11 research outputs found

    Developing novel therapeutic agents for Acanthamoeba infection and investigating the process of encystment

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    A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.Acanthamoeba Keratitis (AK) is a vision-threatening disease which can lead to blinding corneal tissue infection. Many patients who have been infected with Acanthamoeba in their eye do not respond to the current medical treatments involving polyhexamethylene biguanide or chlorhexidine despite the in vitro sensitivity of Acanthamoeba to these drugs. There is an urgent need for new therapeutic agents to eradicate the AK infection. This study focuses on the mechanism by which Acanthamoeba may distinguish between trophozoite, cyst and the newly identified lifecycle known as protocyst. The current study has tested 56 novel and existing therapeutic agents for their activity against Acanthamoeba spp. and their toxicity against a human epithelial cell line. The results of this research have revealed several compounds of interest for further study on their potential use in the treatment of AK. These compounds included, octenidine hydrochloride, alexidine, miltefosine and quaternary ammonium (didecyldimethylammonium chloride). The anti-amoebic effect of benzalkonium chloride, povidone iodine and tetracaine are superior to the current diamidines and slightly lower to the biguanides applied in the treatment for AK. The formulation of novel amidoamine compounds including myristoleyl-amidopropyl-dimethylamine (MOPD) and palmitoleyl-amidopropyl-dimethylamine (POPD) into contact lens solutions showed complete kill at a 4.5-log reduction against trophozoites compared with myristamidopropyl dimethylamine (MAPD) as an existing compound. The combination of biguanide compounds with lipid‚Äďbased carriers has improved the antimicrobial activity from 1-fold to around 7-fold against cysts of Acanthamoeba spp. compared with the use of biguanides alone. The findings of encystment investigation (the transformation of trophozoites into cysts) showed that the agonists in particular the ő≤ ultra-long against indacaterol stimulated the encystment and the antagonists ő≤‚āĀ metoprolol blocked the formation of cysts and protocysts. Two different herbicides including 2,6-dichlorobenzonitrile (DCB) and isoxaben were tested to target the biosynthesis of cellulose in the cyst form and also to evaluate their effects on the formation of protocyst of Acanthamoeba. The results of this study showed that the DCB at a high concentration of 500 őľM, reduced encystment to 17.7% and protocyst production of Acanthamoeba at 24.6%, whereas isoxaben inhibited the transformation of trophozoites into cysts to only 45% and the percentage was decreased for protocyst formation by 37.2%. The test results for DCB and isoxaben individually at concentration of 100 uM showed 31.8% and 68.8% respectively for the conversion of trophozoites into cysts. In addition, a similar concentration of both DCB and isoxaben was evaluated for protocyst formation and the inhibition was observed at 36.9% for DCB and a much higher rate of protocysts production was recorded at 63 % for isoxaben. The combination of both isoxaben and DCB at a concentration of 100 őľM caused a reduction in encystment to 49.1% and lowered the transformation of trophozoites into protocysts to 45.7%, these findings suggested that an antagonistic effect was occurred relative to the use of DCB alone. Finally, the data from LC/MS analysis for sugars suggested that the protocyst and cyst are different stages of Acanthamoeba, as the analysis of cyst walls indicated the presence of cellulose while the protocyst wall analysis showed the existing of cellulose and methylated sugar possibly corresponded to a methylated analogue of N-acetylglucosamine.Iraqi Cultural Attach√© in London the official representative of the Iraqi Ministry of Higher Education and Scientific Research in the United Kingdom

    Encystace a Ňĺivotn√≠ cyklus volnńõ Ňĺij√≠c√≠ch am√©b rodu Acanthamoeba spp.

