Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: When in close contact with the Yellow Dog Tick, Haemaphysalis leachi, dogs show clear signs of disgust or even nausea. It is accepted that the secretion is produced by the tick in order to prevent the dog from removing the tick from its body with its teeth, thereby increasing the chances for the tick's survival. The composition of this secretion was studied in order to identify the chemical compounds responsible for the repellent action of the secretion. Because of the dog's keen sense of smell, the tick only needs to produce minute quantities of this repellant, which significantly complicated the detection of the different components by conventional GC-methods. Several sampling and sample enrichment methods were explored, including solvent extraction, SPME sample enrichment, adsorption on active charcoal, cryoprecipitation, sorption in a phasecoated open tubular trap, as well as solventless sampling, in order to determine which method would ensure an appropriate amount of sample for gas chromatographic detection. Two of these sampling methods yielded acceptable results: The first method consisted of rinsing irritated female ticks with dichloromethane and, after concentrating the sample by evaporation, GC-MS analysis using normal splitless injection. The second method entailed collecting secretions by wiping irritated ticks with glass micro fibre filter paper after which the paper was inserted directly into the inlet liner of the GCMS system, where thermal desorption of the volatile constituents of the secretion preceded gas chromatographic separation and mass spectral detection. Live dogs were needed for the evaluation of the isolated material to determine whether the samples had an aversive effect on them. The extract was separated into different fractions, which were further separated into subfractions. After each separation process, all the fractions were tested for efficacy in order to determine which fraction contained the active components, narrowing down the number of candidate target compounds. It was concluded that a combination of aldehydes, namely hexanal, heptanal, octanal, nonanal, decanal, undecanal and dodecanal, is responsible for the dog repelling action of the secretion. In most cases the semiochemicals of insects and mammals are secreted in a more complex matrix to ensure prolonged activity. For this reason some of the other compounds in the complex cuticular secretion of this tick species were also identified during the course of this study. / AFRIKAANSE OPSOMMING: Honde toon duidelike tekens van afkeer of selfs naarheid wanneer hulle in noue kontak kom met die afskeiding van 'n sekere bosluisspesie, die geelhondebosluis, Haemaphysalis leachi. Daar word aangeneem dat die bosluis die afskeiding produseer om te verhoed dat die hond dit met sy bek van sy liggaam verwyder. Hierdeur word die oorlewingskanse van die bosluis verbeter. 'n Studie is gemaak van die samestelling van die vlugtige komponente van hierdie afskeiding om die chemiese verbindings, wat verantwoordelik is vir die afweer van honde, te identifiseer. As gevolg van die sensitiwiteit van 'n hond se reuksintuig, is dit vir die bosluis nodig om slegs uiters klein kwantiteite van hierdie afweerstof af te skei, wat die waarneming van die verskillende komponente deur middel van konvensionele GC-metodes baie bemoeilik het. Ten einde die mees effektiewe metode te vind wat 'n gepaste hoeveelheid monster vir waarneming op 'n gaschromatograafdetektor sou verseker, is verskeie monsternemings en -verrykingsmetodes ondersoek, naamlik oplosmiddelekstraksie, SPME-monsterverrykingsmetodes, adsorpsie op aktiewe koolstof, kriopresipitasie, sorpsie in 'n fase-belaagde oopbuisval en oplosmiddellose monsterneming, Twee monsternemingsmetodes het aanvaarbare resultate gelewer: Met die eerste metode is geïrriteerde wyfiebosluise met dichlorometaan afgespoel en die ekstrak is na indamping met behulp van monsterinspuiting sonder inlaatstroomverdeling deur middel van GC-MS geanaliseer. Met die tweede metode is die afskeiding van geïrriteerde bosluise met mikroglasveselpapier afgevee en die papier is direk in die binnebuis van die inlaat van die GC-MS-sisteem geplaas, waar die vlugtige komponente termies gedesorbeer is vir gaschromatografiese skeiding en massaspektrometriese waarneming. Om die aktiwiteit van die geïsoleerde materiaal te evalueer, is van lewende honde gebruik gemaak, om vas te stel of hulle aversie teenoor die betrokke monsters toon. Die ekstrak is in verskillende fraksies geskei, wat weer in subfraksies verder geskei is. Alle fraksies is na elke skeidingsproses getoets vir effektiwiteit om vas te stel watter van die fraksies die aktiewe verbindings bevat. Sodoende is die aantal moontlikhede vir die teikenverbindings met elke skeidingstap verminder. Daar is gevind dat 'n reeks aldehiede, naamlik heksanaal, heptanaal, oktanaal, dekanaal, undekanaal en dodekanaal, gesamentlik verantwoordelik is vir die afweer van honde. Insekte en soogdiere skei dikwels semioverbindings in 'n draermateriaal af om daardeur meer langdurige werking te verseker. In hierdie ondersoek is dus ook 'n begin gemaak met die identifisering van die ander verbindings wat in die besonder komplekse kutikulêre afskeiding van hierdie bosluisspesie aanwesig is.

