Comparative electron impact and fast atom bombardment mass spectrometric studies of some HMPA adducts of phenyltin and phenyllead halides and studies of strong hydrogen bonding by FAB-MS

The fragmentation patterns and mass spectra of some phenyl tin and -lead halide adducts with hexamethylphosphoramide are compared by subjecting them t~ electron impact and fast atom bombardment ionization in a mass spectrometer. This comparison is restricted to the metal-containing ions. Ligand-exchange mechanisms of some of the metal-containing species are explored by FAB-MS. Several moisturesensitive organo-metallics and H-bonded systems have been examined by FAB for attempted characterization, but without any success. Scavenging and trapping of water molecules by complex aggregates in solutions of quaternary ammonium fluorides and hydroxides are investigated by FAB to complement previous NMR-studies.

Interactions of diploid embryonal carcinoma cells and normal cell mass cells with 2.5 day mouse embryos following aggregation

Spontaneous teratocarcinomas are ovarian or testicular tumors which have their origins in germ cells. The tumors contain a disorganized array of benign differentiated cells as well as an undifferentiated population of malignant stem cells, the embryonal carcinoma or EC cells. These pluripotent stem cells in tissue culture share many properties with the transient pluripotent cells of the early embryo, and might therefore serve as models for the investigation of developmental events ill vitro. The property of EC cells of prime interest in this study is an in vivo phenomenon. Certain EC cell lines are known to be regulated ill vivo and to differentiate normally in association with normal embryonic cells, resulting in chimeric mice. These mice have two genetically distinct cell populations, one of which is derived from the originally malignant EC cells. This has usually been accomplished by injection of the EC cells into the Day 3 blastocyst. In this study, the interactions between earlier stage embryos and EC cells have been tested by aggregating clumps of EC cells with Day 2 embryos. The few previous aggregation studies produced a high degree of abnormality in chimeric embryos, but the EC cells employed had known chromosomal abnormalities. In this study, two diploid EC cell lines (P19 and Pi0) were aggregated with 2.5 day mouse embryos, and were found to behave quite differently in the embryonic environment. P19 containing aggregates generally resorbed early, and the few embryos recovered at midgestation were normal and non-chimeric. Pi0 containing aggregates survived in high numbers to midgestation, and the Pi0 cells were very successful in colonizing the embryo. All these embryos were chimeric, and the contribution by the EC cells to each chimera was very high. However, these heavily chimeric embryos were all abnormal. Blastocyst injection had previously produced some abnormal embryos with high Pl0 contributions in addition to the live born mice, which had lower EC contributions. This study now adds more support to the hypothesis that high EC contributions may be incompatible with normal development. The possibility that the abnormalities were due to the mixing of temporally asynchronous embryonic cell types in the aggregates was tested by aggregating normal pluripotent cells taken from 3.5 day embryos with 2.5 day embryos. Early embryo loss was very high, and histological studies showed that the majority of these embryos died by 6.5 days development. Some embryos escaped this early death such that some healthy chimeras were recovered, in contrast to recovery of abnormal chimeric embryos following Pl0-morula aggregations, and non-chimeric embryos following P19-morula aggregations. This somewhat surprising adverse effect on development following aggregation of normal cell types suggests that there are developmental difficulties associated with the mixing of asynchronous cell types in aggregates. However, the greater magnitude of the adverse effects when the aggregates contained tumor derived cells suggests that EC cells should not be considered the complete equivalent of the pluripotent cells of the early embryo.

Polyglutamine monomer structure and its implications for molecular self-assembly

Polyglutamine is a naturally occurring peptide found within several proteins in neuronal cells of the brain, and its aggregation has been implicated in several neurodegenerative diseases, including Huntington's disease. The resulting aggregates have been demonstrated to possess ~-sheet structure, and aggregation has been shown to start with a single misfolded peptide. The current project sought to computationally examine the structural tendencies of three mutant poly glutamine peptides that were studied experimentally, and found to aggregate with varying efficiencies. Low-energy structures were generated for each peptide by simulated annealing, and were analyzed quantitatively by various geometry- and energy-based methods. According to the results, the experimentally-observed inhibition of aggregation appears to be due to localized conformational restraint placed on the peptide backbone by inserted prolines, which in tum confines the peptide to native coil structure, discouraging transition towards the ~sheet structure required for aggregation. Such knowledge could prove quite useful to the design of future treatments for Huntington's and other related diseases.

The Fast Regulation of Photosynthesis in Diatoms: an inquiry into the physiological and physical origins of non-photochemical chlorophyll fluorescence quenching.

