulin 1 induced ErbB receptor tyrosine kinase signaling in PC-12 cells. We used transcriptionally coupled split TEV protein complementation assays to monitor ErbB receptor dimerization and phosphorylation dependent interaction with downstream signaling proteins. In addition, we employed 30 different cis-regulatory elements to assess the downstream signaling. All assays were coupled to unique EXTs and analyzed by microarrays. By analyzing three different receptor complexes (ErbB 2/2, 2/3 and 2/4), we were able to measure receptor specific differential signaling effects and demonstrate that EXTassays can be applied for the quantitative profiling of activated signaling pathways

The certification of the absorbed energy (150 J nominal) of charpy V-notch reference test pieces: ERM®-FA415v

This certification report describes the processing and characterisation of ERM®-FA415v, a batch of Charpy V-notch certified reference test pieces certified for the absorbed energy (KV). Sets of five of these test pieces are used for the verification of pendulum impact test machines according to ISO 148-2 (Metallic materials - Charpy pendulum impact test - Part 2: Verification of testing machines). The absorbed energy (KV) is procedurally defined and refers to the impact energy required to break a V-notched test piece of standardised dimensions, as defined in ISO 148-1 [2]. The certified value of ERM®- FA415v is made traceable to the SI, via the SI-traceable certified value of the master batch ERM®-FA415b, by testing samples of ERM®- FA415v and ERM®-FA415b under repeatability conditions on an impact pendulum verified and calibrated with SI-traceably calibrated tools. The certified value is valid only for strikers with a 2 mm tip radius. The certified value is valid at (20 ± 2) °C.JRC.D.2-Standards for Innovation and sustainable Developmen

Polymer Micro- and Nanofluidic Systems for In Vitro Diagnostics: Analyzing Single Cells and Molecules

Polymer micro- and nanofluidic systems, with their critical dimensions, offer a potential to outperform conventional analysis techniques and diagnostic methods by enhancing speed, accuracy, sensitivity and specificity. In this work, applications of microfluidics have been demonstrated to address the existing challenges in stroke diagnosis, by mRNA expression profiling from whole blood within \u3c20 min. A brief overview of various biomarkers for stroke diagnosis is given in chapter 1 followed by design and testing of individual microfluidic modules (chapter 2 and 3) required for the development of POC diagnostic strategy for stroke. We have designed and evaluated the performance of polymer microfluidic devices for the isolation of leukocyte subsets, known for their differential gene expression in the event of stroke. Target cells (T-cells and neutrophils) were selected from with greater purities, from 50 µl whole human blood by using affinity based capture in COC devices within a 6.6 min processing time. In addition, we have also demonstrated the ability to isolate and purify total RNA by using UV activated polycarbonate solid phase extraction platform. Polymer-based nanofluidic devices were used to study the effects of surface charge on the electrodynamic transport dynamics of target molecules. In this work, we report the fabrication of mixed-scale micro- and nanofluidic networks in poly(methylmethacrylate), PMMA, using thermal nanoimprint lithography using a resin stamp and surface modification of polymer nanoslits and nanochannels for the assessment of the associated electrokinetic parameters – surface charge density, zeta potential and electroosmotic flow. This study provided information on possible routes that can be adopted to engineer proper wall chemistry of polymer nanochannels for the enhancement or reduction of solute/wall interactions in a variety of relevant single-molecule studies

Investigating the mechanism of a novel glycine-dependent riboswitch and a putative non-coding regulatory RNA in Streptococcus pyogenes

We investigated gene expression regulation by a putative glycine(Gly) riboswitch located in the 5′-UTR of a SAF protein gene in the S.pyogenes. Gly-dependency was studied using a luciferase reporter gene system. Maximal reporter gene expression happened in the presence of low Gly concentrations. RT-qPCR showed that in the presence of Gly (≥1 mM), expression of the gene was downregulated. Growth in the presence of 0.1 mM Gly led to the production of a full-length transcript. We conclude that the Gly riboswitch in S. pyogenes represses gene expression in the presence of high Gly concentrations

Plasmid-Mediated Expression of Foreign Genes in Ehrlichia Canis

Ehrlichia canis is the etiologic agent of “tropical canine pancytopenia”, or canine ehrlichiosis. The impetus for this research was to overcome the lack of any reliable means of elucidating the genetic profiles of these illusive and historically difficult to manipulate organisms. The use of a broad-host range plasmid greatly facilitated the determination of an electro-transformation protocol. The transforming plasmid possesses a chloramphenicol antibiotic resistance gene marker (chloramphenicol acetyltransferase [CAT] gene), and a visual reporter gene marker, the Green Fluorescent Protein (GFP) gene. With primer sets designed to specifically amplify these two plasmid encoded gene markers, thus verifying the presence of the transforming plasmid, and an additional primer set designed to specifically amplify the p28 gene of E. canis, a multi-copied and species specific genome encoded epitope, PCR verification of electro-transformed E. canis was made possible. Once this PCR protocol was established and accompanied by gel electrophoresis and amplification product sequencing, the presence of electro-transformed E. canis could also be verified by demonstrable antibiotic resistance in tissue culture, as well as fluorescent, light and transmission electron microscopy. Western blot was also performed to verify the expression of GFP in tissue culture by the electro-transformed E. canis

Investigating a BIR-containing plasmodium falciparum protein and identifying its binding partners

