Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl b-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 µM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 µM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.

Conservation ecology and breeding biology of the white-browed treecreeper, Climacteris affinis

The White-browed Treecreeper Climacteris affinis is one of many woodland-dependent birds that are at risk from the encroachment of human-dominated land-uses into natural landscapes. The White-browed Treecreeper inhabits semi-arid woodlands in north-west Victoria, Australia, a vegetation community that has undergone extreme modification in the last century due to the expansion of agriculture in the region. Extant woodlands represent only 10% of the original woodland cover in the region, and are highly fragmented and disturbed in many districts. Thus, the survival of the White-browed Treecreeper may depend on active management. However, current knowledge of the ecology and biology of this species is virtually non-existent, and inadequate for informed and effective conservation actions. The aim of this thesis is to redress this situation and provide the ecological basis for sound conservation management of the species. The thesis consists of two parts: an investigation of habitat use at three spatial scales and a study of the social organization, nesting requirements, breeding behaviour and reproductive success of a population of White-browed Treecreepers. Fifty-six patches of remnant woodland in north-west Victoria were surveyed to determine the factors affecting the occurrence of the White-browed Treecreeper at the regional scale. It was detected in 16 patches, and was largely confined to two core districts - Yarrara and, Wyperfeld (Pine Plains). The floristic composition of the dominant tree species was an important determinant of patch occupancy, with the results providing quantitative support for the previously suspected affinity for Belah Casuarina pauper and Slender Cypress-pine Callitris gracilis — Buloke Allocasuarina luehmannii woodlands. However, the absence of the White-browed Treecreeper from several districts was due to factors other than a lack of appropriate habitat. Demographic isolation - the distance from the focal patch to the nearest population of the White-browed Treecreeper - was the most important variable in explaining variation in patch occupancy. Patches isolated from other treecreeper populations by more than 8.3 km in landscapes of non-preferred native vegetation, and 3 km in agricultural landscapes, were unlikely to support the White-browed Treecreeper. The impact of habitat loss and fragmentation on the capacity of individuals to move through the landscape (i.e. functional connectivity) is considered in relation to disruption to dispersal and migration, and the potential collapse of local metapopulations. Habitat use was then examined in a network of patches and linear strips of Belah woodland embedded in a predominantly cultivated landscape. A minimum area of 18.5 ha of Belah woodland was identified as the most important criterion for patch occupancy at the local scale. This landscape appeared to be permeable to movement by the White-browed Treecreeper, facilitated by the extensive network of linear habitat, and clusters of small to medium fragments. The third scale of habitat use investigated the frequency of use of 1-ha plots within tracts of occupied woodland. It is important to discriminate between habitat traits that operate at the population level, and those that act as proximate cues for habitat selection by individuals. Woodlands that have high tree density, extensive cover of low-stature shrubs, abundant lichen, a complex vertical structure, and relatively low cover of grass and herbs are likely to support larger populations of the White-browed Treecreeper. However, individuals appeared to be using tree dominance (positive) and tall shrub cover (negative) as proximate environmental stimuli for habitat selectivity. A relatively high cover of ground lichen, which probably reflects a ground layer with low disturbance and high structural complexity, was also a reliable indicator of habitat use. Predictive models were developed which could be used to plan vegetation management to enhance habitat for the White-browed Treecreeper. The results of the regional, landscape and patch-scale investigations emphasise that factors operating at multiple spatial scales influence the suitability of remnant vegetation as habitat for the White-browed Treecreeper. The White-browed Treecreeper is typical of many small Australian passerines in that it has high annual survival, small clutches, a long breeding season, multiple broods and relatively low reproductive rates. Reproductive effort is adjusted through the number of clutches laid rather than clutch size. They occupy relatively large, all-purpose territories throughout the year. However, unlike many group territorial birds, territory size was not related to the number of occupants. The White-browed Treecreeper nests in tree hollows. They select hollows with a southerly orientation where possible, and prefer hollows that were higher from the ground. At Yarrara, there was considerable spatial variation in hollow abundance that, in concert with territorial constraints, restricted the actual availability of hollows to less than the absolute abundance of hollows. Thus, the availability of suitable hollows may limit reproductive productivity in some territories, although the magnitude of this constraint on overall population growth is predicted to be small. However, lack of recruitment of hollow-bearing trees would increase the potential for hollow availability to limit population growth. This prospect is particularly relevant in grazed remnants and those outside the reserve system. Facultative cooperative breeding was confirmed, with groups formed through male philopatry. Consequently, natal dispersal is female-biased, although there was no skew in the sex ratio of the fledglings or the general adult population. Helpers were observed performing all activities associated with parenting except copulation and brooding. Cooperatively breeding groups enjoyed higher fledgling productivity than simple pairs, after statistically accounting for territory and parental quality. However, the difference reflected increased productivity in the 1999-breeding season only, when climatic conditions were more favourable than in 1998. Breeding commenced earlier in 1999, and all breeding units were more likely to attempt a second brood. However, only breeders with helpers were successful in fledging second brood young, and it was this difference that accounted for the overall discrepancy in productivity. The key mechanism for increased success in cooperative groups was a reduction hi the interval between first and second broods, facilitated by compensatory reductions in the level of care to the first brood. Thus, females with helpers probably achieved significant energetic savings during this period, which enabled them to re-lay sooner. Furthermore, they were able to recommence nesting when the fledglings from the first brood were younger because there were more adults to feed the dependent juveniles. The current utility, and possible evolutionary pathways, of cooperative breeding is examined from the perspective of both breeders and helpers. Breeders benefit through enhanced fledgling productivity in good breeding conditions and a reduction in the burden of parental care, which may impart significant energetic savings. Further, breeders may facilitate philopatry as a means for ensuring a minimum level of reproductive success. Helpers benefit through an increase in their inclusive fitness in the absence of opportunities for independent breeding (i.e. ecological constraints) and access to breeding vacancies in the natal or adjacent territories (i.e. benefits of philopatry). However, the majority of breeding unit-years comprised unassisted breeders, which suggests that pairs are selectively favoured under certain environmental or demographic conditions.

