The maintenance of cooperation and altruism in the face of manipulation by exploitative cheaters that reap the benefits of cooperative acts without paying the associated costs is a conundrum in evolutionary biology. Cheaters should spread through a population causing it to crash, yet cooperation is common. There are many models and theories that attempt to explain this apparent contradiction. The social amoeba Dictyostelium discoideum, like many microbial species has been used as a model organism to test these theories and to begin to understand the genetic mechanisms behind social behaviours. The aim of this PhD project is to quantify the interactions that occur between naturally-occurring genotypes during social competition in order to identify the types of cheating behaviours and to understand the evolutionary consequences of such behaviours. I first demonstrate that there is a social hierarchy of genotypes and that cheaters can increase their own fitness by increasing their own spore allocation or decreasing their partner's allocation the precise nature of which is dependent upon unique interactions between each competing pair. I also show that the outcome of social competition is dependent upon the physical environment where it can be significantly reduced, or even avoided by segregation of genotypes during development. Finally, it is demonstrated in a collaborative project that much of the observed social behaviour can be explained in terms of the production of and response to developmental signals.

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. It was initially found that comparing to wild type cells, gsk3- cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.

No description available.

Many classes of non-coding RNAs (ncRNAs; including Y RNAs, vault RNAs, RNase P RNAs, and MRP RNAs, as well as a novel class recently discovered in Dictyostelium discoideum) can be characterized by a pattern of short but well-conserved sequence elements that are separated by poorly conserved regions of sometimes highly variable lengths. Local alignment algorithms such as BLAST are therefore ill-suited for the discovery of new homologs of such ncRNAs in genomic sequences. The Fragrep tool instead implements an efficient algorithm for detecting the pattern fragments that occur in a given order. For each pattern fragment, the mismatch tolerance and bounds on the length of the intervening sequences can be specified separately. Furthermore, matches can be ranked by a statistically well-motivated scoring scheme.

Indiana University-Purdue University Indianapolis (IUPUI) / The lipid profile of Dictyostelium discoideum, a cellular slime mold found evolutionarily between plants and animals, has never been clearly defined. To address this, the fatty acid content of vegetative cells was analyzed by gas chromatography-mass spectrometry of fatty acid methyl esters and their identities verified with synthesized authentic standards. The synthetic scheme developed to produce the unusual fatty acids found in D. discoideum was engineered to afford the labeling of compounds (2H) for use in feeding studies to elucidate the fatty acid elongation and desaturation pathways present in D. discoideum. After establishing the fatty acid profile and acyl metabolic pathway, an initial understanding the complex lipids present in D. discoideum, chiefly sphingolipids, was sought. Triple quadrupole and quadrupole time-of flight mass spectrometers equipped with electrospray ionization sources were used to identify these complex lipids.

Modeling or simulating Complex Adaptive Systems (CASs) is both important and challenging. As the name suggests, CASs are systems consisting of large numbers of interacting adaptive compartments. They are studied across a wide range of disciplines and have unique properties. They model such systems as multicellular organisms, ecosystems, social networks, and many more. They are complex, in the sense that they are dynamical, nonlinear, and heterogeneous systems that cannot be simply scaled up/down. However, they are self-organized, in the sense that they can evolve into specific structures/patterns without guidance from outside sources. Modeling/Simulating CASs is challenging, not only because of the high complexity, but also because of the difficulty in explaining the underlying mechanism behind self-organization. The goal of this research is to provide a modeling framework as well as a simulation platform to advance the study of CASs. We argue that there are common principles behind self-organization processes of different systems across different domains. We explore, analyze, and perform experiments into these principles. We propose and implement modeling templates such as short-term and long-term adaptivity. We incorporate techniques from systems theory, employing computing paradigms, including multi-agent system and asynchronous message passing. We also consider an application from the biological domain to model and simulate under our framework, treating it as a CAS for validation purposes. / Ph. D.

The expression of the <I>glycogen phosphorylase- 2</I> gene (<I>gp2</I>) is initiated during early development and regulated by the extracellular morphogens cAMP and Differentiation Inducing Factor (DIF-1) [1-3]. Glycogen phosphorylase- 2 catalyzes the breakdown of glycogen reserves in developing cells to generate glucose precursors required for the synthesis of the end products of differentiation [4-6]. Thus, the expression of <I>gp2</I> is a significant event for cellular differentiation. The sequence of the <I>gp2</I> promoter, like other <I>Dictyostelium</I> promoters, has an AT-rich bias (88%) [7]. Previous deletional analyses of the promoter provided a map of the regions that contained transcriptional regulatory elements. The regions thus identified contained either "TAAAAATGGA" or C-rich repeat sequences [2]. These regions were dissected further by site-directed mutagenesis (SDM) to better define the physical boundaries of the regulatory elements. It was shown that the mutation of either one of the C-rich repeats resulted in a dramatic drop of about 95% in reporter gene levels. These data strongly suggested that both the C-rich repeats of <I>gp2</I> functioned as transcriptional regulatory elements. I have identified and purified a factor called TF2 that demonstrates a high specificity for a C-rich transcriptional regulatory element, the 5' C box. TF2 was first detected with electrophoretic mobility shift assays of DEAE chromatographic fractions of cell-free extracts. The specificity of TF2 for the 5' C box was tested by competition analysis using six other oligonucleotides. Purification of TF2 was achieved by ion-exchange chromatography, DNA affinity chromatography, gel filtration chromatography, and preparative SDS-PAGE. SDS-PAGE analysis indicated an apparent subunit molecular weight of 28 kDa. The apparent molecular weight of the native protein as estimated by gel filtration was about 53 kDa. This suggested that TF2 binds gp2 as a homodimer. A cDNA clone of the tf2 gene was provided by the Japanese <I>Dictyostelium</I> cDNA project. This allowed me to synthesize probes for Southern and Northern blot analyses. Southern blot analysis indicated that there is only one form of the <I>tf2</I> gene. Northern analysis showed little or no expression of <I>tf2</I> in undifferentiated cells. During development <I>tf2</I> expression increases up to a maximum at 8 h, then decreases in later stages. Attempts to disrupt the gene suggest that <I>tf2</I> mutation may be lethal. / Ph. D.

