Academic literature on the topic '069999 Biological Sciences not elsewhere classified'

Insect communities in tropical forests tend to be structured vertically and with respect to tree fall gaps and edges. Furthermore, insect communities vary over time. Insight into such habitat specificity and temporal variation is needed to design and interpret biodiversity surveys and to compare conservation value among habitats. Some aspects of tropical insect community structure, such as the proportion of canopy specialists and temporal variation, vary among biogeographical regions and climatic zones. To date, few regions have been sampled systematically, so generalization remains difficult. We compared fruit-feeding butterfly communities among understory, canopy, natural treefalls, and forest edge, in a tropical forest of the Western Ghats, a strip of rainforest that is isolated from Sundaland, the large rainforest block of South-East Asia. During a yearlong study, we captured 3018 individuals belonging to 32 species and representing 14 genera. While some butterflies were captured in the canopy, no species was significantly more abundant in the canopy than in the understory. This observation was contrary to studies elsewhere in the tropics where 14–55% of the species could be classified as canopy specialists. Even though the largest number of species was captured at forest edges, species diversity was highest in the gaps. The communities at the forest edge differed importantly from those in treefall gaps: at the forest edge, we caught grassland species in addition to the forest species. Larger treefall gaps had higher butterfly abundance than smaller gaps. Both abundance and diversity peaked during the late monsoon season, and all common species in our sample also peaked during this period. The spatiotemporal community structure appears to depend on biogeography (less vertical stratification further from large forest blocks) and climate (more synchrony among species in seasonal abundance when there is a more severe dry season).

Signet-ring cell carcinoma of the prostate gland (SRCCP) an uncommon and aggressive malignant tumour of the prostate gland which is characterized by histopathology examination features of compression of the nucleus into the form of a crescent by a large cytoplasmic vacuole. SRCCPs that have so far been reported have been either (a) primary tumours, metastatic tumours with the primary tumour elsewhere with the gastro-intestinal tract being the site of the primary tumour but the primary tumour could originate elsewhere, and additionally some reported SRCCPs have been classified as carcinoma of unknown primary. SRCCP could be a pure tumour or a tumour that is contemporaneously associated with other types of tumour including various variants of adenocarcinoma. SRCCP can manifest in various ways including: Incidental finding following prostatectomy that has been undertaken for a presumed benign prostatic hyperplasia, lower urinary tract symptoms, visible and non-visible haematuria, raised levels of serum PSA but some SRCCPs have been diagnosed with normal / low levels of serum PSA, there may be a history of dyspepsia in cases of metastatic signet-ring cell carcinoma in association with contemporaneous primary signet-ring cell carcinoma of the stomach or there may be a past history of surgical treatment for signet-ring cell carcinoma of the gastrointestinal tract, or bleeding from the gastrointestinal tract in cases of upper gastrointestinal tract and rectal bleeding as well as change in bowel habit for primary tumours of the anorectal region, retention of urine, and rarely a rectal mass in the case of SRCCP with an anorectal primary tumour. In order to exclude a primary signet ring cell carcinoma elsewhere, a detailed past medical history is required as well as radiology imaging including contrast – enhanced computed tomography (CECT) scan and contrast-enhanced magnetic resonance imaging (CEMRI) scan as well as upper gastrointestinal endoscopy and colonoscopy to exclude a primary lesion within the gastrointestinal tract. Diagnosis of SRCCP requires utilization of the histopathology and immunohistochemistry examination features of prostate biopsy, prostatic chips obtained from trans-urethral resection of prostate specimen or radical prostatectomy specimen. SRCCPs upon immunohistochemistry staining studies tend to show tumour that tend to exhibit positive staining for the following tumour markers as follows: PSA – positive staining for PSA has been variable in some studies, AE1/AE3, CAM 5.2, Ki-67 with a mean of 8%, PAS-diastase, Mucicarmine (50%), Alcian blue (60%), Alpha-methyl-acyl coenzyme A racemase (P504S), and Cytokeratin 5/6. SRCCPs also tend to exhibit negative staining for: Bcl2 (rare positive), and CEA (80%). Traditionally the treatment of Primary Signet-Ring Cell Carcinoma of the Prostate Gland has tended to be similar to the treatment of the traditional adenocarcinoma of the prostate gland which does include: hormonal treatment, radiotherapy, and surgery. Nevertheless, considering that primary SRCCPs and metastatic SRCCPs that have been reported in the literature have generally tended to be associated with an aggressive biological behaviour, even though there is no consensus opinion on the treatment of the disease it would be strongly recommended that these tumours that tend to be associated with rapid progress of the disease and poor survival there is an urgent need to treat all these tumours with aggressive surgery including radical prostatectomy plus adjuvant therapies including: radical radiotherapy, combination chemotherapy, selective prostatic angiography and super-selective embolization of the artery feeding the tumour including intra-arterial infusion of chemotherapy agents directly to the tumour, radiofrequency ablation of the tumour as well as irreversible electroporation of the tumour which should form part of a global multicentre study of various treatment options. With regard to metastatic signet-ring cell carcinomas of the prostate gland with a contemporaneous primary tumour elsewhere the primary tumour should also be treated by radical and complete excision of the primary tumour plus radical surgery and aggressive adjuvant therapy. Considering that SRCCPs have tendered not to respond well to available chemotherapy agents, there is need for urologists, oncologists, and pharmacotherapy research workers to identify new chemotherapy medicaments that would more effectively and safely destroy signet-ring cell tumours in order to improve upon the prognosis.

