abstract: Due to heterogeneity at the cellular level, single cell analysis (SCA) has become a necessity to study cellomics for the early detection of diseases like cancer. Development of single cell manipulation systems is very critical for performing SCA. In this thesis, electrorotation (ROT) chips to trap and rotate single cells using electrokinetic forces have been developed. The ROT chip mainly consists of a set of closely spaced metal electrodes (60µm interspacing between opposite electrodes) that forms a closed electric field cage (electrocage) when driven with high frequency AC voltages. Cells were flowed through a microchannel to the electrocage where they could be precisely trapped, levitated and rotated in 3-D along the axis of interest. The dielectrophoresis based ROT chip design and relevant electrokinetic effects have been simulated using COMSOL 3.4 to optimize the design parameters. Also, various semiconductor technology fabrication process steps have been developed and optimized for better yield and repeatability in the manufacture of the ROT chip. The ROT chip thus fabricated was used to characterize rotation of single cells with respect to the control parameters namely excitation voltage, frequency and cell line. The longevity of cell rotation under electric fields has been probed. Also, the Joule heating inside the ROT chip due to applied voltage has been characterized to know the thermal stress on the cells. The major advantages of the ROT chip developed are precise electrorotation of cells, simple design and straight forward fabrication process. / Dissertation/Thesis / M.S. Electrical Engineering 2012

Botrytis species affect over 300 host plants in all climate areas of the world, at both pre and post-harvest stages, leading to significant losses in agricultural produce. Therefore, the development of a rapid, sensitive and reliable method to assess the pathogen load of infected crops can help to prescribe an effective curing regime. Growers would then have the ability to predict and manage the full storage potential of their crops and thus provide an effective disease control and reduce post-harvest losses. A highly sensitive electrochemical immunosensor based on a screen-printed gold electrode (SPGE) with onboard carbon counter and silver / silver chloride (Ag/AgCl) pseudo-reference electrode was developed in this work for the detection and quantification of Botrytis species. The sensor utilised a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with a monoclonal antibody against Botrytis immobilised on the gold working electrode. Two immobilisation strategies were investigated for the capture antibody, and these included adsorption and covalent immobilisation after self-assembled monolayer formation with 3-dithiodipropionic acid (DTDPA). A polyclonal antibody conjugated to the electroactive enzyme horseradish peroxidase (HRP) was then applied for signal generation. Electrochemical measurements were conducted using 3,3’, 5,5’-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB/H2O2) as the enzyme substrate system at a potential of -200 mV. The developed biosensor was capable of detecting latent Botrytis infections 24 h post inoculation with a linear range from 150 to 0.05 μg fungal mycelium ml-1 and a limit of detection (LOD) as low as 16 ng ml-1 for covalent immobilisation and 58 ng ml-1 for adsorption, respectively. Benchmarked against the commercially available Botrytis ELISA kits, the optimised immuno-electrochemical biosensor showed strong correlation of the quantified samples (R2=0.998).

The purpose of this study was to utilise lignin as a partial substitute for phenol in PF resins. To achieve this, initially brown rot lignin was produced by a bioconversion technique. During the course of the study, it became clear that the production of brown rot lignin had a limited success. Since brown rot lignin could not be obtained in sufficient quantity and purity by a bioconversion method, other alternative lignin production methods, as well as commercially available lignin, were chosen; namely production of lignin from black liquor and Alcell® (organosolv) lignin. Before performing production of resin formulations, the lignin sources were characterised in terms of reactivity and physical properties of lignins. Both lignins had a similar reactivity, but organosolv lignin was found to be more pure, with a low ash content. Since isolation of lignin from black liquor in laboratory conditions is more complex and requires more time, it was decided to use organosolv lignin for subsequent production of lignin-based reSIns. The lignin was introduced to the resin in two different ways. The first method was the replacing of a certain percentage of phenol with lignin (as supplied) directly into resins. In the second method, lignin was modified prior to resin manufacture by phenolation. Different degrees of phenol substitution (from 5% to 60%) were tried for the production of lignin-based resins. Bond qualities of lignin-phenol-formaldehyde (LPF) , phenolated-ligninformaldehyde, commercial phenol-formaldehyde (PF _com) and laboratory made phenol-formaldehyde (PF _made) resins were assessed by using an Automatic Bonding Evaluation System (ABES), prior to production of particleboards, in order to eliminate some of the poor quality resins. The effect of press temperature and time on bond strength appeared to be highly significant, as the lignin substitution levels increased. Up to 30% phenol substitution was achieved without sacrificing bond strength. The bond strength values of phenolated-lignin-formaldehyde resins were similar to commercial phenol-formaldehyde and laboratory made phenolformaldehyde resins, but better than the LPF resins. It was apparent that resins containing a high level of lignin substitution gave the poorest bond strength values. From these results, some of the resins were eliminated, prior to particleboard production. In order to evaluate the quality of lignin-based resins, particleboards were produced and mechanical and physical tests performed. Effect of press platen temperature (140°C, 160°C, 180°C) and press cycle time (5 min, 8 min, IS min) on the mechanical properties of particleboard, produced by using lignin-based resins, were investigated. It was found that particleboards bonded with up to 30% lignin content resins gave similar mechanical and physical properties to commercial phenolformaldehyde resin, as long as a sufficient heating regime and time were applied.