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    Am√©by rodu Acanthamoeba jsou celosvńõtovńõ rozŇ°√≠Ňôen√°, volnńõ Ňĺij√≠c√≠, fakultativnńõ patogenn√≠ jednobunńõńćn√° eukaryota. Jejich nebezpeńćnost pro ńćlovńõka spońć√≠v√° ve schopnosti pronikat do organismu, odolat obrann√Ĺm mechanismŇĮm, mnoŇĺit se, poŇ°kozovat napaden√© tk√°nńõ a t√≠m vyvolat onemocnńõn√≠, proti kter√Ĺm chyb√≠ kauz√°ln√≠ l√©ńćba a kter√° nejńćastńõji postihuj√≠ oko a centr√°ln√≠ nervovou soustavu (CNS). Jedn√≠m z hlavn√≠ch dŇĮvodŇĮ ńćasto ne√ļspńõŇ°n√© terapie je schopnost akantam√©b vytv√°Ňôet v postiŇĺen√Ĺch tk√°n√≠ch cysty, vysoce rezistentn√≠ klidov√° st√°dia. Kromńõ cyst, kter√© akantam√©by tvoŇô√≠ jak v pŇô√≠rodńõ, tak v infikovan√Ĺch tk√°n√≠ch pod vlivem dlouhodob√©ho stresu, se tyto organismy vlivem akutn√≠ho ohroŇĺen√≠ rychle transformuj√≠ v m√©nńõ odoln√° klidov√° st√°dia, pseudocysty. PŇôedkl√°dan√° pr√°ce se zamńõŇôuje na dosud nezn√°m√© aspekty odolnosti obou rezistentn√≠ch st√°di√≠ akantam√©b, cyst a pseudocyst, a souńćasnńõ si klade za c√≠l popsat dalŇ°√≠ charakteristiky odliŇ°uj√≠c√≠ cysty a pseudocysty a procesy spojen√© s jejich tvorbou a rezistenc√≠. Jedn√≠m ze studovan√Ĺch aspektŇĮ odolnosti klidov√Ĺch st√°di√≠ akantam√©b byla pŇô√≠tomnost cukern√©ho alkoholu manitolu a neredukuj√≠c√≠ho cukru trehal√≥zy, sacharidŇĮ, kter√© se v buŇąk√°ch mnoha organismŇĮ √ļńćastn√≠ obrann√Ĺch reakc√≠ vŇĮńći abiotick√©mu stresu. Ańćkoli v genomu A. castellanii jsou enzymy pro synt√©zu obou cukrŇĮ pops√°ny,...Amoebae of the genus Acanthamoeba spp. are free-living unicellular organisms found in disparate ecosystems all over the world. Due to their ability to invade human body, evade its defensive mechanisms and cause extensive tissue damage, Acanthamoeba infection can lead to serious, if rare, diseases, affecting most commonly the eye and the central nervous system. Specific therapy for Acanthamoeba infections is not available. A major reason for therapeutic failure in ameobiasis is the ability of the protist to differentiate into resistant stages. These are cysts, known to be formed under prolonged unfavorable conditions, both in the environment and the infected tissues, and the pseudocysts, less durable but rapidly formed under acute stress. The present thesis focuses on as yet unexplored mechanisms of resistance of cysts and pseudocysts. Moreover, further characteristics distinguishing cysts and pseudocysts as well as the processes involved in their formation are investigated. One of the issues addressed is a presence of protective carbohydrate compounds mannitol and trehalose that participate in defensive reactions against abiotic stress in many organisms. Although putative genes for enzymes of the trehalose and mannitol synthetic pathways are present in the genome of Acanthamoeba, only one of the...Klinika infekńćn√≠ch a tropick√Ĺch nemoc√≠ 1. LF UK a Nemocnice Na BulovceDepartment of Infectious and Tropical Diseases First Faculty of Medicine and Na Bulovce HospitalFirst Faculty of Medicine1. l√©kaŇôsk√° fakult

