Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 148-151). Also available in electronic version. Access restricted to campus users.

Thesis (M.S.)--University of Iowa, 2007. / Supervisor: Janet M. Guthmiller. Includes bibliographical references (leaves 68-76).

Thesis (M.S.)--University of Iowa, 2007. / Supervisor: Kim A. Brogden. Includes bibliographical references (leaves 77-88).

Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC-101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC-101 and PG1 accumulated up to 32-38% and 17~26% of the total soluble protein. Both RC-101 and PG1 were cleaved from GFP by corresponding proteases in vitro and Factor Xa like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6 fold higher yield of RC 101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to TMV infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further pre-clinical studies

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Wine quality is greatly influenced by the number of microorganisms, which occur throughout the winemaking process. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are responsible for malolactic fermentation (MLF), while acetic acid bacteria (AAB) are responsible for converting ethanol to acetic acid. These microorganisms are present on the grapes and in the cellar and these consequently serve as gateways to the fermentation tanks where they will affect the wine quality. However, these microorganisms can be seen either as beneficial or as wine spoilage microorganisms, depending on the conditions that prevail throughout the winemaking process. It is thus very important to prevent any process that could lead to the lowering of the wine quality. In this regard, some of the factors that should always be evaluated include the quality of the grapes, winemaking techniques and quality control. One of the measures that have been implemented during winemaking to ensure the microbial stability is the use of chemical preservatives. Sulphur dioxide (502) has been, and is, used widely as primary preservative in winemaking. However, an ever-increasing consumer resistance against the use of chemical preservatives has developed as it poses possible health risks and decreases the sensorial quality of wine. An alternative approach to chemical preservation that has triggered numerous new investigations, is biological preservation or biopreservation. This is the use of the natural microbial flora and/or their antimicrobial products, such as bacteriocins, to inhibit or destroy the other sensitive microorganisms that are unwanted in the same environment. Evidence in the wine industry has shown that bacterial spoilage still is a very common problem in many wineries. This bacterial spoilage can lead to, amongst other, two main problems, which are of great concern to winemakers. This include high levels of volatile acidity, resulting in the wine having a vinegary off-flavour, and sluggish/stuck fermentation, which is the result of compounds such as acetic- and other fatty acids that causes inhibition of the yeast's growth. With acetic acid being the common link in both cases, it became evident that investigations should be performed on the main producer of acetic acid, namely AAB. As a result, AAB turned out to be one of the main spoilage microorganisms associated with winemaking. Most of the research on biopreservation in the food and beverage industry has been performed on the Gram-positive LAB. The fact that their spectrum of inhibition currently excludes most Gram-negative bacteria, specifically AAB, indicated that AAB should be screened in search of possible antimicrobial compounds that could be applied to control their cell numbers during winemaking. No evidence of antimicrobial action amongst AAB could be found in literature, therefore this work was considered novel. The main objectives of this study were to screen wine isolates of AAB for the production of antimicrobial compounds. This was followed by the isolation and preliminary characterisation of the antimicrobial substances produced. Various attempts to optimise the production of the antimicrobial compounds and isolation procedures, were also included. This study forms part of a larger research programme that has been initiated at the Institute for Wine Biotechnology at Stellenbosch University on the biopreservation in wine. Our results indicated that possible antimicrobial compounds of proteinaceous nature, produced by AAB isolated from wine, do exist. It was found that two different species of AAB, namely Acetobacter aeeti and Gluconobacter frateurii, produced antimicrobial compounds that inhibited other species of AAB. Preliminary results indicated that these compounds are heat sensitive and stable in a wide pH range. It was also shown that after the action of proteolytic enzymes, such as proteinase K and a-chemotrypsin, all inhibitory activity was lost. This study also revealed the existence of the species Gluconobacter frateurii, which have not yet been associated with the winemaking environment. This study made a valuable contribution to the limited amount of information and understanding of AAB, not only in the wine environment, but also elsewhere. The results and findings of this research would