Influence of mechanical mixing rates on sludge characteristics and membrane fouling in MBRs

The influence of shear intensity (G) induced by mechanical mixing on activated sludge characteristics as well as membrane fouling propensity in membrane bioreactors (MBRs) was investigated. Four MBRs were operated at different mechanical mixing conditions. The control reactor (MBR0) was operated with aeration only supplemented by mechanical stirring at 150, 300, and 450 rpm in MBR150, MBR300, and MBR450, respectively. It was found that the MBR300 demonstrated minimum rate of membrane fouling. The fouling potential of the MBR300 mixed liquor was lowest characterized by the specific cake resistance and the normalized capillary suction time (CSTN). Moreover, it was found that the mean particle size reduced with an increase in the shear intensity. These results reveal that membrane fouling can be significantly mitigated by appropriate shear stress on membrane fibers induced by mechanical mixing condition.

Metabolic rate and membrane fatty acid composition in birds : a comparison between long-living parrots and short-living fowl

Both basal metabolic rate (BMR) and maximum lifespan potential (MLSP) vary with body size in mammals and birds and it has been suggested that these are mediated through size-related variation in membrane fatty acid composition. Whereas the physical properties of membrane fatty acids affect the activity of membrane proteins and, indirectly, an animal's BMR, it is the susceptibility of those fatty acids to peroxidation which influence MLSP. Although there is a correlation between body size and MLSP, there is considerable MLSP variation independent of body size. For example, among bird families, Galliformes (fowl) are relatively short-living and Psittaciformes (parrots) are unusually long-living, with some parrot species reaching maximum lifespans of more than 100 years. We determined BMR and tissue phospholipid fatty acid composition in seven tissues from three species of parrots with an average MLSP of 27 years and from two species of quails with an average MLSP of 5. 5 years. We also characterised mitochondrial phospholipids in two of these tissues. Neither BMR nor membrane susceptibility to peroxidation corresponded with differences in MLSP among the birds we measured. We did find that (1) all birds had lower n-3 polyunsaturated fatty acid content in mitochondrial membranes compared to those of the corresponding tissue, and that (2) irrespective of reliance on flight for locomotion, both pectoral and leg muscle had an almost identical membrane fatty acid composition in all birds.