Submitted by Eduardo Henrique Bizari (edubizari@ig.com.br) on 2018-04-17T11:51:15Z No. of bitstreams: 1 Tese Eduardo FINAL.pdf: 1202549 bytes, checksum: fc1e9272c4cd0e5f7c88384fa75d52bb (MD5) / Rejected by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br), reason: Solicitamos que realize correções na submissão seguindo as orientações abaixo: Prezado se faz necessário que a tese em questão possua um resumo geral e um abstract geral também a parte dos mesmos referentes aos artigos ou capítulos, favor inserir os mesmos no arquivo pdf e submeter novamente ao repositório. Agradecemos a compreensão on 2018-04-18T17:25:50Z (GMT) / Submitted by Eduardo Henrique Bizari (edubizari@ig.com.br) on 2018-04-18T20:21:23Z No. of bitstreams: 2 Tese Eduardo FINAL.pdf: 1202549 bytes, checksum: fc1e9272c4cd0e5f7c88384fa75d52bb (MD5) Tese Eduardo FINAL.pdf: 1202549 bytes, checksum: fc1e9272c4cd0e5f7c88384fa75d52bb (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-04-19T12:48:48Z (GMT) No. of bitstreams: 1 bizari_eh_dr_jabo.pdf: 1202549 bytes, checksum: fc1e9272c4cd0e5f7c88384fa75d52bb (MD5) / Made available in DSpace on 2018-04-19T12:48:48Z (GMT). No. of bitstreams: 1 bizari_eh_dr_jabo.pdf: 1202549 bytes, checksum: fc1e9272c4cd0e5f7c88384fa75d52bb (MD5) Previous issue date: 2018-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A podridão de carvão, causada pelo fungo Macrophomina phaseolina (Tassi) Goid, tem sido uma doença de crescente importância na cultura da soja. Essa doença ocorre principalmente em regiões tropicais e subtropicais e sabe-se que sua maior incidência e severidade são observadas principalmente quando há estresse hídrico e altas temperaturas. Na tentativa de compreender o comportamento da resistência desse patógeno, objetivou-se o estudo da herança genética por meio de uma abordagem quantitativa, sendo utilizada uma análise de componentes de médias e variâncias. O experimento foi conduzido em casa de vegetação no delineamento inteiramente casualizado utilizando as seis gerações de estudo (P1, P2, F1, F2, RCr e RCs). As populações foram inoculadas com o fungo Macrophomina phaseolina por meio de sementes de sorgo infectadas. Após o estádio R8, as plantas foram avaliadas utilizando como critério a Severidade em Raiz e Caule (SRC). Foram estimados componentes de média (por meio do método dos mínimos quadrados ponderados), além das variâncias e parâmetros genéticos associados. Foi confirmado o caráter quantitativo da herança da resistência, com estimativa de 5,62 genes envolvidos na resistência. O modelo aditivo-dominante foi adequado, assim como a estimativa de parâmetros genéticos, havendo predominância de efeitos gênicos aditivos, tanto nos modelos de médias como nos de variância. / Charcoal rot disease, caused by Macrophomina phaseolina, is a disease of significant economic importance in the soybean crop. This disease occurs mainly in tropical and subtropical regions and it is known that the incidence and severity occur mainly when there is stress of water and heat. The evaluation of this pathogen is difficult due to the lack of reliable and efficient methods for the evaluation of soybean genotypes. The present work had the objective of evaluating different methods of inoculation and evaluation in ten soybean genotypes, besides discovering the degree of resistance of the genotypes studied. Infected sorghum seeds, pre-germinated seed in culture medium and infected toothpick test were the tested methods. Colony forming units and root and stem severity were evaluated. There was good correlation between evaluations of colony forming units and root and stem severity (0.87). The infected sorghum seed method was the most efficient for inoculating the pathogen in soybean genotypes. Genotype PI594302 presented higher resistance in the two evaluated parameters, being indicated its introduction in programs of genetic improvement aiming the resistance to the charcoal rot in soybean.