serve as platform for further projects. This might soon lead to the development of antimicrobial substances or tailored wine-yeasts with antimicrobial abilities, which can be applied during winemaking to assist the winemaker in combatting high cell numbers and subsequent spoilage by AAB. / AFRIKAANSE OPSOMMING: Wynkwaliteit word beïnvloed deur 'n verskeidenheid van mikroorganismes wat regdeur die wynrnaakproses teenwoordig is. Die giste is vir die alkoholiese fermentasie, die melksuurbakterieë (MSB) vir die appelmelksuurgisting, terwyl die asynsuurbakterieë (ASB) vir die omskakeling van etanol na asynsuur verantwoordelik is. AI hierdie mikroorganismes is teenwoordig op die druiwe en in die kelder, en dit dien gevolglik as 'n weg waardeur hulle in die fermentasietenke kan kom om sodoende die wynkwaliteit te beïnvloed. Hierdie mikroorganismes kan egter gesien word as óf voordelig óf as wynbederfmikroorganismes, afhangende van die heersende kondisies gedurende die wynrnaakproses. Dit is daarom baie belangrik om enige proses te voorkom wat tot 'n verlaging in wynkwaliteit kan lei. Wat laasgenoemde aanbetref, is daar sekere faktore wat altyd geëvalueer moet word, naamlik die druifkwaliteit, wynrnaaktegnieke en kwaliteitsbeheer. Een van die maatreëls wat geïmplementeer is om mikrobiologiese stabiliteit tydens die wynrnaakproses te handhaaf, is die gebruik van chemiese preserveermiddels. Swaweidioksied (S02) word algemeen gebruik as primêre preserveermiddel tydens wynrnaak. Daar is egter 'n toenemende verbruikersweerstand teen die gebruik van chemiese preserveermiddels, aangesien dit moontlike gesondheidsrisiko's kan inhou, asook tot 'n verlaging in sensoriese kwaliteit van die wyn kan lei. 'n Alternatiewe benadering vir chemiese preservering, wat reeds tot verskeie nuwe ondersoeke gelei het, is biologiese preservering of biopreservering. Dit is die gebruik van die natuurlike mikroflora en/of hulle antimikrobiese produkte, soos bv. bakteriosiene, om die sensitiewe mikroorganismes wat in dieselfe omgewing voorkom, se groei te inhibeer óf om hulle dood te maak. Aanduidings vanuit die wynbedryf dui daarop dat bakteriese bederf steeds 'n algemene probleem is wat in baie kelders ondervind word. Hierdie bakteriese bederf kan onder andere twee hoofprobleme veroorsaak, wat 'n groot bekommernis vir verskeie wynmakers is. Dié probleme sluit in hoë vlakke van vlugtige suurheid, wat gevolglik die wyn 'n asyn-afgeur gee, en slepende/gestaakte fermentasies, wat die gevolg is van komponente soos asynsuur en ander vetsure, wat die gis se groei inhibeer. Die feit dat asynsuur die gemeenskaplike faktor in beide gevalle was, het daarop gedui dat 'n ondersoek rakende die hoofproduseerder van asynsuur, naamlik ASB, benodig word. ASB word gevolglik as een van die hoofbederforganismes wat met die wynrnaakproses geassosieer word, beskou. Die meeste navorsing oor biopreservering in die voedsel -en drank bedryf is op die Gram-positiewe MSB gedoen. Die spektrum van inhibisie van die bakteriosiene van MSB sluit egter die meeste Gram-negatiewe bakterieë uit, veral ASB, en dit dui daarop dat ASB gesif moet word in 'n soektog na antimikrobiese substanse wat moontlik gebruik kan word om hul getalle tydens die wynrnaakproses te beheer. Geen bewyse kon tot dusver uit die literatuur gekry word met betrekking tot antimikrobiese aktiwiteit teen ASB nie, daarom word hierdie navorsing dus as nuut beskou. Hierdie studie se hoofdoelwittewas om die wyn-isolate van ASB vir die produksie van antimikrobiese peptiede te sif. Dit is gevolg deur die isolasie en voorlopige karakterisering van die geproduseerde antimikrobiese komponente. Daar is ook verskeie pogings aangewend om die produksie van die antimikrobiese substanse, asook die isolasieprosedures, te optimiseer. Hierdie studie vorm deel van 'n groter navorsingsprogram oor biopreservering van wyn wat deur die Instituut vir Wynbiotegnologie by die Universiteit van Stellenbosch geïnisieer is. Die resultate het daarop gedui dat antimikrobiese substanse van proteïenagtige aard, afkomstig vanaf wyn-isolate van ASB, wel bestaan. Daar is gevind dat twee veskillende spesies, naamlik Aeefobaefer aeefi en Glueonobaefer frafeurii, antimikrobiese peptiede produseer, wat ander spesies van ASB kan inhibeer. Voorlopige resultate het getoon dat hierdie substanse hitte-sensitief is en ook stabiel is oor 'n wye pH-reeks. Daar was ook aanduidings dat, ná die aksie van proteolitiese ensieme, soos bv. proteïnase K en a-chemotripsien, al die inhibitoriese aktiwiteit verlore gegaan het. Hierdie studie het ook die voorkoms van die spesies Glueonobaefer frafeurii aangedui, wat nog nie tot dusver met die wynrnaakomgewing geassosieer is nie. Hierdie studie maak 'n waardevolle bydrae tot die beperkte hoeveelheid inligting oor en begrip van ASB, nie net in die wynomgewing nie, maar ook in die algemeen in die natuur. Die bevindinge en resultate van hierdie navorsing sal as basis dien vir verdere projekte wat sal volg. Dit kan moontlik binnekort lei tot die ontwikkeling van antimikrobiese substanse, en ook pasgemaakte wyngiste met antimikrobiese vermoëns, wat tydens die wynrnaakproses gebruik kan word om sodoende die wynmaker in staat te stelom die hoë bakteriese getalle en die gevolglike bederf deur ASB, te beheer.