BackgroundThe diet of the koala (Phascolarctos cinereus) is comprised almost exclusively of foliage from the genusEucalyptus(family Myrtaceae).Eucalyptusproduces a wide variety of potentially toxic plant secondary metabolites which have evolved as chemical defences against herbivory. The koala is classified as an obligate dietary specialist, and although dietary specialisation is rare in mammalian herbivores, it has been found elsewhere to promote a highly-conserved but low-diversity gut microbiome. The gut microbes of dietary specialists have been found sometimes to enhance tolerance of dietary PSMs, facilitating competition-free access to food. Although the koala and its gut microbes have evolved together to utilise a low nutrient, potentially toxic diet, their gut microbiome has not previously been assessed in conjunction with diet quality. Thus, linking the two may provide new insights in to the ability of the koala to extract nutrients and detoxify their potentially toxic diet.MethodThe 16S rRNA gene was used to characterise the composition and diversity of faecal bacterial communities from a wild koala population (n = 32) comprising individuals that predominately eat either one of two different food species, one the strongly preferred and relatively nutritious speciesEucalyptus viminalis, the other comprising the less preferred and less digestible speciesEucalyptus obliqua.ResultsAlpha diversity indices indicated consistently and significantly lower diversity and richness in koalas eatingE. viminalis. Assessment of beta diversity using both weighted and unweighted UniFrac matrices indicated that diet was a strong driver of both microbial community structure, and of microbial presence/absence across the combined koala population and when assessed independently. Further, principal coordinates analysis based on both the weighted and unweighted UniFrac matrices for the combined and separated populations, also revealed a separation linked to diet. During our analysis of the OTU tables we also detected a strong association between microbial community composition and host diet. We found that the phyla Bacteroidetes and Firmicutes were co-dominant in all faecal microbiomes, with Cyanobacteria also co-dominant in some individuals; however, theE. viminalisdiet produced communities dominated by the generaParabacteroidesand/orBacteroides, whereas theE. obliqua-associated diets were dominated by unidentified genera from the family Ruminococcaceae.DiscussionWe show that diet differences, even those caused by differential consumption of the foliage of two species from the same plant genus, can profoundly affect the gut microbiome of a specialist folivorous mammal, even amongst individuals in the same population. We identify key microbiota associated with each diet type and predict functions within the microbial community based on 80 previously identifiedParabacteroidesand Ruminococcaceae genomes.

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. These enzymes have great economical potential in industrial, agricultural, chemical, pharmaceutical, and biotechnological applications. Thus, the halophiles isolated from Chott Tinsilt offer an important potential for application in microbial and enzyme biotechnology. In addition, these halo bacterial biosurfactants producers isolated from this Chott will help to develop more valuable eco-friendly products to the pharmacological and food industries and will be usefulness for bioremediation in marine environment and petroleum industry.ACKNOWLEDGMENTSOur thanks to Professor Abdelhamid Zoubir for proofreading the English composition of the present paper.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Akbari, S., N. H. Abdurahman, R. M. Yunus, F. Fayaz and O. R. Alara, 2018. Biosurfactants—a new frontier for social and environmental safety: A mini review. Biotechnology research innovation, 2(1): 81-90.Association, A. P. H., A. W. W. Association, W. P. C. 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Nowadays visual information is extracted using a variety of physical methods and cannot always be easily perceived by the human eye, properly recognized and classified. Such large amounts of information provided by the modern image-making equipment is difficult and sometimes impossible objectively evaluate. Research of digital images carried out at the Klaipėda University with a focus on marine research topic. The combination of new underwater technology as remotely operating vehicles, high-resolution video imagery, and seafloor mosaics creating provides new opportunities for marine geological or biological studies. While these underwater techniques are now well-engineered, there is still a lack of methods for the automatic analysis of the acquired image data. In this paper, the video mosaic quality and processing problems discussed. Believes that the versatility of abstract techniques to tailor the fields of biomedical sciences, applied robotics and elsewhere. This could be listed as a main purpose of presented work.