On the growth and reproductive biology of asp, Aspius vorax, population from the middle reaches of Euphrates river

Endemic asp, Aspius vorax, from the middle section of the Euphrates River flowing through eastern Syria were studied to determine the main characteristics of their population structure, morphological parameters and reproductive biology. Samples ranged between 0+ and 4+ years of age and were dominated by 2+ years old group. Total length (TL) ranged between 19 and 70 cm corresponding with 46 to 2824.5 g weight, respectively. Fish growth has isometric pattern and the overall sex ratio was unbiased. Seasonal changes in the condition factor were related with the water temperature as well as the spawning season. Annual cycle of gonadosomatic index (GSI) readings indicated that spawning season occur around March when fish longer than 36 cm can mate. Average pre-spawning GSI was greater in individuals older than 2 years. Meanwhile, female fecundity was highly related to TL and weight. These findings did not always concur with previous observations from other asp populations, mainly in southern and northern Mesopotamia. Our results highlighted basic biological aspects of the local population and indicated differences between populations which can assist in fisheries management, conservation and commercial culture of the investigated species.

Effect of feed temperature and membrane orientation on pre-treatment sludge volume reduction through forward osmosis

This study focuses on volume reduction of pre-treatment sludge as well as on dilution of reverse osmosis (RO) concentrate through emerging forward osmosis (FO) technology where RO concentrate draws water from the pre-treatment sludge (feed solution) in order to reduce pre-treatment sludge volume and increase the RO water recovery. Experiments were carried out using two different types of sludge i.e. (1) synthetic pre-treatment sludge (Lab sludge) which has lower salinity and (2) actual sludge from Perth Seawater Desalination Plant, Australia (Perth Seawater Desalination Plant (PSDP) sludge) which has higher salinity. Effect of membrane orientation (FO and pressure-retarded osmosis (PRO) modes) and temperature of pre-treatment sludge on permeate water flux was investigated. There was a significant increase in water flux from 3.2 to 10.2 LMH (i.e. ~3 times higher) when temperature increased from 20 to 40°C for Lab sludge in PRO mode. However, there is no significant effect of temperature on water flux in FO mode for Lab sludge. On the contrary for PSPD sludge, there was no effect on water flux with increase in temperature at PRO mode. Dissolved ions in the porous side increased the severity of concentrative internal concentration polarization; hence, it could reduce the flux. There was no significant change in water flux when temperature increased from 20 to 40°C for PSDP sludge in FO mode. However, higher amount of water has permeated from Lab sludge compared to PSDP sludge in FO mode. © 2014 © 2014 Balaban Desalination Publications. All rights reserved.

Reuse of car wash wastewater by chemical coagulation and membrane bioreactor treatment processes

Car wash wastewater contains significant concentrations of contaminants such as nutrients, organics, particulate matter, sand, oil, grease, diesel detergents and so on. A range of treatment processes such as a membrane bioreactor (MBR), coagulation and ozonation were investigated to treat car wash wastewater. Ozonation was effective in removing the chemicals and suspended solids; the removal efficiency was greater than the coagulation process. Once the MBR system was acclimatised, 100% of suspended solids, 99.2% of COD, 97.3% of TOC and 41% of ammonia were removed. This study demonstrates that MBR is a potentially promising treatment system for recycling car wash wastewater which could be reused in the same car wash station.

Breeding biology, adult survival and territoriality of the white-browed treecreeper (climacteris affinis) in north-west Victoria, Australia

This is the first study to present empirical data describing the social organisation and breeding biology of the White-browed Treecreeper (Climacteris affinis). The species is typical of many small Australian passerines in that it has high annual survival (~80%), small clutches (mean = 1.95 ± 0.05), long breeding seasons (eggs laid August to November) and long incubation (17–18 days) and nestling periods (25–26 days), corrected for body weight. Reproductive effort is modified in response to variation in climatic conditions by adjusting the commencement of breeding and number of clutches laid per season, which is facilitated by an extended breeding season. White-browed Treecreepers occupied relatively large (mean = 8.4 ± 0.8 ha), all-purpose territories throughout the year. However, unlike many group territorial birds, territory size was not related to the number of occupants. The role of food limitation and climatic variability in relation to territory dispersion and life-history traits is explored. Facultative cooperative breeding was confirmed. Cooperative groups were formed through male philopatry, with usually only one, but up to three, male helpers present in a moderate fraction (35%) of breeding units. Thus, all species of Climacteris are now confirmed as facultative cooperatively breeding species, which provides further evidence for the aggregation of cooperative breeders at the generic level in mixed (i.e. cooperative and pair breeders) phylogenetic clades. In C. affinis, males may attain breeding positions through inheritance of their natal territory or by filling vacancies in nearby territories. Females obtained breeding positions by ‘floating’ as non-breeding residents in established territories, waiting for a vacancy to arise.