Thesis (Ph. D.)--Lehigh University, 2006. / Includes vita. Includes bibliographical references (leaves 117-125).

No description available.

Th.--Chim. phys.--Nancy--I.N.P.L., 1985.

Thesis (Ph. D.)--Lehigh University, 2000. / Includes vita. Includes bibliographical references (leaves 146-152).

Th. doct.-ing.--Toulouse 3, 1978. N°: 628.

Th. doct.-ing.--Nancy, I.N.P.L., 1978.

No description available.

The first part of this thesis consists of a study of the transport of alkali metal cations across model biological membranes mediated by ionophoric antibiotic cationomycin. The transport rates were measured by 23Na and 39K NMR using large unilamellar vesicles (LUVs) prepared from phosphatidylcholine and were compared with results obtained using red blood cells. These kinetic studies have established that there is a 1:1 complex between alkali cations and the cationomycin during the transport process. The transport rate measured are comparable to those of other ionophoric antibiotics studied previously. Cationomycin transports potassium cation more rapidly than sodium cation, and forms a more stable complex with potassium cation. The rate limiting step in the transport process is release of the cation at the membrane surface. The second part involves the study of spirotetrahydrofuran analogues as potential alkali cation transporters. A set of new synthetic ionophoric materials designed to resemble to some degree the naturally occurring ionophoric antibiotics was investigated. The kinetic rates of these compounds were measured in phosphatidylcholine LUVs by an NMR study of 7Li/23Na exchange. However, these compounds are much poorer transporters than the ionophoric antibiotics which are 10 times faster under similar conditions. The third part of this project deals with the study of the transport of halide anions by steroid-based anionophores. The transport rates of these compounds were measured by an NMR study of 35Cl/81Br exchange in phosphatidylcholine LUVs. This preliminary study indicates a close relationship between the lipophilicity of the different anionophores and the transport rates of different halides anions through the model membrane. The final part of this thesis involves the total assignment of the 1H and 13C NMR spectra of the ionophoric antibiotic tetronasin by utilising two dimensional NMR techniques such as COSY 45, Relay COSY, DQF COSY, TOCSY and HSQC.

The crnA gene of A.nidulans is confirmed to encode a transport protein, possibly for nitrate. The single open reading frame of 1449 nt encodes a polypeptide of 483 AAs with a molecular weight of 51,769 Da. The high percentage of clustered apolar AAs suggests a membrane protein with ten membrane spanning domains. No similarity between the crnA encoded polypeptide and those of other transport genes or any other gene was observed. The regulation of the A.nidulans nitrate transporter was investigated by studying levels of mRNA transcribed from the crnA gene. The crnA cDNA hybridised to two mRNAs on Northern blots. The 1.8 kb message corresponding to the size of the crnA gene is inducible with nitrate and nitrite. The 1.1 kb, constitutively synthesized message is believed to be a second transcript from the crnA gene rather than from a gene elsewhere in the genome. The expression of the crnA gene, determined from Northern blotting experiments, is shown to be at the level of mRNA accumulation. A requirement for both positive acting regulatory genes, areA and nirA is observed. The expression of the niiA and niaD genes, encoding nitrite reductase and nitrate reductase respectively, is shown to be regulated at the level of mRNA accumulation by a mechanism involving both the areA and nirA gene products. In addition, the nitrate reductase apoenzyme influences the expression of all three structural genes crnA, niiA and niaD. This effect is also exerted at the level of mRNA accumulation. Sequences upstream of the niaD and niiA genes from Aspergillus oryzae, A.nidulans and A.niger were compared, in addition to a similar region of the crnA gene. In particular, one sequence of ten nucleotides was found to share substantial similarity between each gene. It is possible that this motif is a cis-acting transcription factor recognised by a regulatory protein. The consensus TATCTA, a possible receptor site for the areA gene product, was identified several times upstream of each gene. The nit-4: gene of Neurospora crassa is shown to complement the A.nidulans nirA loss of function mutant via genetic transformation. The three transformant strains each have multiple integrates of the nit-4 gene, although none are present at the homologous site. The regulation of A.nidulans nitrate reductase by the N.crassa nit-4 gene is observed to be essentially normal, i.e. wild type.

Planococcus C.C.M. 316, a gram-positive facultative marine halophile, was studied with respect to growth and membrane composition of cells grown in media containing 0.5%, 3% and 10% sea salt. Membranes were prepared from cells grown in the three sea salt concentrations and analysed to determine any changes which may have been caused by the increasing concentrations of salt in the growth media. The three membrane preparations were found to have similar compositions to those reported for other gram-positive cocci. Cells grown in the 3% sea salt concentration contained membranes with a higher protein:lipid ratio and RNA content than the membranes from cells grown in the 0.5% and 10% concentrations. Amino acid analysis of the membrane proteins showed that the composition remained virtually unchanged in the three membrane preparations. The ratio of acidic: basic amino acid residues was nearer to the figures reported for non-halophiles than for those of the extreme halophiles. Examination of the lipids showed that phospholipids predominated to the extent of about 70% of the total lipids. Cardiolipin and lysocardiolipin were the major phospholipids, with phosphatidyl ethanolamine, phosphatidyl glycerol and phosphatidyl serine present as minor components. Glyco-lipids were found to decrease with increasing sea salt concentration in the medium, and in all three membrane preparations constituted only a very small proportion of the total lipids. Neutral lipids contained long chain alcohols, mono-, di- and tri-glycerides, as well as relatively large amounts of the isoprenoid compound squalene. The major fatty acid associated with the lipids was a branched saturated C15 acid which constituted 50 - 7% of the total fatty acids in most fractions. Although increasing salt in the medium produced changes within the proteins and lipids in the membranes, these changes were not such that they could be interpreted as an increase in the halophilic nature of the membrane. The carotenoids were shown to be derived from beta carotene and to consist mainly of 3'hydroxy 4' oxo compounds, although the extent of polar substitutions was dependent on both culture age and the concentration of salt in the medium.