    Molecular manipulation and new antimicrobial identification in Acanthamoeba spp

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    Date on title page is date of submission (November 2021). Date of award is 2022.Acanthamoeba spp. are causative agents of a painful and severe sight-threatening corneal infection that can lead to blindness known as Acanthamoeba keratitis and a subacute disease in the brain which is usually fatal known as granulomatous amoebic encephalitis. Over the last few years, there has been a notorious increase in the number of infections due to Acanthamoeba spp. Poor diagnosis, problems of side effects, toxicity of the current drug treatment and the lack of gene editing tools as potential future therapy contribute to a high mortality rate. Strathclyde Minor Groove Binders (S-MGBs), compounds that bind to the minor groove of the DNA that designed and synthesised at University of Strathclyde were evaluated as potential alternative inhibitors against Acanthamoeba infections. Through cell viability microplate alamarBlue assays 42 S-MGBs were screened from which S-MGB 235 showed the most potent inhibitory effect with IC50 in the nanomolar range against five Acanthamoeba isolates after 24 h and 96 h incubation. Confocal microscopy of trophozoites labelled with fluorescent S-MGB 363 (analogue of S-MGB235) showed this compound in the nucleus, nucleolus and distributed over the granuloplasm causing cell lysis, supporting the potent effect observed in vitro by S-MGB 235. Furthermore, conditions were standardised to establish Galleria mellonella larvae as a new in vivo infection model for A. castellanii Neff infections to assess the efficacy and toxicity of voriconazole, miltefosine and S-MGB 235. Voriconazole and miltefosine did not protect larvae from trophozoite infection, however S-MGB 235 significantly protected larvae when compared with the negative control. It was attempted to establish a CRISPR-Cas9 system for gene editing in Acanthamoeba. The plasmids pBRFPT7NeoCas9 and pBRFPT7PhleoCas9 (containing genes for the red fluorescent protein, T7 RNA polymerase, Cas9 along with the neomycin and phleomycin resistance genes, respectively, were constructed and transfected into A. castellanii Neff trophozoites using Xfect. Expression of RFP was confirmed by fluorescence microscopy and fluorescence-activated cell sorting.Acanthamoeba spp. are causative agents of a painful and severe sight-threatening corneal infection that can lead to blindness known as Acanthamoeba keratitis and a subacute disease in the brain which is usually fatal known as granulomatous amoebic encephalitis. Over the last few years, there has been a notorious increase in the number of infections due to Acanthamoeba spp. Poor diagnosis, problems of side effects, toxicity of the current drug treatment and the lack of gene editing tools as potential future therapy contribute to a high mortality rate. Strathclyde Minor Groove Binders (S-MGBs), compounds that bind to the minor groove of the DNA that designed and synthesised at University of Strathclyde were evaluated as potential alternative inhibitors against Acanthamoeba infections. Through cell viability microplate alamarBlue assays 42 S-MGBs were screened from which S-MGB 235 showed the most potent inhibitory effect with IC50 in the nanomolar range against five Acanthamoeba isolates after 24 h and 96 h incubation. Confocal microscopy of trophozoites labelled with fluorescent S-MGB 363 (analogue of S-MGB235) showed this compound in the nucleus, nucleolus and distributed over the granuloplasm causing cell lysis, supporting the potent effect observed in vitro by S-MGB 235. Furthermore, conditions were standardised to establish Galleria mellonella larvae as a new in vivo infection model for A. castellanii Neff infections to assess the efficacy and toxicity of voriconazole, miltefosine and S-MGB 235. Voriconazole and miltefosine did not protect larvae from trophozoite infection, however S-MGB 235 significantly protected larvae when compared with the negative control. It was attempted to establish a CRISPR-Cas9 system for gene editing in Acanthamoeba. The plasmids pBRFPT7NeoCas9 and pBRFPT7PhleoCas9 (containing genes for the red fluorescent protein, T7 RNA polymerase, Cas9 along with the neomycin and phleomycin resistance genes, respectively, were constructed and transfected into A. castellanii Neff trophozoites using Xfect. Expression of RFP was confirmed by fluorescence microscopy and fluorescence-activated cell sorting

    Glycolytic Inhibitors as Leads for Drug Discovery in the Pathogenic Free-Living Amoebae