AbstractThe fungal pathogens of spruce are well known in Europe and elsewhere. Therefore, it was surprising to discover a new fungal species and genus in Central Europe that attacks the pollen cones of three spruce species. The new ascomycete forms apothecia on stromatized pollen cones of Norway spruce (Picea abies) and Serbian spruce (Picea omorika) in mountain areas and on West Himalayan spruce (Picea smithiana) planted in urban lowland regions of Switzerland, Germany, and Italy. It was also detected in France, based on metabarcode sequences deposited in the GlobalFungi database. Its sudden appearance and the different origins of the host trees in Europe and Asia leave the origin of the fungus unclear. The new fungus might be a neomycete for Europe. A phylogenetic analysis using SSU, LSU, ITS, RPB2, and TEF1 sequences classified the fungus as a member of Sclerotiniaceae (Helotiales, Leotiomycetes). However, it differs morphologically from the other genera of this family in having an ascus without apical apparatus containing four mainly citriform spores with 16 nuclei each. Furthermore, it is the only known cup fungus that parasitizes pollen cones of conifers by stromatizing their tissue and infecting pollen grains. The fungus does not seem to cause major damage to the spruce populations, as only a few pollen cones per tree are affected. All this leads us to describe the newly discovered fungus as the new species and new genus Microstrobilinia castrans, the fungus that castrates pollen cones of spruce.

The use of lipid profiles from the immobilised alga Selenastrum capricornuizim was investigated as a potential indicator of heavy metal pollution in freshwater environments. The toxicity of Cu", Zn 2 and Cd2t on algal growth was determined and the effective concentration inhibiting specific growth rate by 50 % (EC5 0) for each metal was found to be 124 pM Cu, 20 pM Zn and 5.7 pM Cd respectively. The Cu 24 EC50 value for immobilised cells was also shown to be 124 j.tM, suggesting that Cu exhibits similar toxic effects on growth in both free and immobilised cells. Studies of the effects of temperature and heavy metal exposure (Cu21, Zn 2 and Cd2 +) on S. caprzcornutum demonstrated that these factors altered the fatty acid and free sterol composition of free algal cells in batch culture. A shift in temperature from 25°C to 10°C led to an increase in the relative proportion of oleate and decrease in linoleate and parinate (18:4), together with a significant increase in the composition of ergostenol. Exposure to heavy metal ions led to an increase in oleate (with all three metals) and altered relative proportions of linoleate and parinate (changes being metal specific). Metal ion treatment also increased a22 desaturation of chondrillasterol. This characteristic lipid signature when S. capricornutum was exposed to heavy metal ions was significantly different from changes associated with other environmental factors. These changes in lipid composition upon heavy metal treatment were also observed during exposure of S. capricornuiwn to lower metal concentrations typically found in polluted environments. Studies of cells immobilised within alginate beads showed that gel confinement significantly affected the biochemistry and physiology of algal cells, with a reduction in growth rate and final cells numbers. Scanning electron microscopy demonstrated that growth was mainly limited to the bead periphery. Immobilisation altered the lipid of composition of cells as a consequence of alterations in membrane fluidity and membrane disruption. The Cu uptake from solution was greater in immobilised cells than free cells, thus gel confinement did not confer any protection to cells. The characteristic and significant changes within the lipid composition of free cells with Cu treatment were similarly observed in immobilised cells but were only apparent at 124 pM Cu. Thus lipid profiles, especially sterol composition of immobilised algal cells, may potentially be utilised as sensitive and novel indicators of heavy metal pollution in freshwater environments.

