Master of Science / Department of Agronomy / P. V. Vara Prasad / Grain sorghum (Sorghum bicolor L. Moench) is the fourth most important cereal crop grown throughout the semi-arid regions of the world. It is a staple food crop in Africa and Asia, while it is an important feed crop in the United States (US). More recently it is increasingly becoming important as a potential bioenergy feedstock crop around the world. The state of Kansas is the largest producer of grain sorghum in the US and contributes 40% of the total production. Drought is one of the major environmental factors limiting sorghum production in the semi-arid regions of the US, Asia and Africa. It is estimated that global crop losses due to drought stress exceed $10 billion annually. In crop production, drought stress can be classified into pre- or post-flowering. Even though the world collections of sorghum contain over 35,000 accessions, the genetic base currently used in breeding programs is very small (about 3%). Thus, it is important to identify diverse breeding lines for crop improvement. The diversity (association) panel consisting of 300 sorghum lines from all over the world was assembled for trait evaluation and association mapping. In this research these lines were grouped into the five major races (Figure 1) and 10 intermediate races of sorghum. The objectives of the research are to: (i) quantify the performance of the diversity panel under field conditions in Kansas, (ii) identify critical physiological traits affected by drought at both pre- and post-flowering stages of sorghum development, (iii) identify the most sensitive stage to drought stress during the reproductive phase of sorghum development and, (iv) test the feasibility of using a chlorophyll fluorescence assay (CVA) as a tool for identifying stay-green lines in grain sorghum during early stages of crop development. Field experiments were conducted in 2006 and 2007 in two locations in Kansas (Manhattan and Hays) under rain fed and irrigated conditions for the association panel. Objectives (iii) and (iv) were achieved with controlled environment experiments conducted in the greenhouse at the agronomy department, Kansas State University in 2006 and 2007. Results showed that there was large genetic variability among and within different races in the diversity panel for growth, physiological traits and yield components. Some genotypes showed yield stability across the different environments that were investigated. Drought significantly decreased seed number and harvest index across genotypes and races. In grain sorghum the period prior to flowering (panicle initiation) was the most sensitive stage to drought stress, in terms of its effect on seed-set, during reproductive development. A cell viability assay showed that there were significant differences in the loss of cell viability between leaf sample of stay green and non-stay green genotypes when leaf samples are collected in the morning and subjected to high respiratory demand. Therefore the chlorophyll fluorescence assay has potential as a tool for stay green trait screening at early stages of growth in grain sorghum.

The enzyme 11β-Hydroxysteroid Dehydrogenase 1 (11β-HSD1) catalyses the intracellular biosynthesis of the active glucocorticoid cortisol. Tissue specific dysregulation of the enzyme has been implicated in the development of metabolic syndrome and other associated diseases. Experiments with transgenic mice and prototype inhibitors show that inhibition of 11β-HSD1 in visceral adipose tissue and liver leads to a resistance of diet-induced hyperglycemia and a favourable lipid and lipoprotein profile as compared to controls. 11β-HSD1 inhibition has thus been proposed as an effective strategy to decrease intracellular glucocorticoid levels without affecting circulating glucocorticoid levels that are essential for stress responses. The clinical development of selective and potent drugs has therefore become a priority. In this research, a process of virtual screening employing the novel algorithm UFSRAT (Ultra Fast Shape Recognition with Atom Types) was used to discover compounds which had specific physicochemical and spatial atomic parameters deemed essential for inhibition of 11β-HSD1. The top scoring compounds were assayed for inhibitory activity against recombinant human and mouse enzyme, using a fluorescence spectroscopy approach. In addition, HEK-293 cell based assays with either human, mouse or rat enzymes were carried out using a scintillation proximity assay (SPA). The most potent compound competitively inhibited human 11β-HSD1 with a Kiapp value of 51 nM. Recombinant mouse and human enzyme were expressed, purified and characterised and used in a series of ligand binding assays. Further to this, an X-ray crystal structure of mouse 11β-HSD1 in complex with a tight binding inhibitor – carbenoxolone was solved.