Thesis (PhD)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: The black wildebeest, Connochaetes gnou, is a terriorial animal and although it is not generally accepted, it is believed that it defines its territory by scent marking, using interdigital and preorbital secretions, faeces, and urine. The aim of this study was to characterize the chemical constituents of the interdigital secretion. Due to the complexity of the secretion, only one hundred and ten of the approximately 350 compounds could be determined with known techniques. Gas chromatography, low resolution GC-MS and retention-time comparison were the main analytical techniques used. Classes of compounds identified in the interdigital secretion include the following: • Hydrocarbons - Aliphatic (saturated and unsaturated) and aromatic • Alcohols - Aliphatic (saturated, unsaturated, cyclic and diols) • Phenols and Phenylalkanols • Aldehydes - Aliphatic (saturated and unsaturated) and aromatic • Ketones - Aliphatic (saturated, unsaturated, cyclic and diketones) and aromatic • Hydroxy ketones - Aliphatic and cyclic • Carboxylic acids - Aliphatic (saturated, unsaturated and cyclic) and aromatic • An anhydride • Esters - Methyl esters, ethyl and higher esters, unsaturated esters and aromatic esters • Lactams • A steroid Only small qualitative and quantitative differences were found between the male and female interdigital secretions. / AFRIKAANSE OPSOMMING: Die swartwildebees, Connochaetes gnou, is 'n territoriale dier en alhoewel dit nie algemeen aanvaar word nie, word vermoed dat hierdie bokke hul gebied afbaken met behulp van interdigitale en preorbitale afskeidings, en deur faeces en urine. Die doel van hierdie studie was om die chemiese samestelling van die interdigitale afskeiding te karakteriseer. As gevolg van die kompleksiteit van die afskeiding, kon slegs eenhonderd-en-tien van die ongeveer 350 verbindings met bekende bestaande tegnieke geïdentifiseer word. Gaschromatografie, lae resolusie GC-MS en retensietyd-vergelyking was die belangrikste analitiese tegnieke wat gebruik is. Klasse van verbindings wat bepaal is, sluit die volgende in: • Koolwaterstowwe - Alifaties (versadig en onversadig) en aromaties • Alkohole - Alifaties (versadig, onversadig, siklies en diole) • Fenole en Fenielalkanole • Aldehiede - Alifaties (versadig en onversadig) en aromaties • Ketone - Alifaties (versadig, onversadig, siklies en diketone) en aromaties • Hidroksiketone - Alifaties en siklies • Karboksielsure - Alifaties (versadig, onversadig en siklies) en aromaties • 'n Anhidried • Esters - Metiel esters, etiel en hoër esters, onversadigde esters en aromatiese esters • Laktame • 'n Steroïed Slegs klein kwalitatiewe en kwantitatiewe verskille is gevind tussen die bul en koei interdigitale afskeidings.