Despite improved knowledge and advanced treatments of high-grade gliomas, the overall survival rate of glioma patients remains low due to the recurrences and locations of the tumour. Evidence shows that the existence of a subpopulation of cells - cancer stem cells (CSCs) may be the major obstacle in treating gliomas. CD133 and nestin have been suggested as the markers of CSCs and natural stem cells. The primary focus of this study was to identify CD133+/nestin+ stem-like cells and discover their association with multidrug resistance (MDR) related genes, i.e. multiple drug resistance I (mdrl) gene and anti-apoptotic gene (bcl-2) in human glioma compared to normal brain tissues and cell lines. Glioma and normal astrocyte cell lines have been employed for CD133 isolating purposes to characterise the association with MDR related genotype and phenotype. The chemosensitivity of the isolated CD 133 population was investigated using chemosensitivity assay. Meanwhile, a serum deprivation method was established in this study to enrich and select CD 133+ CSCs in a glioma (GOS-3) cell line. As a secondary focus of this project, the possibility of immortalisation enzyme hTERT being a discriminative masker between normal and cancer brain stem cells and the transcriptional correlation between cd133 and bmi-lIc-myc oncogenes were investigated. For the first time, findings of the current study demonstrated that 1) there was an evident increase of CD133 gene expression in glioma compared to normal brain tissues where the latter expressed low levels of CD133, P-gp and Bcl-2 than glioma tissues, with an exception of nestin expression, 2) serum deprivation enriched CD133 expression and demonstrated a direct coexpression between CD133 and drug resistance in GOS-3 cells, 3) hTERT may not be a discriminative marker for normal and cancer brain stem cells, 4) although there was a strong transcriptional association between bmil and cmyc, there was an inverse transcriptional association between these genes and cd133 in serum deprived glioma cells, suggesting that bmil may not be essential for the maintenance of glioma stem cells, and 5) CD133+ glioma and normal brain cells showed a significantly high resistance towards chemotherapeutic drugs compared to the autologous CD133- cells. In conclusion, an improved understanding of molecules contributing to the maintenance of CSCs may lead to a combined treatment, targeting both CSCs and their protective MDR phenotypes leading eventually to attractive strategies for the treatment of gliomas.

In the mammal, anaesthetics are known to act via two distinct mechanisms, either increasing inhibition via GABAA receptors (eg. Na-thiopentone) or decreasing excitation via NIvIDA receptors (eg. ketamine). The aim of this thesis is to investigate the effects of both increased inhibition and decreased excitation at the synaptic level within an invertebrate model system, something which cannot readily be done in vertebrate systems. This was achieved by carrying out experiments using both the above mentioned anaesthetics on the whole animal, isolated brain and cultured neurons. In invertebrates it has been shown that GABA and Glutamate can be both excitatory and inhibitory, and injection of GABA into Lymnaea has been shown to result in behavioural changes in feeding, locomotion, escape reactions, male mating and respiration. Injection of Na-thiopentone into the whole animal was carried out in this investigation, in order to establish the anaesthetic response of the animal model to this barbiturate. The presence of gamma-aminobutyric acid (GABA) receptors has been demonstrated in a respiratory interneuron (RPeD1) both electrophysiologically and via molecular techniques, however inmiunostaining has proved negative in RPeD1 and follower cells VD2/3 (unidirectional excitatory synapse) and VD4 (mutual inhibitory synapse). This suggests that these neurons are not themselves GABAergic, although this investigation shows the responses of these neurons to bath and direct application of GABA. Na-thiopentone did not reliably anaesthetise Lymnaea upon injection into the sole of the foot, suggesting that Na-thiopentone binds to proteins within the snail, andlor has a low affininty for the GABAA receptor in Lymnaea. Other anaesthetic studies using propofol and ketaniine have also demonstrated a lack of anaesthetic response. RPeD1 hyperpolarised and became quiescent in response to the application of high concentrations of GABA (10 3-104M), however at lower doses (1O 8-1O 5M), no effect was observed (p < 0.05). Under these conditions simultaneous recordings from VD4 showed hyperpolarisation in response to the application of GABA, whereas VD2 and VD3 exhibited excitatory responses. Presynaptic picoinjection of GABA also resulted in hyperpolarisation and quiescence in RJeD1, but the simultaneous response in VD3 was not observed. Postsynaptic application of GABA directly to 'VD2, and VD4 however, resulted in responses similar to those seen in the whole brain. VD2 and 3 also receive input 2, which hyperpolarises RPeD1 and elicits an excitatory EPSP in VD2 and 3 as this is similar to the response observed in this experiment it is possible that the effects of input 2 on RPeD1 and VD2 & 3 are mediated by GABA. As RPeDI does not stain positively for GABA and hyperpolarises in response to the drug, it seems unlikely that the postsynaptie effects are due to presynaptic release of GABA. RPeD1 has been shown to form reciprocal synapses with VD4 both in vivo and in vitro. When perfused with GABA (lmJ'i4), both cells hyperpolarised reversibly. The postsynaptic response could be due to the action of GABA presynaptically inhibiting RPeDI, or directly on postsynaptic GABA receptors. However VD4 forms connections with other cells in the brain such as input 3 which may also have resulted in this inhibitory response. RPeD1 would however have received a simultaneous excitatory input from this interneuron. Attempts were made to establish the nature of the RPeD1JVD4 synapse in these experiments, but no synapses were evident. These experiments therefore confirm the presence of GABA receptors in RPeD1 and suggest theft presence in VD2, and VD4. This investigation confirms the findings of previous studies, that injection and bath perfusion of barbiturates does not lead to responses in Lymnaea comparable to that of the mammal. In addition to it's main target site, ketamine (a frequently used intravenous anaesthetic) has also been shown to act at cholinergic receptors. The effects of ketamine on learning and memory and apoptosis in the mammalian CNS are well recognised. Within the Lymnaea CNS, VD4 and LPeD1 form a unidirectional excitatory cholinergic synapse, and this was chosen to investigate the effects of ketamine on excitatory synaptic transmission, short term potentiation and synapse formation in the invertebrate animal model. Ketamine decreased synaptic transmission between VD4 and LPeD1 in a concentration dependent manner, but did not significantly affect short term synaptic plasticity (pc0.05). While neurite outgrowth remained extensive, actual sprouting was diminished by all doses of ketamine. Cells exhibited extensive veiling, which was not present in control cells. Percentage chemical synapse formation was reduced by all doses of ketamine, and in some cases inappropriate inhibitory chemical synapses were formed. Furthermore acute, clinically relevant levels of ketamine reduce excitatory cholinergic transmission between VD4 and LPeD1, but short term plasticity is unaffected. Nerve regeneration was seriously compromised, and formation of appropriate chemical synapses greatly reduced. This data has serious implications for the clinical - use of ketamine, particularly in pregnant women, children or critical care patients where nerve regeneration and synapse formation are of great importance and long term exposure common practice. In conclusion, this work supports that of other studies which have showed that invertebrates appear to be relatively insensitive to barbiturates, whereas ketamine appears to effect excitation in a manner similar to that in the mammal.