Pathogenic bacteria pose serious threats to public health and safety. They can cause illness, death, and substantial economic losses. The most widely used bacterial detection methods include cell culturing, antibody-based assays, and nucleic acid amplification techniques, such as polymerase chain reaction (PCR). Unfortunately, these techniques are not well suited for point-of-care application, especially in the resource-limited regions of the world, as they require highly trained personnel to perform the test, they take a long time to complete (especially culturing), and they require sophisticated lab equipment. Thus, there is a great need for simpler, faster, and more accurate methods for bacterial detection. In this thesis, we present a simple, low-cost assay for detecting pathogenic bacteria that is based on the immobilization of a bacteria-specific RNA-cleaving DNAzyme (DNAzyme) onto a surface. If the target bacteria is present, a fluorescently labelled piece of DNA (FDNA) is released through the activity of the DNAzyme; if the target bacteria is not present, the FDNA remains attached to the surface as part of the DNAzyme construct. This method allows untrained users to determine whether a target bacteria is present by simply monitoring the fluorescence intensity in the liquid phase with a hand-held fluorimeter. The first step in this work was to experimentally evaluate different surfaces (including reduced graphene oxide and different beads) onto which the DNAzyme could be immobilized. These tests determined that agarose beads, covered with streptavidin, were ideally suited for DNAzyme immobilization. Next, we conducted a comparative evaluation of the kinetics/activity of the DNAzyme that had been immobilized onto the beads and the free DNAzyme in solution; the results of this evaluation revealed virtually identical reaction rates for the two cases, suggesting no loss of activity after immobilization. Finally, we explored how the DNAzyme sequence length influenced the assay. Specifically, we analyzed a full-length DNAzyme (Full DNAzyme) sequence and a truncated alternative (Short DNAzyme) and found that the full-length construct resulted in faster signal generation. Therefore, it was determined that the long version should be used in the assays. When coupled with a filtration step, the immobilization of biotinylated DNAzymes onto the surface of streptavidin-coated agarose beads enabled the sensitive detection of E. coli in both water samples and complex matrices, such as milk and apple juice. The bead-based assay was able to produce a strong fluorescence signal readout in as little as 2.5 min following contact with E. coli, and it was capable of achieving a detection limit of 1,000 colony-forming units (CFUs) without sample enrichment. As DNAzyme probes can be generated through in vitro selection to react to different bacteria, the RNA-cleavage based detection mechanism described in this work can be adapted for the detection of a wide range of bacterial targets. Overall, this research has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that is highly attractive for field applications, especially in resource-limited regions. / Thesis / Master of Applied Science (MASc)

RNAs have become increasingly recognized as possible drug targets due to their involvement in important biochemical functions, as well as their unique but well-defined structures. Recently published crystal structures depict the binding of a series of aminoglycosides- or more specifically- 2-deoxystretamine (2-DOS), the most preserved central scaffold of aminoglycosides, to a conserved 5'-GU-3'region on their target RNAs. A novel unnatural γ-amino acid, 1, has been synthesized using 2-deoxystreptamine as a template through structure-based rational design. The unnatural amino acid has been designed to replace a glycosidic linkage with an amide bond, which may limit the promiscuous binding characteristics of aminoglycosides through increased rigidity of the ligands and additional hydrogen bonding. The binding selectivity and affinity will be studied in the future through a fluorescence assay.

Kronisk parodontit orsakar vävnadsdestruktion till följd av matrixmetalloproteinasaktivitet. Dessa enzym härrör från värdcellerna och är en del av det immunologiska svaret på bakteriella virulensfaktorer. Grönt te har studerats för sina hälsofrämjande egenskaper, som omfattar bland annat anti-inflammatoriska effekter. Effekten beror delvis på enzyminhibering av tepolyfenoler. Syftet med denna studie var att ytterligare undersöka den inhiberande effekten av grönt te, med fokus på enzymatisk aktivitet i gingivalvätska från patienter med parodontal sjukdom. Patienter med kronisk parodontit valdes ut för att delta i studien. Gingivalvätska extraherades med mikropipetter från patienternas gingivala sulci. Proverna behandlades med grönt te och jämfördes med obehandlade prover från samma försöksperson. Fluorescens proteasanalys med kasein som substrat utfördes på fjorton prover för att detektera skillnader i kaseinolytisk aktivitet. Zymogramanalys med användning av gelatin som substrat utfördes på fyra prover, för att undersöka skillnader i gelatinolytisk aktivitet och analysera molekylvikter för de olika enzymerna. Den fluorometriska analysen visade en signifikant lägre enzymaktivitet i prover med tillsatt grönt te jämfört med obehandlade prover (p<0.001). Zymogramanalysen visade en skillnad i enzymaktivitet som var mest uttalad i banden för molekylär vikt runt 255 kDa, analogt med komplex av matrixmetalloproteinas-9. Sammanfattningsvis har det i denna studie påvisats att grönt te har en hämmande effekt på kaseinolytisk aktivitet och en mindre, mer specifik, hämmande effekt på gelatinasaktivitet. / Chronic periodontitis involves tissue destruction by matrix metalloproteinase, derived from the host cells, as part of the immunological response to bacterial virulence factors. Green tea has been studied for its health promoting properties, which includes anti-inflammatory effects. The effect is in part due to enzyme inhibition by tea polyphenols. The aim of this study was to further investigate the inhibitory effect of green tea, focusing on enzymatic activity in gingival crevicular fluid from patients with periodontal disease. Patients with chronic periodontitis were selected for participation in the study. Gingival crevicular fluid was extracted with micropipettes from the gingival sulci of the patients. Samples were treated with green tea and compared with untreated samples from the same subject. Fluorescence protease assay with casein as substrate was made using fourteen samples for detecting differences in caseinolytic activity. Zymogram assay using gelatin as substrate was done using four samples to test gelatinolytic activity and analyse molecular weights of the different enzymes. The fluorometric assay showed a significantly lower enzyme activity in samples mixed with green tea than untreated samples (p<0.001). The zymogram assay showed a difference in band strength which was most pronounced in the bands of molecular weight around 255 kDA, analogous to complexes of matrix metalloproteinase-9. In conclusion, green tea has been shown in this study to have a strong inhibitory effect on caseinolytic activity and a lesser, more specific, inhibitory effect on gelatinase activity.