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    The free-living amoeba, Naegleria fowleri, can cause a rare yet usually lethal infection of the brain called primary amebic meningoencephalitis. Because of poor diagnostics and limited treatment options, the mortality rate associated with the disease is \u3e97%. Due to our finding that glucose is critical for trophozoite growth in culture, we have been interested in exploiting amoebae glucose metabolism to identify new potential drug targets. We have characterized the first enzyme of the glycolytic pathway, glucokinase (Glck), from N. fowleri and two other pathogenic free-living amoeba, Acanthamoeba castellanii and Balamuthia mandrillaris. We have assessed their biochemical properties and tested potential inhibitors on the recombinant Glcks, which revealed that these enzymes are sufficiently different from one another that developing pan-amoeba inhibitors may be challenging. However, their individual differences from the human host enzyme suggests that species-specific Glck inhibitors could be identified. We have also explored targeting the glucose metabolizing enzyme enolase in N. fowleri using a series of phosphonate human enolase 2 (ENO-2) specific inhibitors that were developed to treat human cancer. These compounds are curative for ENO-1 deleted glioblastoma in a rodent model, can cross the blood-brain barrier, and are of limited toxicity to non-human primates. The phosphonate inhibitors were toxic to N. fowleri in vitro with (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) being the most potent, with an EC50 value of 0.21 ¬Ī 0.02 ¬ĶM, almost 1500-fold lower than the concentration required to impact human cells. Unbiased metabolomics indicates that glycolytic intermediates upstream of NfENO accumulate in HEX treated amoebae. In an effort to genetically validate new targets for therapeutic intervention, we have initiated efforts to develop molecular tools for use in N. fowleri. We have designed a vector for transient transfection of the amoebae that harbors portions of the 5‚ÄôUTR of actin 1 (NF0111190) upstream of both eYFP and a hygromycin resistance gene, termed pJMJM1. We have tested a variety of approaches used in other parasite systems for plasmid delivery including the transfection reagent SuperFect, Amaxa Nucleofector technologies, and various electroporation settings. Transfection of N. fowleri flagellates with 5 ¬Ķg pJMJM1 by electroporation (100 V, 500 ¬ĶF, 400 ő©) yielded a population of fluorescent cells seven days after being treated with 300 ¬Ķg/mL hygromycin, but this expression of eYFP was lost over time. More recently, we have used CRISPR/Cas9- mediated gene editing to successfully introduce an eYFP repair template into a predicted protein locus. While fluorescent cells were not noted in the culture, editing was confirmed by PCR analysis. Development of these molecular techniques will provide an important tool for uncovering potential target genes and allow for a better understanding of amoeba biology

    Prevalence of clinically relevant acanthamoeba and bacteria in a South African paediatric burns unit

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    Abstract: The presence of Acanthamoeba and select clinically relevant bacteria contribute immensely to acquiring hospital-acquired infections (HAIs) in hospitals globally. The World Health Organisation (WHO) has emphasized the following organisms: Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. These organisms are collectively referred to as 'ESKAPE" organisms, mainly due to their infamous resistance to antibiotics, which serve as the standard primary treatment line against the HAIs they are known to cause. The interaction of amoeba and these organisms has been documented as playing an under-reported but significant role in their pathogenicity (Boyce, 2017). Children are generally at a higher risk of contracting HAIs because of their immune immaturity that is naturally worsened by pre-existing medical conditions (Brooks and Mias, 2018). Acanthamoeba and other harmful pathogens have been isolated in water, soil, air, humans, and animals. The exposure risk for acquiring Healthcare-associated infections is directly linked to Infection Prevention and Control (IPC) practices by healthcare workers, patients, and visitors that they come into physical contact with...M.Tech. (Environmental Health

    Avalia√ß√£o da atividade antiamebiana de compostos presentes no Covid-Box¬ģ contra trofozo√≠tos de Acanthamoeba castellanii

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    TCC(gradua√ß√£o) - Universidade Federal de Santa Catarina. Centro de Ci√™ncias da Sa√ļde. Farm√°cia.Free-living amoebas are ubiquitous protozoa that can be isolated from various environmental niches, such as soil, water and vegetation. The genus Acanthamoeba spp. has the characteristic of being amphizoic amoebas, being able to behave in the free and parasitic form. Acanthamoeba spp. it has two stages in its life cycle, the trophozoite, characterized as the metabolically active form and cyst, its active form. The drug repositioning is one of the strategies for the emergent infection‚Äôs treatment such as those occurred by the free-living ameba of the genus Acanthamoeba. In this context, this paper aimed to evaluate the anti-Acanthamoeba activity of the compounds present in the Covid-Box¬ģ in order to search for new therapeutical options for the amoebic keratitis and disseminated infections. The tests of the amebicidal activity were performed using the strain of the Acanthamoeba castellanii (ATCC 50492). In order to carry out the amebicity activity, the Acanthamoeba spp‚Äôs trophozoite were exposed in the concentration of the 10 ¬ĶM of each compound present in the Covid-Box¬ģ. Moreover, the compounds that showed better amebicidal activity were evaluated in the concentrations of 20 ¬ĶM, 15 ¬ĶM, 10 ¬ĶM, 5 ¬ĶM and 2,5 ¬ĶM to determine the IC50. Furthermore, to determine the trophozoites‚Äô viability, it was used the viability indicator alamarBlue¬ģ. Also, the cytotoxicity assay against the SIRC of selected cells was performed. Ciclosenide and promethazine were able to considerably decrease the number of the A. castellanii‚Äôs trophozoites such as a viability decrease of the 35,01 ¬Ī 5,088% and 34,66 ¬Ī 5,165% respectively. Both ciclosenide and promethazine had lower ocular toxicity that the gold standard drug used in the amoebic keratitis treatment. The results allowed to identify two active compounds against the A.castellanii‚Äôs trophozoites with low cytotoxic potential which could be used as a study prototype of new active molecules, synergy studies, aiming to improve the treatments of the infections caused by Acanthamoeba spp