Antiulcer agents, notably inhibitors of gastric acid secretion, have been the most successful category of drugs to be discovered in recent years; and moreover, there are currently four such agents in the world list of top 25 best selling drugs. Histamine H2 antagonists have been the number one selling pharmaceutical product for more than a decade and inhibitors of the parietal cell HIC-ATPase, so called "proton pump inhibitors" (PPI), look set to continue this success. The proposed study was designed to establish the relative efficacy and mechanisms of action of three novel agents using both in vitro and in vivo models. The three compounds namely AG-1749 (Lansoprazole), PD-136450 and transforming growth factor alpha (TGF(x) were studied to evaluate their antisecretory and antiulccr activities. Lansoprazole, the second PPI to be developed for clinical use, is a non-competitive inhibitor of the H1C-ATPase and has recently been launched in a number of countries. PD-136450 is a competitive antagonist of central and peripheral cholecystokinin-B (CCK-B) receptors (gastrin receptor) and it under clinical development as an anxiolytic but which has actions on the stomach and pancreas. Anxiolytic drug is otherwise known as anti-anxiety drugs, which are used to treat anxiety disorders, like depression, panic disorders, phobias and many personality disorders. TOFu is a polypeptide growth factor, which acts at the EGF receptor and displays potent mitogenic and antisecretory activity. The initial study deals with the comparison of the three compounds with omeprazole and ranitidine in terms of their ability to inhibit acid secretion and their activity in a range of experimental ulcer models. Potency, duration of action and activity against a range of stimulants of acid secretion (histamine, pentagastrin and basal) was determined in anaesthetized rat models by establishing dose-response relationships. The compounds represent a spectrum of activities in as much as lansoprazole is a potent, long acting inhibitor, PD-136450 is an orally active but selective inhibitor, while TOFu has a very short duration and is only active after parenteral administration. In a view to find out the mechanism of action of these drugs on gastric acid secretion, isolated gastric glands from rabbits were employed as an in vitro technique using radiolabeled 14C-aminopyrine as a marker. The results show that lansoprazole was the most potent antisecretory agent compared to other two drugs. The second phase of the study deals with the activity of the three compounds against gastric ulcers induced by acid hypersecretion, indomethacin and stress. This study enabled us to assess the extent to which antisecretory activity per se compared with other actions such as wound healing (TGFa) or anxiolytic activity (PD-136450) contribute to ulcer healing. As other workers already established that prostaglandins and nitric oxide are involved in the cytoprotective activity, the present study investigated the influence of prostaglandin and nitric oxide by using indomethacin and L-NAME pretreatment on the cytoprotective activity of lansoprazole, PD-136450 and TGFcz. Moreover, the three drugs were tested for their activities on the mucus and bicarbonate production in the stomach. It was found that lansoprazole and TGFc increased the gastric mucus secretion while PD-136450 did not show any change. Moreover it was evidenced from this study that the protective activity of PD-136450 is associated with the influence of bicarbonate secretion from the pancreas. In conclusion, the results of this study have indicated that lansoprazole, PD- 136450 and TGFct are potent antisecretory and antiulcer agents which have great therapeutic importance.