Micro RNA sind nicht-kodierende dsRNA ~22 Nukleotiden lang, die eine wichtige Rolle in der Entwicklung und Regulation in beinahe allen Eukaryoten spielen. MiRNAs binden target mRNA, was zu einer Blockierung der Proteintranslation führt. Viele Krankheiten sind bekannt, die durch veränderte miRNA Expressionsmuster entweder beeinflusst oder verursacht werden. Demnach könnte eine Manipulation der miRNA-Bildung einen therapeutischen Ansatz darstellen. MiRNA werden im Zytoplasma von längeren haarnadelförmigen prekursor RNA (pre-miRNA) durch das Enzym Dicer freigsetzt. Inhibition dieser Spaltung könnte durch spezifische pre-miRNA-bindende Moleküle erfolgen. Selektive Binder der pre-miRNA als Inhibitoren der miRNA-Reifung können durch testen von Substanzbibliotheken mit Hochdurchsatzscreening (HTS) gefunden werden. Diese Arbeit beschreibt den ersten homogenen Assay der miRNA-Reifung. Eine Fluoreszenzsonde in Form einer pre-miRNA wurde benutzt, die einen 5´-Fluorophor (FAM, Cy3, oder TMR) und einen 3´-Quencher (DABCYL) aufweist. Durch die nahe Nachbarschaft von Fluorophor und Quencher in der nativen Haarnadelstruktur erfolgt Fluoreszenzlöschung. Dicer spaltet diese Struktur effizient, was zur Dissoziation von Fluorophor und Quencher und somit zu einem Fluoreszenzanstieg führt. Der Assay wurde für HTS optimiert. Die ersten Verbindungen wurden auf deren Inhibition der miRNA-Reifung getestet. Mit einem Duplexassay, wobei zwei unterschiedliche pre-miRNA Sonden mit verschiedenen Fluorophoren eingesetzt wurden, konnte etwas spezifische Inhibition gezeigt werden. Der Assay wurde in Zellen durchgeführt und der Fluoreszenzanstieg mit Fluoreszenzmikroskopie detektiert. Somit ist ein Zell-basiertes Screening von Inhibitoren möglich. Eine einfachere Synthese der Sonde mittels in vitro Transkription und anschließender enzymatischen Ligation wurde entwickelt. Verwendung des Assays um hoch selective Inhibitoren der miRNA-Reifung zu entdecken könnte zu therapeutischen Ansätzen führen. / Micro RNAs (miRNAs) are non-coding dsRNAs of ~22 nucleotides that play a vital role in development and regulation in nearly all eukaryotes. MiRNAs bind target mRNA, thus blocking protein translation. Many diseases have been found to be influenced or caused by aberrant expression of miRNAs. A manipulation of miRNA formation may have therapeutic potential. MiRNAs are cleaved from longer hairpin precursor RNA (pre-miRNA) in the cytoplasm by the enzyme Dicer. It might be possible to inhibit this cleavage through specific pre-miRNA binding molecules. Selective binders of pre-miRNA as inhibitors of miRNA maturation can be found by testing large libraries of substances through high throughput screening (HTS) using an appropriate assay. This work describes the first homogenous assay of miRNA maturation. A fluorescent probe in the form of a pre-miRNA containing a 5´-fluorophore (FAM, Cy3, or TMR) and a 3´-quencher (DABCYL) was used. This ‘beacon’ in its native hairpin formation brings the fluorophore and quencher moieties into close proximity, resulting in fluorescence quenching. Dicer efficiently cleaves this structure, leading to dissociation of fluorophore and quencher and thus a fluorescence increase. In the presence of an RNA ligand that blocks cleavage, a lower fluorescence increase is seen. The assay was optimized for HTS. The first compounds were tested for their inhibition of miRNA maturation. Using a duplex assay, with two different pre-miRNA probes each containing a different fluorogenic group, some specific inhibition was shown. The assay was performed in cells using fluorescence microscopy to measure the fluorescence. This would allow for a cell-based screening of inhibitors. A simpler approach of beacon synthesis using in vitro transcription followed by enzymatic ligation was also established. Use of this assay to discover highly selective inhibitors of miRNA maturation may lead to disease therapeutics.

