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No description available.

No description available.

No description available.

<p><i>Saprolegnia parasitica</i> and <i>Aphanomyces astaci </i>are parasitic water moulds belonging to the Oomycetes. Despite their importance as parasites they are very little studied at the molecular level and the work described in this thesis was aimed at increasing the molecular knowledge of these organisms by cloning and characterising genes of potential importance for reproduction and pathogenicity.</p><p>Stage-specific transcripts from<i> Saprolegnia parasitica</i> were isolated by differential display RT-PCR. One of the markers, <i>puf1 </i>encodes a putative mRNA binding protein which may be involved in post-transcriptional regulation of gene expression. <i>S. parasitica puf1 </i>is expressed exclusively in spore cysts that have not been determined for germination or repeated zoospore emergence indicating that the cyst stage has two phases, of about equal duration, which are physiologically and transcriptionally distinct. A similar expression pattern is observed in <i>Aphanomyces </i>spp. with different regulation of spore development and in the transcript is detected in both primary and secondary cysts.</p><p>A putative chitinase <i>AaChi1</i>, was cloned from the crayfish plague fungus, <i>Aphanomyces astaci. </i>Analysis of chitinase activity and <i>AaChi1</i> expression showed that chitinase in <i>A. astaci </i>is constitutively expressed in growing and sporulating mycelia, but absent in zoospores, a pattern which reflects the infectious life cycle of <i>A. astaci</i>. This expression pattern is conserved between the four known genotypes of <i>A. astaci</i>, in contrast to saprophytic and fish-pathogenic <i>Aphanomyces </i>spp. </p><p>Genetic and physiological analysis were conducted on five strains of <i>Aphanomyces, </i>isolated from suspected outbreaks of crayfish plague in Spain and Italy. The strains are not virulent against freshwater crayfish, and RAPD PCR and ITS sequence analysis show that they are unrelated to the crayfish plague fungus, <i>A. astaci.</i></p>

Saprolegnia parasitica and Aphanomyces astaci are parasitic water moulds belonging to the Oomycetes. Despite their importance as parasites they are very little studied at the molecular level and the work described in this thesis was aimed at increasing the molecular knowledge of these organisms by cloning and characterising genes of potential importance for reproduction and pathogenicity. Stage-specific transcripts from Saprolegnia parasitica were isolated by differential display RT-PCR. One of the markers, puf1 encodes a putative mRNA binding protein which may be involved in post-transcriptional regulation of gene expression. S. parasitica puf1 is expressed exclusively in spore cysts that have not been determined for germination or repeated zoospore emergence indicating that the cyst stage has two phases, of about equal duration, which are physiologically and transcriptionally distinct. A similar expression pattern is observed in Aphanomyces spp. with different regulation of spore development and in the transcript is detected in both primary and secondary cysts. A putative chitinase AaChi1, was cloned from the crayfish plague fungus, Aphanomyces astaci. Analysis of chitinase activity and AaChi1 expression showed that chitinase in A. astaci is constitutively expressed in growing and sporulating mycelia, but absent in zoospores, a pattern which reflects the infectious life cycle of A. astaci. This expression pattern is conserved between the four known genotypes of A. astaci, in contrast to saprophytic and fish-pathogenic Aphanomyces spp. Genetic and physiological analysis were conducted on five strains of Aphanomyces, isolated from suspected outbreaks of crayfish plague in Spain and Italy. The strains are not virulent against freshwater crayfish, and RAPD PCR and ITS sequence analysis show that they are unrelated to the crayfish plague fungus, A. astaci.

With ever-increased quality and quantity of vertebrate genomic sequences, gene finding remains problematic, and predicting gene function from sequence remains a tremendous challenge. Nevertheless, similarity of sequence and patterns of gene expression are rapid methods for providing insight into potential functional roles of novel genes. For developmental studies, microarrays offer a means for screening a large number of sequences for expression within defined tissues and/or organs, but each experiment profiles expression level for only one anatomical region at one specific developmental stage. Whole mount in situ hybridization (ISH) offers an alternative approach that gives precise spatial and temporal resolution of gene expression throughout an embryo. I predicted that combined high throughput analysis of expressed sequence tags (ESTs) and whole mount ISH analysis of novel clones would effectively identify novel developmentally regulated avian genes. 5' ESTs were generated from randomly chosen cDNA clones of a chick late gastrula endoderm-mesoderm library. Following screening to remove ubiquitously expressed clones, internal clustering, and comparison to GenBank sequences, remaining cDNAs (both similar and dissimilar to known genes) were screened for expression in HH stage 4-12 embryos by automated high throughput whole mount ISH. Comparison to GenBank sequences by blastn and blastx (e < 0.05) revealed that one quarter of all 955 ESTs represented novel genes. Of these novel clones, ISH showed that about one quarter (60 clones) exhibited patterned expression during embryogenesis. EST sequences, ISH images and corresponding blast reports were assembled into a freely accessible Web database at http://geisha.biosci.arizona.edu.