Mature skeletal muscle fibres can be classified as type I, type IIa, type IIx or type IIb fibres according to the myosin heavy chain (MHC) isoform that they express. More broadly, type I fibres are classified as slow fibres and type IIa, IIx and IIb fibres as fast fibres. However, the phenotype of an adult skeletal muscle fibre is not fixed: it displays plasticity being capable of adapting to changing activity and loading levels by either transition towards a slower phenotype or transition towards a faster phenotype. Overall, the aims of these studies were to further investigate and define the signal transduction pathways implicated in the control of skeletal muscle fibre phenotype. The ability of a fast muscle to undergo a transition towards a slower phenotype in response to chronic low-frequency stimulation (CLFS) was assessed, via metabolic enzyme activity assays and NADH-TR staining, following blockade of the calcineurin signalling pathway. Metabolic enzyme assays and northern blots were employed to assess the changes in enzyme activities and MEC isoform expression levels following blockade of the calcineurin and ERK1/2 signalling pathways in primary cultures of rat myotubes. Differences in the levels of various signal transduction proteins/transcription factors between slow and fast muscle were investigated using western blotting. The nuclear translocation kinetics of NFAT and NF-κB in response to treatment with the calcium ionophore A23187 were investigated in L6 myotubes using immunocytochernistry. Calcineurin blockade using cyclosporin A failed to prevent a decrease in lactate dehydrogenase activity and an increase in NADH-TR staining intensity, both characteristics of a transition towards a slower phenotype, following CLFS of the fast rat tibialis anterior muscle. Blockade of the ERK1/2 pathway in primary cultures of rat myotubes using U0126 significantly decreased MHC Iβ mRNA levels and significantly increased MIHC IIx, MEC IIb and perinatal MHC mRNA levels. Calcineurin blockade significantly decreased MHC Iβ and embryonal mRNA levels and significantly increased MHC IIx mRNA levels. Calcineurin blockade also significantly increased the activities of lactate dehydrogenase and creatine kinase while ERK1/2 blockade significantly increased the activities of lactate dehydrogenase, creatine kinase, hexokinase, malate dehydrogenase and β-hydroxyacyl-CoA deydrogenase. ERK1/2 and NF-κB levels were found to be higher in slow muscle compared to fast muscle while calcineurin and p38α,β levels were higher in fast muscle compared to slow muscle. No nuclear translocation of NF-κB and only limited evidence for NFAT nuclear translocation was seen in L6 myotubes following treatment with A23187. Overall these studies further characterize the roles of the ERK1/2 and calcineurin pathways in the regulation of muscle phenotype suggesting that each pathway controls some, but not all, of the genes that are differentially expressed between slow and fast muscle fibres. Western blotting suggests further signal transduction protein/transcription factor targets, the functions of which may be explored in the future.

