Functional Characterisation Of Novel Plasmodium falciparum Proteins And Their Role In Erythrocyte Invasion

Malaria caused by Plasmodium falciparum is responsible for the deaths of hundreds of thousands of children every year, and imparts an overwhelming economic burden on the world’s poorest countries. All symptoms of malaria are caused during the asexual blood-stage of the P. falciparum lifecycle, which is reliant on merozoite invasion of host red blood cells (RBCs). Due to its essentiality for parasite survival, and its exposure to the host immune system, merozoite invasion is an attractive target for the development of antimalarial therapeutics. Merozoite invasion is coordinated by a series of secretory organelles, of which the largest and most well characterised are the rhoptries. Previous studies into rhoptry proteins have been strongly foccused on antigens that are secreted from the rhoptries during invasion; primarily due to their promise as vaccine candidates. As such, very little is known about proteins that coordinate rhoptry biogenesis, structure, or function. Prior to this study the P. falciparum proteins Pf3D7_0210600 and Pf3D7_0405200, hereafter referred to as P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting proteins (PfCERLI) 1 and 2, were largely uncharacterised proteins that previous studies had suggested may play a role in merozoite invasion. We hypothesised that PfCERLI1 and PfCERLI2 were rhoptry proteins that shared an evolutionary relationship and were both essential for merozoite invasion. We aimed to test these hypotheses through bioinformatic analyses, immunofluorescence microscopy, and gene disruption or inducible knockdown. Bioinformatic and phylogenetic analyses showed that cerli1 and cerli2 arose through an ancestral gene duplication of cerli1 that was present in the most recent common ancestor of haematozoa and coccidia. Analysis of the structure of CERLI proteins revealed they possess a conserved motif with the consensus sequence PHISE/DxxP that we have termed PHIS, along with conserved C2 and Pleckstrin homology (PH) domains that are likely ito have a role in membrane association. Using selection linked integration targeted gene disruption (SLI-TGD) we determined that both Pfcerli1 and Pfcerli2 were refractory to gene deletion and likely important for blood-stage growth. To assess their functions, we used an inducible protein knockdown system whereby the addition of glucosamine (GLCN) results in specific mRNA degradation prior to translation. Knockdown of either CERLI1 or CERLI2 resulted in growth inhibition caused by an inability of merozoites to invade RBCs. Immunofluorescnce microscopy and biochemical techniques revealed that both proteins are peripheral membrane proteins that localise to the cytosolic face of the rhoptry bulb. Rhoptry secretion assays showed that knockdown of PfCERLI1, but not PfCERLI2, leads to a defect in the secretion of key rhoptry antigens. By contrast, electron microscopy analysis of rhoptry size indicated a significant increase in rhoptry length following PfCERLI2 knockdown, but no change with PfCERLI1 knockdown. Semi-quantiative super-resolution microscopy analysis determined that knockdown of PfCERLI1 alters rhoptry antigen distribution, and it was shown that both PfCERLI1 and PfCERLI2 knockdown inhibit processing of key rhoptry antigens. The findings of these studies show that the previously uncharacterised proteins, PfCERLI1 and PfCERLI2, are related rhoptry proteins whose functions are essential for maintaining rhoptry morphology, rhoptry secretion, and rhoptry antigen processing.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 202

Expansion Microscopy Reveals Plasmodium falciparum Blood-Stage Parasites Undergo Anaphase with A Chromatin Bridge in the Absence of Mini-Chromosome Maintenance Complex Binding Protein

The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of the P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing the visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles, and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP-deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum

Structure-Based Identification and Functional Characterization of a Lipocalin in the Malaria Parasite Plasmodium falciparum

Highlights: • Crystal structure of the malaria parasite lipocalin • Comparative analysis of lipocalin superfamily members in alveolate genomes • Localization of PfLipocalin to the parasitophorous vacuole and food vacuole • Reverse genetics reveal PfLipocalin function in oxidative damage control Summary: Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers; hence we name it P. falciparum lipocalin (PfLCN). We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress-induced cell damage during multiplication of parasites within erythrocytes

Retargeting azithromycin analogues to have dual-modality antimalarial activity

Background: Resistance to front-line antimalarials (artemisinin combination therapies) is spreading, and development of new drug treatment strategies to rapidly kill Plasmodium spp. malaria parasites is urgently needed. Azithromycin is a clinically used macrolide antibiotic proposed as a partner drug for combination therapy in malaria, which has also been tested as monotherapy. However, its slow-killing 'delayed-death' activity against the parasite's apicoplast organelle and suboptimal activity as monotherapy limit its application as a potential malaria treatment. Here, we explore a panel of azithromycin analogues and demonstrate that chemical modifications can be used to greatly improve the speed and potency of antimalarial action. Results: Investigation of 84 azithromycin analogues revealed nanomolar quick-killing potency directed against the very earliest stage of parasite development within red blood cells. Indeed, the best analogue exhibited 1600-fold higher potency than azithromycin with less than 48 hrs treatment in vitro. Analogues were effective against zoonotic Plasmodium knowlesi malaria parasites and against both multi-drug and artemisinin-resistant Plasmodium falciparum lines. Metabolomic profiles of azithromycin analogue-treated parasites suggested activity in the parasite food vacuole and mitochondria were disrupted. Moreover, unlike the food vacuole-targeting drug chloroquine, azithromycin and analogues were active across blood-stage development, including merozoite invasion, suggesting that these macrolides have a multi-factorial mechanism of quick-killing activity. The positioning of functional groups added to azithromycin and its quick-killing analogues altered their activity against bacterial-like ribosomes but had minimal change on 'quick-killing' activity. Apicoplast minus parasites remained susceptible to both azithromycin and its analogues, further demonstrating that quick-killing is independent of apicoplast-targeting, delayed-death activity. Conclusion: We show that azithromycin and analogues can rapidly kill malaria parasite asexual blood stages via a fast action mechanism. Development of azithromycin and analogues as antimalarials offers the possibility of targeting parasites through both a quick-killing and delayed-death mechanism of action in a single, multifactorial chemotype.Amy L. Burns, Brad E. Sleebs, Ghizal Siddiqui, Amanda E. De Paoli, Dovile Anderson, Benjamin Liffner, Richard Harvey, James G. Beeson, Darren J. Creek, Christopher D. Goodman, Geoffrey I. McFadden, and Danny W. Wilso

Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites

Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.Benjamin Liffner, Juan Miguel Balbin, Gerald J. Shami, Ghizal Siddiqui, Jan Strauss, Sonja Frölich, Gary K. Heinemann, Ella May Edwards, Arne Alder, Jan Stephan Wichers, Darren J. Creek, Leann Tilley, Matthew W.A. Dixon, Tim-Wolf Gilberger, Danny W. Wilso

Small municipalities influence on regional cohesion policy : An explorative case study of small and declining municipalities and their possibilities to create economic growth through regional cohesion policy in Kalmar county

Syftet med uppsatsen är att studera hur kommunpolitiker i Kalmar län upplever möjligheterna att påverka den regionala utvecklingen i länet. Regional utveckling handlar om att skapa hållbar tillväxt i alla delar av EU och omsätts genom de nationella och regionala utvecklingsstrategierna. Sedan den 1 januari 2019 har samtliga tidigare landsting omvandlats till regioner med ansvar för regional utveckling. Många små kommuner saknar dock resurser att satsa på regional utveckling då demografiska och strukturella förändringar har bidragit till en ökad utflyttning och sämre ekonomi. Uppsatsen antar en explorativ ansats och genom semi-strukturerade intervjuer med tre av länets kommunstyrelseordförande försöker studien besvara frågan om kommunernas möjligheter att påverka den regionala utvecklingspolitiken har förändrats efter den 1 januari 2019. De empiriska resultaten bekräftar mycket av den tidigare forskningen och visar att kommunerna inte har tillräckliga resurser att satsa på regional utveckling. Studien visar också vikten av ett tydligt regionalt ledarskap och att regional utveckling till stora delar handlar om infrastruktur för en liten kommun

Small municipalities influence on regional cohesion policy : An explorative case study of small and declining municipalities and their possibilities to create economic growth through regional cohesion policy in Kalmar county

Syftet med uppsatsen är att studera hur kommunpolitiker i Kalmar län upplever möjligheterna att påverka den regionala utvecklingen i länet. Regional utveckling handlar om att skapa hållbar tillväxt i alla delar av EU och omsätts genom de nationella och regionala utvecklingsstrategierna. Sedan den 1 januari 2019 har samtliga tidigare landsting omvandlats till regioner med ansvar för regional utveckling. Många små kommuner saknar dock resurser att satsa på regional utveckling då demografiska och strukturella förändringar har bidragit till en ökad utflyttning och sämre ekonomi. Uppsatsen antar en explorativ ansats och genom semi-strukturerade intervjuer med tre av länets kommunstyrelseordförande försöker studien besvara frågan om kommunernas möjligheter att påverka den regionala utvecklingspolitiken har förändrats efter den 1 januari 2019. De empiriska resultaten bekräftar mycket av den tidigare forskningen och visar att kommunerna inte har tillräckliga resurser att satsa på regional utveckling. Studien visar också vikten av ett tydligt regionalt ledarskap och att regional utveckling till stora delar handlar om infrastruktur för en liten kommun

“I think sometimes we lose sight of the point of what assessments are”

Sammanfattning Det finns två huvudsyften med bedömning. En bedömning med ett formativt syfte är en bedömning för lärande. Det innebär att läraren genom sin bedömning ska hjälpa eleven att utvecklas mot målen. En summativ bedömning är en bedömning av lärande. Syftet är att summera kunskaper. Betyg är ett exempel på ett resultat av en summativ bedömning. Vi har genomfört en kvalitativ intervjuundersökning med pedagoger och elever. Vi har besökt en skola i New York och en skola i Göteborg. På skolorna har vi intervjuat lärare som är verksamma i årskurs tre till fem och elever som går i årskurs fem. Lärarintervjuerna riktade sig mot hur lärarna bedömer. Elevintervjuerna berörde hur de påverkas av lärares bedömningar. Syftet med metodvalet var att få svar på våra frågeställningar: om pedagogerna har en formativ eller summativ syn på bedömning? hur man arbetar med formativ bedömning? Vilka likheter och skillnader det finns mellan skolorna vi har besökt. Resultatet av vår studie visade att de lärare vi intervjuade hade en formativ syn på bedömning. Eleverna på skolan i New York förstod att lärarna har både formativa och summativa syften med sin bedömning. I Göteborg dominerade den formativa synen bland eleverna. Genom att arbeta formativt blev bedömning en kontinuerlig del av undervisningen som användes med syfte att hjälpa eleven. Vår förhoppning med denna studie var att belysa lärares syn på bedömning samt visa exempel hur pedagoger kan arbeta med formativ bedömning. Tidigare forskning visar att formativ bedömning främjar elevens lärande. Formativ bedömning förklaras ofta genom tre processer: var är eleven? vart ska eleven? hur kommer eleven dit? Det finns inte någon allmängiltig mall för hur lärare ska arbeta med formativ bedömning. Det innebär att arbetet med formativ bedömning ser olika ut på olika skolor och klassrum. Det behövs enligt skolverket mer forskning om hur lärare bedömer sina elevers kunskaper