Objectives: To study the expression of cathelicidin antimicrobial peptides (CAMP) and surfactant protein D (SP-D) in patients with chronic rhinosinusitis (CRS) and eosinophilic mucus chronic rhinosinusitis (EMCRS) and by a nasal explant in vitro model cultured with fungal allergens. Methods: Nasal biopsies from 59 CRS and EMCRS patients, stratified into Allergic fungal sinusitis (AFS), Nonallergic fungal eosinophilic sinusitis (NAFES), and Nonallergic nonfungal eosinophilic sinusitis (NANFES) were studied by quantitative real-time (q)RTPCR, Western blot, immunostaining and ELISA. Nasal tissue from CRS and EMCRS patients were cultured with increasing concentrations of fungal allergens in a nasal explant in vitro model for 24 hours and CAMP and SP-D mRNA and protein levels in response to the fungi were determined by qRT-PCR and ELISA. Results: The expression of CAMP mRNA was significantly increased in EMCRS patients compared to CRS patients (p=0.0004). By immunohistochemistry, expression of CAMP was localised to nasal epithelial, submucosal glands and inflammatory subepithelial cells. Western blotting demonstrated the presence of CAMP in the study patients. Culturing nasal explants with fungal allergens demonstrated significant upregulation of CAMP mRNA expression in CRS, but not EMCRS patients, by Aspergillus (mean 4-fold increase) and Alternaria (mean 6-fold increase) extracts with a significant dose-response effect (p<0.001). CAMP protein levels in the nasal tissue from CRS patients increased in response to Alternaria (p<0.05). In contrast, with EMCRS patients the expression of CAMP peptide in nasal tissue increased with Aspergillus (p<0.001) but decreased with Alternaria. Staining for SP-D was detected in the submucosal glands from the nasal biopsies in all patient groups except for AFS. By ELISA, SP-D was undetectable in AFS and decreased in NAFES, NANFES, and CRS compared to controls. CRS patients cultured with Aspergillus and Alternaria allergens in the in vitro nasal explant model induced significant upregulation of SP-D mRNA (p<0.0001). In contrast, NANFES nasal tissue explants cultured with Aspergillus allergens induced downregulation of SP-D and only a modest upregulation of SP-D mRNA to Alternaria allergens. Conclusion: This study demonstrates expression of cathelicidin antimicrobial peptides and surfactant proteins in nasal mucosa supporting its potential role in innate defences against inhaled pathogens. There is significant upregulation of CAMP mRNA in the EMCRS group implying an increased inflammatory state. In vitro, CAMP is significantly upregulated at the mRNA and protein level in CRS tissue explants to Aspergillus and Alternaria allergens. However, EMCRS tissue cultured with Alternaria in vitro does not demonstrate increased CAMP at the mRNA or protein level. The expression of SP-D in nasal tissue is reported for the first time. SP-D expression in the CRS, but not the EMCRS group, is upregulated in vitro by Aspergillus and Alternaria. The EMCRS group compared to CRS group demonstrate abnormal CAMP and SP-D expression to common fungal allergens. These important findings in understanding the pathogenesis of chronic rhinosinusitis are discussed in this thesis and may provide potential novel therapies for chronic rhinosinusitis in the future. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1287042 / Thesis(PhD)-- School of Medicine, 2007