Based on previous studies of the novel Lactobacillus reuteri DPC16 strain, an in vitro investigation on the supernatant antimicrobial activity and the culture safety against normal gastrointestinal microflora and gastric mucus was done in this thesis. DPC16 cell-free supernatants (fresh and freeze-dried, designated as MRSc and FZMRSc) from anaerobic incubations in pre-reduced MRS broth, have shown significant inhibitory effects against selected pathogens, including Salmonella Typhimurium, E. coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes. These effects were mainly due to the acid production during incubation as evidenced by the negation of such activity from their pH-neutral counterparts, and this acidic effect was shown to reduce the pathogen growth rate and decrease the total number of pathogen cells. By incubation of concentrated (11 g/L) resting cells in glycerol-supplemented MRS broth, another DPC16 cell-free supernatant (designated as MRSg) has shown very strong antimicrobial effect against all target pathogens. As indicated by a kinetic profile, this activity developed in a sigmoidal fashion as incubation proceeded, reaching to maximum activity after 6-8h and maintained at the same level thereafter. Further study has shown that the antimicrobial activity of this supernatant was pH-independent, effective across a pH range of 4.6 to 6.5, and acted on both Gram-negative and Gram-positive pathogens. Using the minimum effective dose, a time course investigation has provided evidence that this supernatant affected the growth of the target pathogens by elongating the lag phase and lowering the total cell number at the end of the incubation. Lastly, it was found that the strong antimicrobial effect of MRSg was bactericidal at high concentrations and bacteriostatic at low concentrations. However, it also found that the viability of DPC16 cells also decreased as incubation prolonged, which suggests that this glycerol-derived supernatant had a lethal effect to its own cells. Nevertheless, this lethal effect was exerted to a much lesser extent compared with that to the pathogens. The last DPC16 cell-free supernatant was designated as SGF, which was produced from secondary fermentation of the same resting cells in glycerol-water. SGF did not show a significant antimicrobial activity, which suggests that this specific strain is not capable of utilising glycerol in the absence of fermentable carbohydrates. The antimicrobial activity found in MRSg matched with previously published characteristics of reuterin, which is a unique antimicrobial substance synthesised by L. reuteri when incubated with glycerol. Therefore, a study on the production kinetics of reuterin by DPC16 was carried out. Supernatants of both MRSg and SGF were studied. Results showed that glycerol utilisation occurred in both MRSg and SGF, whereas the bioconversion of glycerol into reuterin was different. In MRSg, glycerol was constantly utilised by DPC16 resting cells, and by the end of an 18h incubation 85.8 mM of glycerol was utilised, where 72.8% was transformed into reuterin. The formation of reuterin initiated with an inclining production and reached the maximum rate of 10.9 mM/h after 6h of incubation, with the total production of 64 mM of reuterin at the end of the 18h incubation. This reuterin production in MRSg followed a similar pattern to that of its antimicrobial activity, which suggests a certain correlation between reuterin formation and the increase of antimicrobial activity in MRSg. Therefore, the major antimicrobial component in MRSg that accounted for its potent antimicrobial activity was very much likely to be reuterin. In SGF, however, detectable reuterin was negligible even though some glycerol may have been absorbed into the highly concentrated DPC16 resting cells. This has responded to the antimicrobial activity assay in that due to the lack of essential carbohydrate nutrient for normal cell metabolism, there was no glycerol utilisation and hence no reuterin synthesis. Having studied the antimicrobial activity of L. reuteri DPC16 and the production of antimicrobial-competent reuterin, two safety issues (the impact on growth on other normal commensal probiotics and mucin degradation activity) of this strain were assessed to further evaluate its probiotic potential. By using similar in vitro assays as in the antimicrobial test, the same set of DPC16 supernatants have demonstrated no adverse effect on the growth of either Lactobacillus acidophilus, Lactobacillus plantarum, Pediococcus acidilactici, or Bifidobacterium lactis DR10. No stimulatory effect was found either. By incorporating purified porcine gastric mucin into classic mucin-degradation assays in both liquid and agar media, DPC16 has not exhibited the same mucinolytic activity as that of the faecal flora cultures. Thus, it can be concluded that L. reuteri DPC16 is as safe to the host as normal commensal microflora.

The ecological importance of introduced mammalian predators is well acknowledged in New Zealand, however, little research has focused on the ecology of native avian predators and their role in communities. This study investigated the ecology of moreporks (Ninox novaeseelandiae) on Ponui Island, Hauraki Gulf, New Zealand between August 2007 and April 2008. The primary aim was to investigate the functional response of moreporks to availability of their prey. The contents of regurgitated morepork pellets were compared with relative abundance of prey taxa (invertebrates, small birds and rodents) over the study period. The diet consisted primarily of a range of invertebrate prey, particularly weta (Anostostomatidae and Raphidophoridae) and beetles (Coleoptera). Small numbers of vertebrate prey were recorded including rodents and birds. A positive relationship between the percentage contribution to pellet samples of certain taxa and their relative availability was found, and there were peaks in the occurrence of seasonally abundant taxa including cicadas (Cicadidae), and huhu beetles (Prionoplus reticularis). The tendency of moreporks to prey on abundant taxa indicates that they are unlikely to depress prey populations to low levels, and may have some degree of stabilising influence. A significant increase in the rodent component of the diet in April indicated that the risk to moreporks of secondary poisoning during mammalian pest control operations may vary considerably with the time of year. The secondary aims were to collect data on roost site characteristics and breeding success. Moreporks roosted at a mean height of 4m, and foliar cover at the 4-6m height tier appeared to be the most important characteristic of roost sites when compared with control sites. These findings suggested that moreporks were selecting roost sites with high overhead cover. Possible reasons for this include predator avoidance, avoidance of mobbing passerines, and the microclimate provided. None of three established pairs and two other birds were observed to establish a nest or breed successfully. Additionally, only three juvenile moreporks were sighted or heard across the 90ha study area suggesting low breeding success in 2007-08. This may have been influenced by a range of factors including 1), predation by the high densities of ship rats on Ponui, or other predators 2), a lack of suitable nest sites such as tree hollows in some areas or 3), competition for invertebrate prey with high densities of ship rats and North Island brown kiwi (Apteryx mantelli).