Yeast plasma membrane transporters play crucial roles in many cellular processes, including detoxification and build-up and maintenance of the plasma membrane potential (ΔΨ). The former development of the diS-C3(3) fluorescence assay by the Biophysics Group of the Institute of Physics, Charles University, enabled us to conveniently study both, including their changes, using a simple fluorescent probe diS-C3(3). Many studies carried out on both animal and yeast cells have revealed that ethanol and other alcohols inhibit the functions of various membrane channels, receptors and solute transport proteins, and a direct interaction of alcohols with these membrane proteins has been proposed. Using the diS- C3(3) assay for multidrug-resistance pump inhibitors in a set of isogenic yeast pdr5 and snq2 deletion mutants we found that n-alcohols (from ethanol to hexanol) exhibit an inhibitory effect on both pumps, increasing with the length of the alcohol carbon chain. The inhibition is not connected with loss of plasma membrane structural or functional integrity and is fully reversible. This supports a notion that the inhibitory action does not necessarily involve only changes in the lipid matrix of the membrane but may entail a direct interaction of the alcohols with the pump proteins. Tok1p is a highly specific...

Under flera månaders tid har ett kandidatexamensarbete utförts med syftet att optimera produktionen av protein-nanofibrer av vassleproteinisolat. Vassleproteinisolat består till stor del av proteinet β-laktoglobulin. Detta protein kan under upphettning bilda nanofibrer i sur miljö. Det var därför med avseende på parametrarna värme, koncentration och inkubationstid som processen optimerades eftersom det redan existerar ett pH-optimum vid pH-värdet 2. Lösningar av vassleproteinisolat med olika koncentrationer inkuberades under 24 timmar vid fyra olika temperaturer. Samtliga lösningar hade pH-värdet 2. För varje temperatur och inkubering togs proverna ut en åt gången för att sedan analyseras. De olika proverna analyserades sedan med Thioflavin T fluorescens för att se indikationer på fibrillering. De erhållna ThT spektrumen visade på fibrillbildning och resultaten för detta experiment visar på att utbytet av fibrilleringsreaktionen blir högre i takt med att hydrolysens hastighetskonstant blir lägre samt att lägre temperaturer kan gynna fibrillbildning . Ytterligare försök, tid och resurser bör läggas ner på detta område för att med säkerhet kunna optimera produktionen av nanofibrer av vassleproteinisolat. / This bachelor’s degree project’s aim was to optimize the production of protein nanofibrils originating from whey protein isolate. Whey protein isolate largely consists of the protein β-lactoglobulin, which can form nanofibrils while immersed in an acidic environment when heated. Therefore, the process was attempted to be optimized with regards to the yield of the final product of protein nanofibrils by varying parameters such as incubation time, initial concentration and temperature, with a constant pH-value of 2. Solutions of the whey protein isolate at different concentrations were incubated during a time interval of 24 hours and at different temperatures. For every temperature and time period of incubation, one sample at a time was taken out to be measured and analyzed, a total of four samples per initial concentration. The samples were analyzed with Thioflavin T fluorescence to see indications of the existence of fibrillation. The obtained ThT spectra showed intensity diagrams that can be related to the amount of formed nanofibrils, and this experiment shows that the yield of fibrils increases while the rate constant of the hydrolysis decreases, and that the fibrillation is favoured by lower temperatures. To optimize the production of nanofibrils of whey protein with certainty, further experiments, time and resources should be invested in this area.