M.Sc. (Botany) / Please refer to full text to view abstract

This diploma thesis deals with acidic proteins contained in the potato (Solanum tuberosum) tubers, or rather about their separation with chromatography techniques. For the analysis were chosen tubers of two czech potato varieties, Adéla and Westamyl. From the concentrated potato juice were eliminated basic and most of the patatin proteins with the gravity column chromatography. In the next step were applied the chromatography techniques on the anion and hydroxyapatite columns by the fast protein liquid chromatography (FPLC) system. On the anion column were separated protein fractions which contained proteins of molecular weight in the range of 15 kDa to 60 kDa (Adéla) and 15 kDa to 100 kDa (Westamyl). The most abundant were the proteins with molecular weight 15 kDa a 20 kDa. In the last step was used the mass spectrometry for the identification of chosen protein fractions.

A defining feature of the non-flying mammal pollinated (NMP) syndrome is inflorescence crypsis whereby flowers are close to the ground and somewhat hidden within the canopy. A number of species in the Cape Proteaceae are NMP, two of which were chosen as focal species for this study: Protea amplexicaulis and Protea humiflora. This study investigated the two previously suggested hypotheses for crypsis: hidden flowers are more difficult for nectarivorous birds to access, or hidden flowers provide greater cover for small mammal pollinators from aerial predators. Using remote triggered cameras, P. amplexicaulis and P. humiflora inflorescences were observed over the 2017 flowering period, noting visitation by birds and small mammals and assessing the legitimacy of birds as pollinators. In the literature, bird visitation to exposed inflorescences is suggested to be rare, but this study showed that it is considerable. Observations of camera footage suggest that birds are in fact illegitimate pollinators and thus nectar rob. Bird visitation to exposed inflorescences was more than tenfold that of hidden inflorescences, suggesting that crypsis is likely a strategy to avoid nectar robbing by birds. Both P. amplexicaulis and P. humiflora have been observed to retain dead leaves, which may contribute to their cryptic nature. Alternative hypotheses for dead leaf retention in Proteaceae – that it may increase flammability or result in a below canopy spike in nutrients post fire (selfish fertilization) – were assessed and rejected. Sampling of eight local Protea species showed that dead leaf retention is not a consequence of prolonged live leaf retention, with P. amplexicaulis retaining dead leaves for up to 6 years. The removal of dead leaves in 30 P. amplexicaulis individuals resulted in a significant decrease in the number of inflorescences hidden from aerial view, thus suggesting that dead leaf retention may be a strategy to enhance crypsis and thus forms part of the NMP syndrome. This research expands on the knowledge of the NMP syndrome; providing evidence in support of an anti- nectar robbing crypsis function, discovering a novel crypsis adaptation regarding dead leaf retention, and casting doubt on the Restricted Distributions hypothesis for the evolution of the syndrome.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The commercial cultivation of Proteaceae is an important industry in the Western Cape, however, farmers are challenged with arthropod infestation which compels them to solely rely on chemical pesticides. Past studies in South Africa have shown that Proteaceae comprise a rich and diverse arthropod fauna. However, as most of these studies were conducted on wild Proteaceae, they may not be representative of cultivated proteas. Moreover, most of these species remained unidentified due to lack of identification expertise. These past studies, however, form a useful baseline for arthropod studies in proteas, e.g. the feeding guilds found in proteas. The aim of this research was to conduct an intensive and extensive survey of the arthropod-fauna associated with commercially-cultivated proteas across an entire year. Specifically, this survey was designed to document the composition of the arthropod fauna (creating a comprehensive reference collection for pest management purposes) and to assess whether the arthropod fauna differed between seasons and pesticide treatments. Infructescences, inflorescences and foliage of mainly commercial Proteaceae were sampled for arthropods seasonally for a period of twelve months by collection of plant material and direct searching. Seven commercial protea blocks, and a wild protea block (remnant patch of fynbos vegetation), were used as the sampling sites, and two sprayed blocks were used for assessing pesticide efficacy. Individual arthropods were identified as far as possible, with 37% identified to species level. A species accumulation curve showed that rare (minor) arthropod species made up of 70% of arthropods occurring in cultivated proteas. More than 8 700 individuals from more than 140 species and about 80 families were collected and identified, revealing that cultivated proteas have a rich and diverse insect fauna. These arthropods represent the full range of plant-feeding guilds: leaf miners, leaf chewers, flower bud borers, sap suckers and seed feeders. Flower visitors/free living guild was the most abundant (72%) and speciose (25%). In addition to phytophages, there was a large suite of insect predators and parasitoids. A large number of the arthropods were endemic to the Cape Floristic Region (CFR) and some (7.86%) have a pest status, in that they cause significant damage to the protea plants (for example, 60% of Safari sunset cultivar (Leucadendron salignum x L. laureolum) new flush stems and leaves were affected by Epichoristodes acerbella (Tortricidae). Capys alphaeus (Lycaenidae) and Phyllocnistis sp. (Phyllocnistidae) appear to be specialist pests, as they attack mainly Protea cynaroides and Susara cultivar (Protea magnifica x P. susannae) respectively. Arthropod abundance did not differ significantly between seasons, although significant seasonal effects were observed in species richness when the protea cultivars were examined separately. Pesticide application did not affect arthropod abundance, but did decrease species richness in sprayed blocks. Pesticides appeared to negatively affect minor (rare) species disproportionately, probably due to their lack of prior exposure to pesticides and hence sensitivity. Due to this inefficacy of pesticides in cultivated proteas, an increasing emphasis on the importance of non-chemical control measures, and our improved knowledge of the predatory and parasitic species in this system, integrated pest management strategies deserve greater research attention. Monitoring and use of threshold values for arthropod pests were suggested here, as well as the use of biological, cultural, physical and chemical (optimal use) control. For instance, in cultural control, polycropping and intercropping in proteas to increase plant diversity in the monocultures to promote a higher density of predators and parasitoids can be used. Certain flowering plants are known to provide greater temporal and spatial distribution of nectar and pollen sources, which can increase parasitoid reproductive potential and abundance of alternative hosts/prey when the pest species are scarce or at an inappropriate stage. / AFRIKAANSE OPSOMMING: Die kommersiële verbouing van Proteaceae (proteas) is 'n belangrike bedryf in die Wes-Kaap. Menige plantasie wemel egter van artropodes, wat boere noop om slegs van chemiese plaagdoders gebruik te maak. Vorige studies in Suid-Afrika toon dat proteas die gasheerplant vir 'n ryke en diverse artropodefauna is. Aangesien die meeste van hierdie studies egter op wilde proteas uitgevoer is, weerspieël dit moontlik nie die stand van sake met verboude proteas nie. Weens 'n gebrek aan kundigheid om die artropodes te eien word baie van die spesies boonop nooit uitgeken nie. Dié studies voorsien egter 'n nuttige grondlyn vir 'n ondersoek na die artropodes op proteas, veral vir die bestudering van die gilde wat van die protea leef (“the feeding guild”). Hierdie navorsing het ten doel om 'n intensiewe en omvattende opname te maak van die artropodefauna wat oor die tydperk van 'n jaar op kommersieel verboude proteas voorkom. Die opname is meer bepaald ontwerp om die samestelling van die artropodefauna te bestudeer (deur 'n omvattende verwysingsversameling vir plaagbestuurdoeleindes te skep), en om vas te stel of seisoene en plaagbehandelings enige beduidende uitwerking op die artropodefauna het. Oor 'n tydperk van 12 maande is seisoenale monsters van die vrug- en bloeistadia, saadkoppe en blare van hoofsaaklik kommersiële proteas gesoek en ingesamel. Sewe kommersiële proteablokke sowel as 'n blok wilde proteas het as proefpersele gedien, en twee bespuite blokke is gebruik om die doeltreffendheid van plaagdoder te beoordeel. Individuele artropodes is so noukeurig moontlik uitgeken – 37% tot op spesievlak. Volgens 'n spesieakkumulasiekurwe maak seldsame (kleiner) artropodespesies sowat 70% van die artropodes uit wat op verboude proteas voorkom. Die meer as 8 700 individue van meer as 140 spesies en sowat 80 families wat ingesamel en uitgeken is, toon die rykheid en diversiteit van die artropodefauna op verboude proteas. Hierdie artropodes verteenwoordig die volle reeks plantvreterspesies – van blaardelwers en blaarkouers tot blomknopboorders, sapsuiers en saadvreters. Blombesoeker-/vrylewende spesies was die volopste (72%) en mees divers (25%). Buiten plantvreters was daar ook 'n groot aantal roofinsekte en parasitoïede. Baie van die artropodes was inheems, en sommige (7,86%) het boonop plaagstatus, aangesien hulle beduidende skade aan die proteaplant aanrig. [By ongeveer 60% van die Safari Sunset-kultivar (Leucadendron salignum x L. laureolum) is nuwe stamme en blare byvoorbeeld deur die Epichoristodes acerbella (Tortricidae) aangetas.] Capys alphaeus (Lycaenidae) en Phyllocnistis sp. (Phyllocnistidae) blyk spesialisplae te wees wat onderskeidelik hoofsaaklik die Protea cynaroides en die Susarakultivar (Protea magnifica x P. susannae) in die visier het. Artropodegetalle het nie juis tussen seisoene gewissel nie, hoewel 'n afsonderlike ondersoek van die proteakultivars 'n beduidende seisoenale uitwerking op spesierykheid aan die lig gebring het. Eweneens het die toediening van plaagdoder nie die artropodegetalle verminder nie, maar wel spesierykheid op die bespuite blokke verswak. Plaagdoders blyk besonder negatiewe uitwerking op kleiner (seldsame) spesies te hê – waarskynlik omdat dié spesies nie voorheen aan plaagdoders blootgestel was nie, en dus gevoelig is daarvoor. Weens die oënskynlike ondoeltreffendheid van plaagdoders op verboude proteas, verg 'n toenemende klem op die belang van niechemiese beheermaatreëls, 'n behoefte aan meer kennis van die roof- en parasitiese spesies in die stelsel, en die vraag na geïntegreerde plaagbeheerstrategieë, meer navorsing. Die studie moniteer en gebruik drempelwaardes vir artropodeplae, sowel as biologiese, kulturele, fisiese én chemiese (‘optimalegebruik’-) plaagbeheer. Met kulturele beheer kan poli- en interverbouing van proteas byvoorbeeld gebruik word om plantdiversiteit in die monokulture te verbeter, ten einde só 'n hoër digtheid van roofspesies en parasitoïede in die hand te werk. Sekere blomplante bied kenmerkend 'n wyer tyd- en ruimtelike verspreiding van nektar- en stuifmeelbronne, wat parasitoïede se voortplantingsvermoë en die getalle van alternatiewe gashere/prooi kan verbeter wanneer die plaagspesies skaars is of in 'n ontoepaslike stadium verkeer.