The sap beetle genus Carpophilus Stephens (Coleoptera: Nitidulidae) is a large genus consisting of over 200 species and are found worldwide. Several species are important pests of crops and stored products, and are frequently intercepted as part of biosecurity operations. The genus is poorly known taxonomically, and there are several species groups that are challenging to identify by morphological methods. In particular, two species found across the Pacific, C. maculatus Murray and C. oculatus Murray are frequently confused with each other. These two species are similar in size and colour, but differ primarily by the shape of the colour pattern on their elytra. However, this colour pattern is highly variable within both species, leading to ambiguity in the indentification of these species. Within C. oculatus, three subspecies have been described based on differences in the male genitalia and pronotal punctation: C. o. oculatus and C. o. gilloglyi Dobson are distributed widely across the Pacific, while C. o. cheesmani Dobson is known only from Vanuatu. A search of literature records and specimen collections revealed 32 species of Carpophilus recorded from the Pacific region. In addition there remain several unidentified specimens representing at least four species, two of which will be described subsequent to this research. A number of species recorded in the literature may have been misidentified, and these require further field collections and inspection of museum specimens to confirm their presence in the Pacific. To test the validity of the subspecies of C. oculatus, and its distinctiveness from C. maculatus, a phylogeny of available specimens of Carpophilus was inferred from one mitochondrial gene (cytochrome c oxidase subunit I (COI)), and two nuclear genes (28S ribsomal RNA (28S) and the internal transcribed spacer 2 (ITS2)). These data show large genetic distances between the three subspecies of C. oculatus of 7-12%. Given these distances are similar to those between other species in the genus, this indicates these subspecies may be elevated to full species. The data also consistently support a monophyletic relationship between C. o. oculatus and C. o. gilloglyi. Nuclear genes also support C. o. cheesmani as part of a clade with the other subspecies, but these relationships are unresolved in COI. Carpophilus maculatus was not supported as being the sister taxon of the C. o. oculatus and C. o. gilloglyi clade. Other relationships within Carpophilus were unresolved, possibly due to a combination of incomplete taxon sampling, and saturation of substitutions within the COI gene. Phylogeographic analysis of specimens collected from several localities within the range of C. oculatus showed that, with only one exception, there were no shared haplotypes between archipelagoes. This result suggests it may be possible to determine the provenence of intercepted specimens, providing further information regarding potential invasion pathways. A degree of geographic structuring was also present within C. o. gilloglyi, being separated into a western clade found in Fiji and Rotuma and an eastern clade distributed from the Kermadec Islands and Tonga to French Polynesia. This separation was most profound in COI data, with a mean pairwise distance between the clades of 7%. ITS2 data also demonstrates a degree of differentiation between the two clades, based on differences in the insertions and deletions between the clades. The variability in the shape and colour of the elytral pattern of C. oculatus was also investigated. Colour was quantified using a method based on Red-Green-Blue (RGB) colour values derived from digital photographs, while an outline analysis of the elytral pattern was conducted using elliptic Fourier analysis (EFA). Principal Components Analysis of the RGB values and EFA coefficients showed no clear separation between subspecies, nor were any trends correlated with host fruit or collection localities. Variation at all levels and all measures studied in this thesis show that this geographic region and this genus of beetles offer intruiging insights into speciation, biogeography and biological invasions. There is much scope for further research on the causes and consequences of this variation and the lives of these interesting insects.

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

Utilizing environmental DNA (eDNA) for the detection of species in the field is a potentially cost-effective and time-saving technology that may be useful in understanding the distribution and abundance of threatened or endangered species such as the Eastern Massasauga (Sistrurus catenatus). I describe the development of an eDNA assay for the species and evaluate its ability to detect eDNA in laboratory and field conditions. In the field samples, I also investigated the potential for abiotic conditions to influence eDNA detection. Species-specific primers and probe were designed to amplify a 152 bp segment of the massasauga COI gene. Target eDNA could be detected in samples containing as little as 100 copies of target DNA/μL. Water samples collected from laboratory housed snakes indicated that eDNA can be detected in water 56 days after massasauga removal. Field samples were taken from crayfish burrows, known overwintering habitat for the species, from four sites that vary in snake use as ascertained by traditional visual surveys. Of the 60 burrows sampled, seven had a positive detection for massasauga eDNA with no difference in detection rate between DNA extracted from burrow water and burrow sediment. Occupancy models fitted to burrow water indicated that larger amounts of total DNA in a sample may increase the probability of detection of a massasauga eDNA. Large confidence intervals in site occupancy (ѱ) and burrow detection (Θ) values suggest that a larger sample size is needed for more reliable occupancy models. Abiotic conditions within crayfish burrows varied among sites but correlation with eDNA detection was not supported. Estimates of qPCR detection within a burrow with eDNA (ρ) suggest that up to 10 qPCR replicates per burrow sample may be necessary. Further studies need to examine eDNA degradation in the field, improve upon the limit of detection, and sample a larger number of sites for eDNA sampling to be a stand-alone survey method for Eastern Massasaugas.