Diatoms are renowned for their robust ability to perform NPQ (Non-Photochemical Quenching of chlorophyll fluorescence) as a dissipative response to heightened light stress on photosystem II, plausibly explaining their dominance over other algal groups in turbulent light environs. Their NPQ mechanism has been principally attributed to a xanthophyll cycle involving the lumenal pH regulated reversible de-epoxidation of diadinoxanthin. The principal goal of this dissertation is to reveal the physiological and physical origins and consequences of the NPQ response in diatoms during short-term transitions to excessive irradiation. The investigation involves diatom species from different originating light environs to highlight the diversity of diatom NPQ and to facilitate the detection of core mechanisms common among the diatoms as a group. A chiefly spectroscopic approach was used to investigate NPQ in diatom cells. Prime methodologies include: the real time monitoring of PSII excitation and de-excitation pathways via PAM fluorometry and pigment interconversion via transient absorbance measurements, the collection of cryogenic absorbance spectra to measure pigment energy levels, and the collection of cryogenic fluorescence spectra and room temperature picosecond time resolved fluorescence decay spectra to study excitation energy transfer and dissipation. Chemical inhibitors that target the trans-thylakoid pH gradient, the enzyme responsible for diadinoxanthin de-epoxidation, and photosynthetic electron flow were additionally used to experimentally manipulate the NPQ response. Multifaceted analyses of the NPQ responses from two previously un-photosynthetically characterised species, Nitzschia curvilineata and Navicula sp., were used to identify an excitation pressure relief ‘strategy’ for each species. Three key areas of NPQ were examined: (i) the NPQ activation/deactivation processes, (ii) how NPQ affects the collection, dissipation, and usage of absorbed light energy, and (iii) the interdependence of NPQ and photosynthetic electron flow. It was found that Nitzschia cells regulate excitation pressure via performing a high amplitude, reversible antenna based quenching which is dependent on the de-epoxidation of diadinoxanthin. In Navicula cells excitation pressure could be effectively regulated solely within the PSII reaction centre, whilst antenna based, diadinoxanthin de-epoxidation dependent quenching was implicated to be used as a supplemental, long-lasting source of excitation energy dissipation. These strategies for excitation balance were discussed in the context of resource partitioning under these species’ originating light climates. A more detailed investigation of the NPQ response in Nitzschia was used to develop a comprehensive model describing the mechanism for antenna centred non-photochemical quenching in this species. The experimental evidence was strongly supportive of a mechanism whereby: an acidic lumen triggers the diadinoxanthin de-epoxidation and protonation mediated aggregation of light harvesting complexes leading to the formation of quencher chlorophyll a-chlorophyll a dimers with short-lived excited states; quenching relaxes when a rise in lumen pH triggers the dispersal of light harvesting complex aggregates via deprotonation events and the input of diadinoxanthin. This model may also be applicable for describing antenna based NPQ in other diatom species.

HG 198.20-198.35 Thin Section

Coarse grained brown sediment with angular-sub-angular grains. Contains mainly medium sized clasts with a few smaller aggregates. A dark organic rich domain can be seen within this sample. Grain crushing (edge-to-edge and into one another) can be seen alongside with grain stacks, and silt caps.

HG 276.80-276.95 Thin Section

Coarse grained sample, with grains ranging from small to large in size. Shapes range from angular to rounded. This sample is abundant in lineations, and rotation structures can also be seen around larger aggregates. Many large grains are fractured and minor grain stacking can also be seen.

GI

Dark brown sediment with clasts ranging from small to large. Clast shape ranges from angular to rounded. Rotation structures are abundant in this sample and mainly occur around medium sized and large aggregates. Comet structures can also be seen throughout the sample. Lineations and minor amounts of grain crushing can also be seen.

AB-2

Brown sediment with clasts ranging from small to large in size. Clast shape ranges from sub-angular to sub-rounded. The clasts are generally well dispersed, with a few areas of closer aggregates. Lineations are common throughout the sample. A few rotation structures and a few areas of edge-to-edge grain crushing can also be seen. Comet structures can also be seen in very minor amounts.

WB-4

Brown sediment with clasts ranging from small to large in size. Clast shape ranges from sub-angular to sub-rounded. Rotation structures are abundant in this sample. They are seen around small and large clasts. A few necking structures can also be seen. These typically occur between larger aggregates. Lineations, comet structures, and grain crushing can also be seen in minor amounts.

WB-6a

Brown sediment with clasts ranging from small to large in size. Clast shape ranges from angular to sub-rounded. Necking structures are abundant within this sample, mainly between larger aggregates. Some rotation structures and lineations can also be seen. Comet structure and grain stacking can be seen in minor amounts.

WB-6b

Dark brown sediment with clasts ranging from small to large in size. Grain shape ranges from sub-angular to rounded. Rotation structures are common throughout the sample. Some necking structures are also present between large and medium sized aggregates. Lineations and grain stacks are also present in minor amounts.

11 PTA0011

Dark grey sediment with clasts ranging from small to large size. Grain shape ranges from angular to rounded. Many grains are fractured and weathered. Necking structures, along with rotation structures, are very common in this sample. They can be seen between many of the larger aggregates. Lineations and grain crushing are also present throughout.

11 PTA0013H

Dark grey sediment sample with clasts ranging from small to large in size. Clast shape ranges from angular to rounded. Lineations and grain crushing are the most abundant. Necking structures and rotation structures can also be seen throughout the sample mainly between/surrounding larger aggregates. A few comet structures can also be seen.

WR-1

Dark brown sediment with clasts ranging from small to large. The sample is mostly made up of large clasts. Clast shape ranges from angular to sub-angular. Necking structures are common throughout this sample and can mainly be seen between larger aggregates. Edge-to-edge grain crushing, crushed grains, and some lineations can also be seen.

WR-2

Dark brown sediment with clasts ranging from small to large in size. Clast shape ranges from angular to sub-rounded. The main domain mainly contains larger aggregates. There is one domain inclusion in this sample. It mainly contains small and medium sized clasts, and contains many lineations. Necking structures can be commonly seen in the main domain between larger aggregates. This sample also contains many elongated clasts and inclusions of clay material.