The liver fluke, Fasciola hepatica, is one of the most important parasites of livestock, and it also infects humans. The proteolytic system of trematodes is critical for their interaction with the host and is a potential target for the development of novel vaccines. This work is focused on proteases secreted by F. hepatica adults and on FheCy2, a new protease inhibitor from the cystatin family. The proteolytic activity of the secreted proteases was analyzed using: (a) chromogenic protein substrates and fluorogenic peptide substrates, (b) selective protease inhibitors, and (c) a fluorescent activity-based probe for visualization of proteases. The results showed that the secreted proteases are cysteine proteases of papain family belonging to cathepsins L and B. These proteases were effectivelly inhibited by FheCy2 as demonstrated by enzymological analysis. It can be assumed that FheCy2 participates in the physiological regulation of endogenous proteases secreted by F. hepatica adults, which makes it attractive candidate protein for vaccination studies. Key words: Fasciola hepatica, cathepsins, proteolytic activity, substrate specificity, protease inhibitors (In Czech)

Introduktion COVID-19 orsakas av viruset SARS-CoV-2. 3-chymotrypsin-liknande cysteinproteaset (3CLpro) är ett enzym som styr coronavirus replikation och är viktigt för viruset livscykel. Därför 3CL pro är en viktig måltavla för att hämma SARS-CoV-2. Syfte Syftet med detta projekt är att undersöka om proteashämmare kan vara en framkomlig väg för att ta fram antivirala läkemedel mot COVID-19. Metod Arbetet är en litteraturstudie och databasen PubMed har använts för att hitta vetenskapliga artiklar med hjälp av olika sökord, såsom SARS-CoV 2 and protease inhibitors, SARS-CoV-2 and 3CL-pro, MERS-CoV and 3CLpro inhibitors och SARS-CoV and 3CLpro. Totalt valdes 10 vetenskapliga artiklar ut som grund för att undersöka om några proteashämmare finns rapporterade och hur de utvärderats, dvs i prekliniska studier eller kliniska studier. Resultat  Peptidomimetiska alfa-ketoamider är effektiva bredspektrum hämmare för att hämma 3CL pro i SARS-CoV-2 och enterovirus, t.ex. alfa-ketoamiderna (13a och 13b) som även visade goda farmakokinetiska egenskaper i prekliniska studier. Även läkemedelskandidater (11a och 11b) har utvärderats i prekliniska studier och både visade hög SARS-CoV-2 3CL-pro hämmande aktivitet. Två nya studier visade att genomsekvensen för SARS-CoV-2 3CLpro är mycket lik den för SARS-CoV-1 än MERS-CoV. Herbacetin, rhoifolin pectolinarin och Biflavonoid amentoflavon 9 visade en effektiv hämning av SARS-CoV-1 3CL pro. Herbacetin, isobavachalcone, quercetin 3 ‐ β ‐ d ‐ glukosid, helikrysetin och enterovirushämmare (6b, 6c och 6d) visat sig ha hämmande aktivitet och kunde hämma den enzymatiska aktiviteten hos MERS-CoV 3CL pro. De redan framtagna HIV-proteashämmarna lopinavir och ritonavir har utvärderats i kliniska prövningar och både visade ingen skillnad mot standardbehandling samt minskade inte dödlighet. Slutsats Slutsatsen som kan dras av studien är att det finns flera proteashämmare i preklinisk fas mot SARS-COV-2 3CL-pro samt finns flera proteashämmare mot SARS-CoV-1 och MERS-CoV. Proteashämmare kan vara ett möjligt läkemedel mot COVID-19 i framtiden, men det tar lång tid för att utveckla ett nytt läkemedel.

This diploma thesis is concerned on protease inhibitors isolated from potato (Solanum tuberosum L.) tubers and evaluation of their antifungal properties. Theoretical part of the thesis deals with protease inhibitors which have an antifungal effect. Tubers of potato cultivars Adéla, Ornella, Eurostarch - were used for protease inhibitors isolation. Antifungal activity of isolated protein fractions were evaluated versus fungi from genus Rhizoctonia and Fusarium that are important pathogens in agriculture. Their activity was also evaluated statistically.