A critical event during the life cycle of Dictyostelium discoideum is glycogen turnover. This process is catalyzed by glycogen phosphorylase-2 (gp-2). Since gp-2 expression is first induced during the transition from growth to differentiation, understanding how this gene is controlled may provide some insight into the process of differentiation. In order to identify the trans-acting factors responsible for activating gp-2 expression, cDNA plasmid libraries of amoebae, and cells at 8 h and 12 h of development were generated. The long-term goals of this project involve screening expression libraries with identified cis-acting elements from the gp-2 promoter to yield the DNA binding proteins responsible for gene regulation. For this approach to succeed, a high-quality cDNA library is essential. The library must contain full-length cDNA that represents the complexity of mRNA present during the developmental stage of interest. Hence, all three libraries were subjected to extensive testing prior to and following cloning. RNA quality and the fidelity of the time points were determined by Northern blot analysis and by RTPCR for several marker genes. Following cDNA synthesis, the cDNA was assessed for complexity and full-length synthesis by PCR and radioactive primer extension, respectively. Ligation of the cDNA into a vector was performed using several ratios of vector:insert in order to ensure that long cDNA species were included in the plasmid library. Finally, the presence of the marker genes was confirmed by PCR amplification of plasmid extracted from bacteria transformed with the plasmid library. / Master of Science

The gene encoding the Dictyostelium replication protein A large subunit (DdRPA1) has been cloned by screening of an EcoR I partial genomic library and a Hind III genomic sub-library. The complete nucleotide sequence, including the promoter region of the gene has been obtained by sequencing. Though the DdRPA1 protein has a size shift during development, 62 kDa in undifferentiated cells and 81 kDa in differentiated cells; they are the products of the same gene. Northern blot analysis revealed that the expression level of the DdRPA1 was constant throughout differentiation and the size of mRNA is the same at all stages, corresponding to a 81 kDa protein. Thus, it seems that the size change between the 62 kDa and 81 kDa is probably due to posttranslational modification, most likely, proteolytic cleavage. The transcription start site for both sizes of DdRPA1 has been identified at 306 bp upstream of the coding sequence by primer extension reaction. A PCR fragment representing 27% of the gene encoding the DdRPA middle size subunit (DdRPA2) has been generated by using the degenerate primers. This PCR fragment has been cloned and sequenced. The mRNA for this subunit corresponds to a protein of about 35 kDa. A decrease of the DdRPA2 mRNA expression level during differentiation was found by comparison between undifferentiated and differentiated cells. In Dictyostelium, replication protein A is a heterotrimeric protein that can bind with specific DNA sequences in a stage-dependent pattern. These DNA sequences were identified as the cis-acting regulatory sites in differentiation-related genes, including the glycogen phosphorylase 2 gene (gp2). Therefore, it is possible that DdRPA is not only a single-stranded DNA binding protein that is used in multiple essential DNA metabolic processes, such as DNA replication, repair and recombination in undifferentiated cells, but also involved in the transcriptional regulation process during differentiation. / Master of Science

Dictyostelium discoideum (Dd) cells are amoeba cells that feed on soil or plant leaf matter bacteria. They divide freely in the presence of food. When starved, cell division is ended, and cells transition from unicellular to multicellular organisms initiated by a chemoattractant, cyclic adenosine monophosphate (cAMP). This study intricately examines cellular sorting within the Dd slug, focusing on individual cell dynamics. Represented as oriented ellipsoids, these cells maintain constant volume through a viscoelastic structure. Applying force to specific semi-axes reveals predictable deformations, highlighting the relationship between force and deformation rate. Manipulating damping coefficients and spring constants uncovers insights into cell viscosity and stiffness. Exploring the effects of a cell's active force and cone angle revealed that a cone angle less than 10 degrees or greater than 30 degrees and an active force less than 2N or greater than 6N of the prestalk cells led to unsatisfactory conditions, resulting in insufficiently directed cell movement, and an inability to achieve the desired cell sorting patterns within the slug. This work guides exploring diverse cellular behaviors, advancing the understanding of complex biological phenomena within a concise framework.