Botrytis species affect over 300 host plants in all climate areas of the world, at both pre and post-harvest stages, leading to significant losses in agricultural produce. Therefore, the development of a rapid, sensitive and reliable method to assess the pathogen load of infected crops can help to prescribe an effective curing regime. Growers would then have the ability to predict and manage the full storage potential of their crops and thus provide an effective disease control and reduce post-harvest losses. A highly sensitive electrochemical immunosensor based on a screen-printed gold electrode (SPGE) with onboard carbon counter and silver / silver chloride (Ag/AgCl) pseudo-reference electrode was developed in this work for the detection and quantification of Botrytis species. The sensor utilised a direct sandwich enzyme-linked immunosorbent assay (ELISA) format with a monoclonal antibody against Botrytis immobilised on the gold working electrode. Two immobilisation strategies were investigated for the capture antibody, and these included adsorption and covalent immobilisation after self-assembled monolayer formation with 3-dithiodipropionic acid (DTDPA). A polyclonal antibody conjugated to the electroactive enzyme horseradish peroxidase (HRP) was then applied for signal generation. Electrochemical measurements were conducted using 3,3’, 5,5’-tetramethylbenzidine dihydrochloride / hydrogen peroxide (TMB/H2O2) as the enzyme substrate system at a potential of -200 mV. The developed biosensor was capable of detecting latent Botrytis infections 24 h post inoculation with a linear range from 150 to 0.05 μg fungal mycelium ml-1 and a limit of detection (LOD) as low as 16 ng ml-1 for covalent immobilisation and 58 ng ml-1 for adsorption, respectively. Benchmarked against the commercially available Botrytis ELISA kits, the optimised immuno-electrochemical biosensor showed strong correlation of the quantified samples (R2=0.998) ... [cont.].

Potato (Solanum tuberosum) is one of the most popular food crops grown as a substitute staple in Zimbabwe. Its production is constrained by pest and diseases which reduce the yield drastically. The blackleg / soft rot disease complex caused by Pectobacterium and Dickeya species (formerly known as Erwinia) has been identified as a potential threat to potato production. The soft rot pathogens can cause systemic and vascular infections in potatoes, which result in the development of various symptoms on the stem and tubers. The symptoms that develop on the plant are not species specific but depend on climatic conditions prevailing at the infection stage. The pathogens affect the crop at all stages of production, which include in the field, storage and in transit. The primary objectives of this study were to identify the pathogens which cause blackleg and soft rot on potatoes in Zimbabwe; document the grower’s knowledge of blackleg and soft rot diseases in Zimbabwe; evaluate cultivars grown in Zimbabwe and South Africa for tolerance to Pectobacterium and Dickeya species; determine the role of calcium in blackleg and soft rot development; and determine the genetic diversity of Pectobacterium carotovorum subsp. brasiliensis isolates from South Africa, Zimbabwe and mini-tubers imported from China. The first chapter introduces the research highlighting the importance of potatoes globally and in Zimbabwe. It discusses production constraints caused by soft rot pathogens, as well as the pathogenesis of Pectobacterium and Dickeya spp. It also summarises the research objectives and thesis outline. Chapter 2 provides an overview of published literature on morphology, pathogenicity and identification of blackleg and soft rot pathogens, epidemiology, host range, defense mechanisms in plants, host nutrition and disease management strategies. Taxonomic revisions have led to the reclassification of pectolytic Erwinia into several genera. Strains formerly described as Erwinia carotovora have been incorporated into the genus Pectobacterium, and strains classified as Erwinia chrysanthemi are now assigned to the genus Dickeya. Several species and subspecies of Pectobacterium and Dickeya have isolated from infected potatoes. In Zimbabwe only Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum have until now been identified as the causal agents of blackleg and soft rot diseases, respectively. Although other species and subspecies of Pectobacterium and Dickeya have been isolated from infected potato plants in Zimbabwe, their potential to cause diseases on potatoes should not be underestimated. In chapter 3 bacterial isolates collected from infected plants showing typical blackleg / soft rot disease symptoms were identified using biochemical and physiological methods, as well as rep-PCR, Amplified Fragment Length Polymorphism (AFLP) and single gene sequencing using two genes, viz. gyrB and recA. Amplified Fragment Length Polymorphisms (AFLPs) were used to study the genetic diversity among potato isolates from Zimbabwe, South Africa and mini-tubers imported from China. Pectobacterium and Dickeya species enter the plant through wounds and natural openings such as lenticels. When they invade the plant they colonise the vascular tissue and spaces between the thin walled parenchyma cells. They remain inside these cells as latent infections and disease symptoms develop when host resistance is impaired or when environmental conditions become conducive for disease development. Chapter 4 investigates the role of defense related enzymes, polyphenol oxidase, phenylalanine ammonia lyase, peroxidases, chlorogenic acid and total soluble phenols in host resistance to potato soft rot. The potato varieties were assayed for activity of polyphenol oxidase and phenylalanine ammonia lyase, peroxidases and concentration of chlorogenic acid and total soluble phenols in tuber tissue. The role of these four components in imparting resistance against the soft rot pathogens was also investigated. Blackleg and soft rot diseases are seed-borne and difficult to control using chemical, physical and cultural methods. Several approaches aimed at controlling blackleg and tuber rot have been studied but the degree of success has been variable. Plant nutrition is an important component of natural disease resistance. The effect of calcium soil amendments in enhancing resistance to potato soft rot pathogens was investigated in Chapter 5. A management strategy based on calcium-induced defense responses of potato to Pectobacterium and Dickeya spp. was investigated. Calcium promotes production of phenolics and strengthens the cell wall, making the plants resistant to maceration by pectolytic enzymes. In Chapter 6 a survey was conducted in nine potato growing regions of Zimbabwe using an informal structured questionnaire. The findings highlighted the distribution and impact of blackleg and soft rot diseases on the Zimbabwean potato industry. A broad spectrum of information on blackleg / soft rot disease complex was gathered and this information can help growers to make informed decisions about control strategies to apply. Each chapter in this thesis has been treated as an independent entity. Thus redundancy between chapters could not be avoided. It is my hope that the results of these studies on soft rot pathogens in Zimbabwe will contribute to a better understanding of the blackleg / soft rot disease complex that they cause. I also hope that these studies will form the basis of detailed and future investigations in epidemiology and disease control strategies. / Thesis (PhD)--University of Pretoria, 2012. / Microbiology and Plant Pathology / unrestricted