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: multimerization; antimicrobial peptides; expression. Includes bibliographical references (p. 66-68).

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 194-221) Also available in print.

Thesis (Ph.D.) - University of St Andrews, April 2010.

Nucleic acids are ubiquitous in nature and modified nucleosides are present in a wide range of anti-viral, anti-cancer drugs and antibiotics. Although a variety of naturally occurring nucleoside analogues exist, few include modifications to the ribose or deoxyribose ring. Intriguingly, the uridyl peptide antibiotics (UPAs), such as pacidamycin, contain a biosynthetically unique 3'-deoxyuridine which resembles synthetic anti-retrovirals. Elucidation of the biosynthesis of this structuraly unique nucleoside motif suggests a degree of substrate flexibility, making it a highly attractive prospect for biosynthetic approaches to nucleoside modification. In order to fully exploit the biotransformative potential, a detailed mechanistic understanding of the individual enzymes involved in the biosynthesis of the nucleoside moiety, and especially the enzyme employed at the installation of the 3'-deoxy modification, is required. Chapter 1, the introduction the thesis, discusses the importance of nucleosides for Chemistry and Biology. The section describes the biosynthesis of the nucleoside antibiotics and reviews the recent advances relating to the synthesis and biosynthesis of 3'-deoxy-nucleosides. The Chapter proceeds to describes the biosynthesis of deoxy-sugars, deoxy-nucleosides and nucleotides, reviewing the most common dehydratase mechanisms in addition to examining unusual dehydratases involved in carbohydrate metabolism. Chapter 2, the study of Pac13, the uridine-5'-aldehyde dehydratase of the pacidamyicin nucleoside cluster, is reported. Through detailed functional, structural and kinetic analysis of the wild-type enzyme as well a series of mutants, Chapter 2 provides insight into the mechanism emplyed by this unusual enzyme. Chapter 3 describes the structural and functional analysis of Pac11, the flavin-dependent oxidoreductase of the nucleoside biosynthetic cluster, while Chapter 4 revolves around Pac5, the PLP-dependent aminotransferase. In Chapter 5, the chemical synthesis of fluorinated nucleosides, as probes for exploring the enzymes' mechanism is investigated. Chapter 7 reports the experimental procedures for the research described in this document. The work described in this thesis broadens the understanding of the biosynthesis of deoxy-nucleosides and constitutes the first structural and mechanistic study of the biosynthesis of the biosynthesis of the valuable yet, synthetically challenging 3'-deoxy nucleosides.