Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Encystation of unicellular organisms is of considerable medical relevance since cysts are encapsulated byresilient cell walls, rendering them resistant to biocides and immune clearance. This survival strategymakes it complicated to produce effective treatment of diseases caused by many protozoan pathogens,e.g. species of Acanthamoeba which causes fatal granulomatous amebic encephalitis (GAE) and keratitis.Due to genetic limitations in most protists, the machinery of encystation is so far little understood.However, the signalling pathways can be investigated in the non-pathogenic social amoebas, Dictyostelia.In this master’s project, five genes in Polysphondylium pallidum were investigated, which might beinvolved in encystation. Research has demonstrated a relationship between encystation and the cyclicadenosine monophosphate (cAMP) signalling pathways in Dictyostelia spore formation. This indicates thatcysts are ancestral to spores, and hence are the sporulation genes: pkaC, yakA, regA, cudA and srfAselected for this study. The genes were individually knocked-out by a standard homologous recombinationapproach and the genes’ contribution to encystation was determined. Five knock-out constructs werecompleted and a preliminary analysis of the role of the intracellular cAMP phosphodiesterase RegA inPolysphondylium pallidum encystation process was performed.

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.