Staphylococcus aureus is a known cause of chronic biofilm infections. Recent studies have demonstrated the importance of proteinaceous material in the biofilm matrix. S. aureus secretes at least ten proteases and there is growing evidence that these enzymes have self-cleavage roles that alter biofilm integrity. The goal of this dissertation is to characterize the function of secreted proteases with respect to S. aureus biofilms. In addition, we aimed to determine whether a known regulator of S. aureus proteases, the Regulator of toxins (Rot), is also a regulator of biofilms. Studies presented in Chapter II utilize a mutation in Sigma factor B (SigB) to analyze the role of secreted proteases in biofilm formation. S. aureus strains with sigB mutations have enhanced protease activity and a biofilm negative phenotype, and this biofilm phenotype was conserved on human plasma coated surfaces. To identify the protease(s) responsible for the phenotype, inhibitor studies revealed that the addition of the cysteine protease inhibitor E-64, or Staphostatin inhibitors that specifically target the S. aureus cysteine proteases SspB or ScpA (Staphopains), could restore biofilm formation. Using gene deletion mutants, we identified that the combined mutation of sspB and scpA also restored ΔsigB biofilm formation. Together these findings indicated that Staphopain enzymes are responsible for the ΔsigB biofilm negative phenotype. To address the Staphopain inhibitory role in biofilms further, regulatory studies indicated that enzymes levels were maintained at a low level during biofilm formation and exogenous addition of purified Staphopains inhibited biofilm formation or disassembled established biofilms across multiple S. aureus strain lineages. Taken together, these findings suggest an unappreciated role of the Staphopains in biofilm maturation. Studies in Chapter III examine the role of the transcriptional regulator rot in biofilm formation. Using both coated and uncoated biofilm assays, Rot was found to be essential for S. aureus biofilm formation. The expression of RNAIII, the major effecter molecule of the agr quorum-sensing system, inversely correlated with intracellular Rot protein concentration. Rot protein levels were found to be high under biofilm conditions and modulation of rot expression demonstrated that intracellular levels of Rot modulate biofilm formation. Examination of protease expression, production, and activity revealed that production of secreted proteases are inhibited by Rot and electrophoretic mobility shift assays demonstrated that inhibition was due to the direct binding of Rot to protease promoters. Chemical and biochemical protease inhibitors were able to restore the capacity to form a biofilm to rot and follow-up mutation of protease genes confirmed these findings. Finally, Δ rot mutant is attenuated in a murine catheter model of infection. These findings suggest an essential role for Rot as an inhibitor of proteases to modulate biofilm formation, and demonstrate the importance of coordinated regulation during infection.