    Biophysikalische Untersuchungen von Infektionen mit Acanthamöben: Kontamination von Kontaktlinsen, Adhäsion und intrazelluläre Dynamik

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    Acanthamoeba castellanii can cause a severe and painful, acanthamoeba keratitis. In this disease, the acanthamoebae reach the eye via contaminated contact lenses. In the eye, they invade cornea cells and kill them. The first step in this process is the formation of a close contact between the acanthamoeba and the target-cell. This contact is mainly mediated by mannose. Subsequently, cytolytic factors move to the contact site and are released leading to the death of the target-cell. The single steps involved in the infection pathway, namely contact lens contamination, carbohydrate-mediated acanthamoeba adhesion, and intracellular transport were investigated in this thesis. As the use of contact lenses is a main risk factor in the infection process, in the first part of this thesis different silicone hydrogel and hydrogel materials were investigated for their potential to be contaminated with A. castellanii. Furthermore, influences of standard contact lens cleaning procedures on the adhesion and proliferation of A. castellanii were evaluated. It was observed that A. castellanii preferentially adhered to lenses with high water content (>60 %). Furthermore, while multipurpose and peroxide solution reduced adhesion of acanthamoeba significantly, only the peroxide solution was able to kill all A. castellanii effectively. If contaminated contact lenses are inserted into the eye, acanthamoebae reach the cornea, move through the epithelial tissue of the cornea and kill target-cells in the underlying cell layers. Thus, in the second part of this thesis interactions of acanthamoebae with proteins in the extracellular matrix, as well as the carbohydrate-mediated adhesion to target-cells were investigated. In order to mimic the extracellular matrix, glass slides were coated with proteins and the number of adhering acanthamoebae was evaluated. Compared to the glass, increased adhesion was observed on matrigel followed by fibronectin. This increase in number of adhering acanthamoeba was presumably mediated by mannose present in the protein coatings. In order to investigate the initial adhesion of acanthamoebae to target-cells, quasi-hexagonally arranged patterns of gold nanodots with different interparticle distances were produced by diblock copolymer micelle nanolithography and functionalized with mannose or glucose, respectively. By passivating the areas between the dots with polyethylene glycol (PEG), unspecific adhesion of the acanthamoebae in the inter-particle space was reduced and therefore only the biofunctionalized gold dots served as anchoring points for the adhesion. The evaluation of the number and the spreading area of A. castellanii showed that already relatively low densities of mannose lead to stable adhesion of the acanthamoebae and that mannose as single ligand is sufficient for adhesion. In order to quantify forces of the binding of A. castellanii to carbohydrate-functionalized surfaces, force spectroscopic measurements were carried out by atomic force microscopy (AFM). The measurements showed that with increasing contact time of acantameobae to mannose coated AFM cantilevers the maximum adhesion force and the adhesion energy increase. After initial adhesion of acanthamoeba to the target-cell, cytolytic factors, move to the contact site, are released and lead to target-cell death. Thus intracellular transport of vesicles was investigated in the last part of this thesis. In order to examine the intracellular dynamics, the transport processes of particles inside acanthamoebae were evaluated and compared after addition of filament depolymerizing substances and molecular-motor inhibiting substances. The intracellular space in A. castellanii is extremely crowded, and most particles were moving actively. This was also the case after the different treatments, which inhibited the