Kv7 Channels Can Function without Constitutive Calmodulin Tethering

9 p.

Regulation of Energy Metabolism by the Extracytoplasmic Function (ECF) σ Factors of Arcobacter butzleri

11 p.

Influence of seagrass landscape structure on the juvenile blue crab habitat-survival function

Organismal survival in marine habitats is often positively correlated with habitat structural complexity at local (within-patch) spatial scales. Far less is known, however, about how marine habitat structure at the landscape scale influences predation and other ecological processes, and in particular, how these processes are dictated by the interactive effect of habitat structure at local and landscape scales. The relationship between survival and habitat structure can be modeled with the habitat-survival function (HSF), which often takes on linear, hyperbolic, or sigmoid forms. We used tethering experiments to determine how seagrass landscape structure influenced the HSF for juvenile blue crabs Callinectes sapidus Rathbun in Back Sound, North Carolina, USA. Crabs were tethered in artificial seagrass plots of 7 different shoot densities embedded within small (1 – 3 m2) or large (>100 m2) seagrass patches (October 1999), and within 10 × 10 m landscapes containing patchy (<50% cover) or continuous (>90% cover) seagrass (July 2000). Overall, crab survival was higher in small than in large patches, and was higher in patchy than in continuous seagrass. The HSF was hyperbolic in large patches and in continuous seagrass, indicating that at low levels of habitat structure, relatively small increases in structure resulted in substantial increases in juvenile blue crab survival. However, the HSF was linear in small seagrass patches in 1999 and was parabolic in patchy seagrass in 2000. A sigmoid HSF, in which a threshold level of seagrass structure is required for crab survival, was never observed. Patchy seagrass landscapes are valuable refuges for juvenile blue crabs, and the effects of seagrass structural complexity on crab survival can only be fully understood when habitat structure at larger scales is considered.

Use of generalised additive models to categorise continuous variables in clinical prediction

13 P.

Structure-function studies of serine hydrolases: synthesis of a gene for ∝-lytic protease and the purification and characterization of a mutant β-lactamase

The author has constructed a synthetic gene for ∝-lytic protease. Since the DNA sequence of the protein is not known, the gene was designed by using the reverse translation of ∝-lytic protease's amino acid sequence. Unique restriction sites are carefully sought in the degenerate DNA sequence to aid in future mutagenesis studies. The unique restriction sites are designed approximately 50 base pairs apart and their appropriate codons used in the DNA sequence. The codons used to construct the DNA sequence of ∝-lytic protease are preferred codons in E-coli or used in the production of β-lactamase. Codon usage is also distributed evenly to ensure that one particular codon is not heavily used. The gene is essentially constructed from the outside in. The gene is built in a stepwise fashion using plasmids as the vehicles for the ∝-lytic oligomers. The use of plasmids allows the replication and isolation of large quantities of the intermediates during gene synthesis. The ∝-lytic DNA is a double-stranded oligomer that has sufficient overhang and sticky ends to anneal correctly in the vector. After six steps of incorporating ∝-lytic DNA, the gene is completed and sequenced to ensure that the correct DNA sequence is present and that no mutations occurred in the structural gene.

β-lactamase is the other serine hydrolase studied in this thesis. The author used the class A RTEM-1 β- lactamase encoded on the plasmid pBR322 to investigate the roll of the conserved threonine residue at position 71. Cassette mutagenesis was previously used to generate all possible amino acid substitutions at position 71. The work presented here describes the purification and kinetic characterization of a T71H mutant previously constructed by S. Schultz. The mutated gene was transferred into plasmid pJN for expression and induced with IPTG. The enzyme is purified by column chromatography and FPLC to homogeneity. Kinetic studies reveal that the mutant has lower k_(cat) values on benzylpenicillin, cephalothin and 6-aminopenicillanic acid but no changes in k_m except for cephalothin which is approximately 4 times higher. The mutant did not change siginificantly in its pH profile compared to the wild-type enzyme. Also, the mutant is more sensitive to thermal denaturation as compared to the wild-type enzyme. However, experimental evidence indicates that the probable generation of a positive charge at position 71 thermally stabilized the mutant.