Generellt i byggbranschen avslutas sällan ett projekt utan att ha stött på hinder. För Region Uppsala har ROT-projekt, som står för renovering, ombyggnad och tillbyggnad, ofta slutat dyrare än förväntat. Det är vanligt att beskylla projekteringen men en kartläggning av anledning till uppkomst av kostnadsdrivande avvikelser finns inte i dagsläget, eller om entreprenadform har någon påverkan. Detta är ett examensarbete inom byggteknik vid Uppsala Universitet på 15 hp och är utfört tillsammans med Region Uppsalas förvaltning Fastighet och Service som ansvarar för Regionens fastighetsbestånd. Deras uppgifter består av uthyrning, ombyggnad, nybyggnad och skötsel för drift av fastigheterna. Projektavdelningen är den avdelning på förvaltningen som driver de större bygg- och installationsprojekten. Deras största beställare är Akademiska sjukhuset. Syftet med arbetet är att kartlägga de kostnadsdrivande avvikelser som uppkommer i ROT-projekt för respektive entreprenadform, och målet är att undvika återkommande avvikelser i Fastighet och Service framtida ROTprojekt. Arbetet består dels av en litteraturstudie för att ge en fördjupning inom ämnet och förståelse för dagens problematik. Vidare har sju ROT-projekt tillhandahållna av Fastighet och Service analyserats i fallstudier. Projekten var begränsade till 15 miljoner i entreprenadpris för att fler projekt skulle kunna analyseras och bestod av både total- och utförandeentreprenader. En jämförelse gjordes mellan dessa. Utöver detta utfördes intervjuer med projektledare hos Fastighet och Service om deras erfarenheter och tankar kring ämnet. I detta arbete har en kartläggning över upprättade avvikelser gjorts och statistiken presenteras för varje projekt samt sammanlagt för entreprenadformerna. Resultatet visar att projekteringsprocessen består av flera skeden och det kan vara svårt att påvisa i vilket projekteringsmissarna grundar sig i. Sammanlagt bestod dessa av en tredjedel av de upprättade avvikelserna men det fanns andra orsaker som bidrog till större kostnader. Utifrån intervjuerna framkom det att upprättade avvikelser inte ligger till grund för val av entreprenadform utan andra parametrar var mer väsentliga. I slutsatsen presenteras för- och nackdelar för båda entreprenadformerna.

Due to considerable loss through root-rot of mother beets while in storage during the winter of 1918-1919 the Amalgamated Sugar Company asked advice in regard to planting beets which had decay lesions on them. Would the beets yield seed if planted? How much seed would these beets yield as compared with healthy beets? Would the seed produced be injured by having been grown on decayed beets? It was felt that an experiment carried out with the above questions in view would bring out many facts upon which to base conclusions in answering the questions of the Sugar Company.

This dissertation is composed of two research projects. The first research project is aimed at using synthetic fluorogenic probes to study the possible or dominant linkages in biomass. These probes that mimic the linkages found in lignin-cellulosic biomass are designed to select the optimal fungi from direct evaluation process or could be tested against other microbials to screen candidates which can break ligno-hemicellulose bonds. For the first stage, these probes would be tested against white rot fungi extract. The white rot fungi are used for the first stage to see if releasing or degrading carbohydrates while keeping lignin largely intact is possible or not. These probes can help to answer fundamental questions, such as what could be the dominant linkages between lignin and hemicellulose, and what are the possible mechanisms for the cleavage of carbohydrates in biomasses. Understanding the linkages in these biomass will enable high efficient degradation or release of carbohydrates, primarily hemicelluloses, from biomass. The second project is focused on synthesizing new aminoglycoside analogs and exploring the potential to revive traditional antibacterial kanamycin as new types of antifungal agents. Aminoglycosides are widely used broad spectrum antibiotics. Although mainly used as antibacterial agents, there have been studies to show amphiphilic aminoglycoside derivatives could be possibly employed as antifungal agents. A concise and novel method for site-selective alkylation of tetra-azidokanamycin has been developed that leads to the divergent synthesis of three classes of kanamycin derivatives. These new amphiphilic kanamycin derivatives bearing alkyl chains length of 4, 6, 7, 8, 9, 10, 12, 14，16 have been synthesized and tested against bacteria and fungi. Surprisingly, the antibacterial effect of the synthesized kanamycin derivatives decline or disappear compared with the original kanamycin A, but some of the compounds show very strong activity as antifungal agents.