motion of the whole amoebae, but not of the intracellular particles.Acanthamoeba castellanii k√∂nnen eine schmerzhafte, das Sehverm√∂gen beeintr√§chtigende Acantham√∂benkeratitis verursachen. Dabei gelangen die Acantham√∂ben h√§ufig √ľber kontaminierte Kontaktlinsen oder Kontaktlinsenbeh√§lter in das Auge. Dort dringen sie in die Hornhaut ein und t√∂ten Zielzellen ab. Dieser Mechanismus wird √ľber die Ausbildung eines engen Kontakts zur Zielzelle initiiert. Die Kontaktbildung selbst wird haupts√§chlich durch das Kohlenhydrat Mannose vermittelt. Nach Kontaktbildung bewegen sich zytolytische Faktoren in der Am√∂be zur Kontaktstelle und werden dort freigegeben. Dies f√ľhrt zum Tod der Zielzelle. In dieser Arbeit wurden drei relevante Phasen der Infektion untersucht: Kontamination von Kontaktlinsen mit Am√∂ben, Kohlenhydrat-basierte Kontaktbildung zwischen Acantham√∂be und Zielzelle, sowie intrazellul√§rer Transport. Da Kontaktlinsen einen Hauptrisikofaktor in der Infektion darstellen, wurden im ersten Teil dieser Arbeit verschiedene Kontaktlinsenmaterialien (Siliconhydrogele sowie Hydrogele) auf ihr Kontaminationspotential untersucht. Zudem wurde der Einfluss von g√§ngigen Reinigungsmethoden auf die Anhaftung und das √úberleben der Acantham√∂be gepr√ľft. Es zeigte sich, dass A. castellanii bevorzugt an Kontaktlinsen mit hohem Wassergehalt haften (> 60 %). Sowohl Pflegel√∂sung als auch Peroxidl√∂sung reduzierten die Adh√§sion der Acantham√∂ben, wobei jedoch nur die Peroxidl√∂sung die Zellen vollst√§ndig abt√∂tete. Werden kontaminierte Kontaktlinsen ins Auge eingesetzt, erreichen die Am√∂ben die Hornhaut, durchdringen das Epithelgewebe und t√∂ten Zellen in den darunterliegenden Zellschichten ab. Daher wurden im zweiten Teil Wechselwirkungen zwischen Acantham√∂ben und extrazellul√§rer Matrix, sowie die durch Kohlenhydrate vermittelte Anhaftung von Acantham√∂ben an Zielzellen untersucht. Um die extrazellul√§re Matrix zu imitieren, wurden Deckgl√§ser mit Proteinen beschichtet und die Anzahl anhaftender Acantham√∂ben bestimmt. Der Vergleich zur unbeschichteten Kontrolle ergab eine erh√∂hte Adh√§sion von Acantham√∂ben auf Matrigel gefolgt von Fibronektin. Zur Untersuchung der initialen Anhaftung von Acantham√∂ben an Zielzellen, wurden Substrate mit quasi-hexagonal angeordneten Goldnanopunkten mit unterschiedlichen Abst√§nden hergestellt und mit Mannose bzw. Glucose funktionalisiert. Unspezifische Anhaftung der Acantham√∂ben zwischen Goldpunkten wurde durch Passivierung dieser Bereiche mit Polyethylenglykol (PEG) verhindert, sodass ausschlie√ülich funktionalisierte Goldpunkte als Haftstellen f√ľr Acantham√∂ben dienten. Die Analyse von Zellzahl und Zellfl√§che ergaben, dass bereits geringe Dichten des Kohlehydrats Mannose zu stabiler Adh√§sion der Acantham√∂ben f√ľhrten, wobei Mannose als einzelner Ligand ausreichte. Das kurzzeitige Anhaftungsverhalten an Kohlenhydrat-beschichtete Oberfl√§chen sowie die Adh√§sionskraft von Acantham√∂ben wurden erstmals mittels kraftspektroskopischer Untersuchungen am Rasterkraftmikroskop (AFM) quantifiziert. Die Messungen ergaben eine deutliche Zunahme von maximaler Adh√§sionskraft und Adh√§sionsenergie mit ansteigender Kontaktzeit zwischen Acantham√∂be und Mannose-beschichteter Oberfl√§che. Das initiale Anhaften der Acantham√∂be an die Zielzelle bewirkt eine Migration von in Vesikeln eingeschlossenen zytolytischen Faktoren zur Kontaktstelle. Daher wurde im letzten Teil dieser Arbeit der intrazellul√§re Transport von Vesikeln n√§her untersucht. Dazu wurden Vesikeltrajektorien analysiert und einzelne zellul√§re Transportstrukturen gezielt mittels Zugabe von Filament-depolymerisierenden Substanzen sowie Substanzen zur Inaktivierung molekularer Motoren gest√∂rt. Es wurden √ľberwiegend aktive Bewegungen von intrazellul√§ren Vesikeln beobachtet. Die verschiedenen Substanzen stoppten zwar nahezu die Bewegung der gesamten Zellen, inhibierten jedoch nicht den Transport der Vesikel in der Acantham√∂be
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