As repetições ricas em leucina (LRR) são domínios presentes em diversas famílias de proteínas com diferentes funções, sendo responsáveis pela formação de uma estrutura capaz de estabelecer interações protéicas. Em decorrência do projeto de caracterização de um segmento do cromossomo 17 de L. amazonensis, identificamos um gene que codifica uma proteína de 72 kDa, contendo em sua região central, seis motivos LRR. Genes ortólogos estão presentes nos genomas de L. major e L. braziliensis. Observamos que o gene LRR17 é regulado de forma distinta ao longo dos ciclos biológicos de L. braziliensis e L. amazonensis. A proteína LRR17 de L. braziliensis e secretada tanto nos estágios promastigota como amastigota. Identificamos também a secreção da proteína LRR17 em promastigotas de L. amazonensis. Obtivemos mutantes hiperexpressores da proteína LRR17 em L. braziliensis e L. amazonensis. As linhagens mutantes foram mais infectivas em infecções de macrófagos in vitro quando comparadas com a linhagem selvagem. A proteína LRR17 parece estar envolvida no processo de invasão do parasita em infecções in vitro e no estabelecimento da infecção da forma amastigota de Leishmania. / Leucine-rich repeats (LRRs) are versatile binding motifs found in a variety of proteins involved in protein-protein interactions. The LaLRR17 gene, identified initially in the L. amazonensis genome, encodes a protein with 6 LRR in its central region, that is secreted to the cytoplasm of L. amazonensis-infected macrophages. An orthologue to LaLRR17 was identified in L. braziliensis chromosome 17. LRR17 gene expression is regulated differentially during the life cycle of L. braziliensis and L. amazonensis. The LbLRR17 protein is secreted in L. braziliensis promastigotes and amastigotes. To characterize the function of the LRR17 protein we obtained transgenic parasite lines of L. amazonensis overexpressing the LaLRR17 gene and of L. braziliensis overexpressing the LbLRR17 gene. The mutants were more infective to macrophages in vitro when compared with the wild type strains, indicating that the LRR17 protein may interact with macrophage molecules, modulating the cellular response to increase parasite survival.

As repetições ricas em leucina (LRR) são domínios presentes em diversas famílias de proteínas com diferentes funções, sendo responsáveis pela formação de uma estrutura capaz de estabelecer interações protéicas. Em decorrência do projeto de caracterização de um segmento do cromossomo 17 de L. amazonensis, identificamos um gene que codifica uma proteína de 72 kDa, contendo em sua região central, seis motivos LRR. Genes ortólogos estão presentes nos genomas de L. major e L. braziliensis. Observamos que o gene LRR17 é regulado de forma distinta ao longo dos ciclos biológicos de L. braziliensis e L. amazonensis. A proteína LRR17 de L. braziliensis e secretada tanto nos estágios promastigota como amastigota. Identificamos também a secreção da proteína LRR17 em promastigotas de L. amazonensis. Obtivemos mutantes hiperexpressores da proteína LRR17 em L. braziliensis e L. amazonensis. As linhagens mutantes foram mais infectivas em infecções de macrófagos in vitro quando comparadas com a linhagem selvagem. A proteína LRR17 parece estar envolvida no processo de invasão do parasita em infecções in vitro e no estabelecimento da infecção da forma amastigota de Leishmania. / Leucine-rich repeats (LRRs) are versatile binding motifs found in a variety of proteins involved in protein-protein interactions. The LaLRR17 gene, identified initially in the L. amazonensis genome, encodes a protein with 6 LRR in its central region, that is secreted to the cytoplasm of L. amazonensis-infected macrophages. An orthologue to LaLRR17 was identified in L. braziliensis chromosome 17. LRR17 gene expression is regulated differentially during the life cycle of L. braziliensis and L. amazonensis. The LbLRR17 protein is secreted in L. braziliensis promastigotes and amastigotes. To characterize the function of the LRR17 protein we obtained transgenic parasite lines of L. amazonensis overexpressing the LaLRR17 gene and of L. braziliensis overexpressing the LbLRR17 gene. The mutants were more infective to macrophages in vitro when compared with the wild type strains, indicating that the LRR17 protein may interact with macrophage molecules, modulating the cellular response to increase parasite survival.