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2018.The majority of the worldwide malaria deaths are caused by the Plasmodium falciparum parasite and unfortunately parasite strains are emerging that are resistant to not only artemisinin, but also the partner drugs used in current antimalarial combination therapy. The intraerythrocytic P. falciparum life stage is characterised by exponential, asexual proliferation that could cause the premature death of the human host before the sexual gametocytes have had enough time to develop and be taken up by the mosquito vector to continue its lifecycle. It is hypothesised that P. falciparum maintains its population at a level low enough to allow for the transmission of these gametocytes by using a form of regulated cell death (RCD). The molecular members of this cell death pathway are currently unclear, but a putative P. falciparum inhibitor of apoptosis protein (PfIAP; PF3D7_0519600) has previously been identified. Metazoan IAP proteins play anti-apoptotic roles in cells by interacting and inhibiting pro-apoptotic caspases, but also perform other functions. Analysis of the PfIAP protein using bioinformatic tools revealed that it contains one conserved baculoviral IAP repeat (BIR) domain and that this P. falciparum BIR domain is structurally similar to the BIR domains of various human IAP proteins. mRNA extracted from asexual P. falciparum parasites was used to construct a biotin-tagged phage display library, which was used in biopanning experiments with two regions of the PfIAP protein, expressed as recombinant GST-tagged proteins. Four binding partners were identified for the N-terminal BIR domain of the protein, while two proteins were identified as interacting partners for the C-terminal region of PfIAP. Of these, a double C2-like domain-containing protein (PfDOC2) and the high molecular weight rhoptry protein 3 (PfRhopH3) were expressed as recombinant His-tagged proteins and verified as PfIAP binding partners by in vitro binding assays. Transgenic P. falciparum parasites were generated expressing a GFP-tagged PfIAP BIR domain, which localised to the cytoplasm under both normal and high temperature conditions which mimic febrile malaria, a physiological trigger of RCD. Knockout experiments of the pfiap gene using the CRISPR-Cas9 genome editing tool suggested that this gene could be essential for the survival of asexual P. falciparum parasites. This study offers the first details of a putative P. falciparum inhibitor of apoptosis protein and suggests that it could have non-apoptotic roles in the parasite, given the diverse functions of the binding partners that comprise the PfIAP protein-protein network.LG201

Studies on proteins from Methylococcus capsulatus Bath : expression in Lactobacillus plantarum WCFS1, purification and large-scale fermentation

The work described in this thesis is part of a larger project investigating the non-commensal methanotroph Methylococcus capsulatus Bath. This species has shown anti-inflammatory and obesity-reducing effects in animal models when given as an extract in the diet. In silico studies of the M. capsulatus Bath genome revealed four genes (mam, tir, mif and sim) that encode proteins which are homologous to proteins with known immunomodulatory functions. They might therefore contribute to the observed effects. These proteins (Mam, Tir, Mif and Sim) were the focus of this thesis. The genetic sequences from M. capsulatus Bath were cloned into the pSIP401 vector of the pSIP inducible gene expression system. They were successfully transformed into the lactic acid bacteria Lactobacillus plantarum WCFS1. Inducible protein expression was successfully accomplished as verified with Western blot. The genetic sequences were also cloned into the pNIC-CH vector for purification of protein. The proteins were successfully produced in Escherichia coli BL21 and purified with immobilized metal ion affinity chromatography. . After successfully producing purified protein and lysate from L. plantarum harbouring the pSIP401-derivatives, in vitro assays were performed. In the first assay, effects of these bacterial stimuli on interleukin (IL)-1b-induced production of IL-8 was investigated. In the second assay, effects on transepithelial electrical resistance when co-incubated with tumour necrosis factor (TNF)a. Neither of these assays were successful, however. Finally, L. plantarum harbouring pSIP401_MAM or empty vector were cultured in 12-liter batch fermentations by using a 15-liter bioreactor. The resulting broth was lysed via French pressing and lyophilized before being shipped away to Copenhagen, to be used in a murine study.Arbeidet som ble gjennomført i denne masteroppgave er en del av et større prosjekt som undersøker den metanotrofe bakterien Methylococcus capsulatus Bath. Denne bakterien, som ikke finnes i tarmfloraen, har fått påvist betennelsesdempende og fedmereduserende effekter i dyreforsøk når den blir gitt som et ekstrakt i fôret. In silico-studier av genomet til M. capsulatus Bath viste at fire gener (mam, tir, mif og sim) koder for proteiner som har homologi til proteiner med kjente immunomodulerende effecter. Disse proteinene (Mam, Tir, Mif og Sim) var fokuset I denne oppgaven. Gensekvensene fra M. capsulatus Bath ble klonet inn i pSIP401-vektoren som er en del av det induserbare genuttrykkssystemet pSIP. De ble deretter transformert inn i melkesyrebakterien Lactobacillus plantarum WCFS1. Induserbar produksjon av protein ble deretter verifisert ved bruk av Western blot. Gensekvensene ble også klonet inn i pNIC-CH-vektoren for rensing av protein. Proteinene ble produsert i Escherichia coli BL21 og renset ved bruk av kolonnekromatografi. Etter at renset protein og lysat fra L. plantarum med pSIP401-derivatene var gjennomført ble to in vitro-forsøk utført. I det første forsøket ble effekter av de bakterielle stimuliene på interleukin (IL)-1b-indusert produksjon av IL-8 undersøkt. I det andre forsøket ble effekter av stimuliene sammen med tumour necrosis factor (TNF)-a på elektrisk motstand over et epitelcellelag undersøkt. Ingen av disse to assayene var vellykket. Til slutt ble L. plantarum med pSIP401_MAM eller tom vektor dyrket i 12-liters batchfermenteringer i en 15-liters bioreaktor. Bakteriemassen ble deretter lysert med French press, før den ble frysetørket og sendt til København for å brukes i et museforsøk.M-MA