Synthetic biomedical implants are used to replace diseased tissues and organs. Unfortunately, these implants often fail due to a lack of biocompatibility and poor integration by the recipient. This implant failure is associated with the formation of an avascular fibrous capsule and chronic inflammatory response. Additionally, small diameter vascular grafts have complications associated with surface thrombogenenicity and intimal hyperplasia. Porous polymers are often incorporated in the construction of biomedical devices because they permit tissue integration and improved biocompatibility. While the inclusion of porosity has enhanced device performance, these devices still do not perform optimally. The incorporation of a vascular network in association with and within the pores of these materials is believed to improve tissue integration and long-term device function. Several approaches are actively being studied for their ability to stimulate new vessel growth, angiogenesis, as well as to improve the direct interaction of cells with material surfaces. The process of angiogenesis involves the coordinated involvement of both soluble and insoluble factors such as growth factors and cytokines, and extracellular matrix proteins respectively. Often, growth factors and cytokines are expressed by the inflammatory cells associated with the biomedical implants, but the microenvironment within the polymer remains unstable with respect to the presence of the appropriate extracellular matrix proteins. The overall hypothesis of this dissertation is that the reestablishment of an extracellular microenvironment on and within a porous polymer will provide the appropriate substrates for promoting angiogenesis and neovascularization of porous polymers. The results of the studies within this dissertation demonstrate that extracellular matrix modifications of commercially available expanded polytetrafluoroethylene (ePTFE) successfully promote new vessel growth in the tissue surrounding the implant, termed angiogenesis, and new vessel growth within the pores of the polymer, termed neovascularization. Furthermore, the extracellular matrix protein laminin 5 was determined to promote human microvessel endothelial cell adhesion to ePTFE as well as support angiogenesis and neovascularization when used as a surface modification of ePTFE. Based on these studies, the extracellular matrix protein, laminin 5, could be utilized in the tissue engineering of biomedical implant devices to promote increased new vessel integration and improve the long-term viability of these devices.

In young rats, the pattern of neuronal ensemble activity correlations expressed among hippocampal pyramidal cells during behavior, persists during subsequent off-line periods, such as quiet wakefulness and slow-wave sleep. This process may facilitate the consolidation of memories. The present study explored the hypothesis that age-related changes in this process might contribute to age-related memory impairments. Neuronal activity was recorded from CAI pyramidal cells in young and old rats during track running and off-line periods before and after track running. Two methods were used to quantify memory reactivation, one which measured similarities in activity patterns without considering the temporal order of neuronal activity (EV) and one that quantified the off-line preservation of temporal asymmetries formed between neurons during behavior (TB). A consistent similarity between the resting epoch activity patterns and those from the preceding behavior epoch was observed in both age groups, using the EV method. This similarity was strongest during sharp wave events. With the EV method, no age differences in the reactivation process were found in experiments using a familiar environment. In addition, the aged group exhibited greater reactivation versus the young group in the novel track experiments. In light of the observed age-related plasticity deficits these results suggest that memory reactivation, measured by EV, is not dependent on such mechanisms. In contrast, the TB method revealed a significant age-related deficit in the off-line preservation of temporal asymmetries. Thus, it appears that newly formed activity sequences are not preserved during off-line reactivation in aged animals, possibly because the new storage of activity sequences requires intact synaptic plasticity.

Cardiac muscle contraction is critically dependent upon the extensive level of organization of cytoskeletal proteins found in the repeating sarcomeric units of individual myofibrils. Within these units, thick and thin filament systems are assembled and aligned to the precision of single molecules. For years, scientists have been challenged to uncover the mechanisms by which this is accomplished. To date, however, these mechanisms remain relatively unclear due in large part to the lack of suitable in vitro models that faithfully recapitulate the events of myofibril assembly observed in vivo. Several years ago, an avian embryo explant system was developed to investigate other aspects of heart development. Within this system, premyocardial cells differentiate in culture and commence beating in a temporal pattern that corresponds with cardiomyocyte differentiation in vivo. We hypothesized that premyocardial explants could also serve as a particularly advantageous system for investigating myofibrillogenesis. To test this, in Chapter 2, we characterized the temporal/spatial relationships between sarcomeric components during assembly using immunofluorescence microscopy. Our results indicated that events of myofibril assembly in explants mirrored those observed in vivo. Furthermore, these cells are accessible to experimental manipulation (Chapter 5). In Chapter 3, we utilized the precardiac explant system to investigate events of actin (thin) filament assembly during development. Immunofluorescence and ultrastructural analyses revealed that thin filament and sarcomere lengths increase gradually as cardiomyocytes mature. FRAP analyses also demonstrated that the thin filament pointed-end capping activity of E-Tmod is more dynamic during early assembly stages, a property that could dramatically affect the rate of actin monomer exchange/addition during myofibrillogenesis. Research continues in an attempt to identify potential mechanisms regulating E-Tmod dynamics. Finally, in Chapter 4, we investigated the function of a unique elastic region of I-band titin called titin-N2B. In this study, GFP-tagged constructs of titin-N2B were overexpressed in cardiomyocytes in an attempt to disrupt the potential interaction of endogenous N2B with an intracellular ligand. Our results suggested that the NH2-terminal domains of N2B are directly or indirectly critical for stabilizing thin filament structure; thus, N2B emerges as a unique region of titin that is critical for the maintenance of cardiac myofibrils.