Miracle-dz/UniFAR-FTUs huggingface dataset:
The checkpoint role of Cdc18 in fission yeast

The highly conserved eukaryotic checkpoints keep tight control over cell cycle progression, arresting the cell in response to incomplete DNA replication or DNA damage. In fission yeast, Rad3 (functional homologue of ATM, mutated in ataxia telangiectasia, and structural homologue of ATR, ataxia telangiectasia and rad3 related) is necessary for activation of both replication and damage checkpoints. However, despite the identification of many checkpoint genes, the actual sequence of upstream events leading to Rad3 activation remains unclear. The aim of my project was to identify and characterise the Rad3- dependent DNA damage/perturbed replication sensors and checkpoint activators. A genetic screen was carried out in fission yeast, using the working hypothesis that overexpression of these sensors/checkpoint activators would ectopically induce a Rad3-dependent block over mitosis in the absence of DNA damage or disturbed replication. The screen identified several genes of which the DNA replication initiation factor Cdc18/CDC6 had the strongest and most reproducible phenotype. Cdc18 is essential to prevent mitosis during S phase. I chose to concentrate on characterisation of the Rad3-dependent checkpoint role of Cdc18. The actual level of Cdc18 is important for producing the Rad3-dependent cell cycle block. A stabilised Cdc18 protein, mutated at the conserved CDK (cyclin dependent kinase) consensus sites also caused a Rad3-dependent cell cycle arrest, and was more stable and easier to manipulate than the screen- derived clone. Genetic crosses demonstrated Cdc18 acts early on in the checkpoint pathway, and through Crb2/Chk1. There was no gross DNA damage or detectable replication intermediates in the presence of elevated or stabilised Cdc18 levels. I also found that artificial depletion of Cdc18 during an S phase block results in loss of the checkpoint but not the replication structures, uncoupling the maintenance of replication forks from the maintenance of the mitotic block. An unexpected consequence of Cdc18 stabilisation was an increase in the size and variability of chromosome III on pulsed field gel electrophoresis. This localised to an expansion of Sfi1 restriction fragments containing the rDNA repeats. In conclusion, Cdc18 stabilisation activates a Rad3-dependent checkpoint in the absence of apparent re-replication, which is associated with an expansion of the rDNA repeats on chromosome III. Two models are proposed. In the first, Cdc18 induces low level genome wide replication, that is undetectable but sufficient for checkpoint activation. This leads to increased recombination with unequal crossover events in the rDNA repeats on chromosome III, with subsequent repeat expansion. In the second, the increased levels of Cdc18 directly activate the cell cycle checkpoint independently of the concurrent expansion of chromosome III.
Standardization of DNA isolation and PCR protocols for ISSR analysis in Cluster bean (Cyamopsis tetragonoloba (L.) Taub.) Genotypes

Cluster bean [Cyamopsis tetragonoloba (L) Taub. ] is one of the most important cash legume crop has played an increasingly important role in wide range of industries. Owing to the significance of molecular marker studies in numerous applications including in genetic improvement of crops, there is an obvious need to undertake such studies in cluster bean. Present research work was carried out with the genomic DNA optimization and PCR standardization for ISSR analysis in cluster bean genotypes. The standard CTAB method for isolation of genomic DNA from cluster bean have their own limitation as it produces gel image with high contamination of yellowish, sticky and viscous matrix. The commonly used CTAB method was modified by giving Polyvinylpyrrolidone (PVP) treatments after grinding fresh young leaves of cluster bean. The yield of genomic DNA was increased in ranged from120 ng/µl to 220 ng/µl of 0.2 g leaf tissue and the purity (ratio) was between 1.5-1.7 indicating minimal levels of contaminating metabolites. ISSR protocol was standardized after implementing various modifications. Based on above findings of present experiment work this modified genomic DNA isolation and PCR protocol may be utilized for ISSR profiling to produce sharp and clear bands in cluster bean genotypes.
Diospyros cuneata Inhibition of Fusarium oxysporum: Aqueous Extract and its Encapsulation by Ionic Gelation

The application of plant extracts to control fungal crop pathogens is an ecological strategy that could potentially be useful in agriculture. Aqueous extracts of some species of the genus Diospyros spp have been tested against fungal pathogens of crops. Nevertheless, there is no information about the inhibitory effect of aqueous extracts of Diospyros cuneata on the micelial growth of Fusarium oxysporum, a pathogen responsible for “Panama disease” and “vanilla stem rot disease”. Therefore, the antifungal activity of aqueous extracts of Diospyros cuneata leaves collected during the dry and rainy seasons was tested in-vitro against spores of Fusarium oxysporum. Only the aqueous extract from leaves collected during the dry season had an inhibitory effect on the micelial growth of asexual spores (2.5% minimum inhibitory concentration). Phytochemical analysis showed that both aqueous extracts contained mainly flavonoids and tannins; the chromatographic profile showed a larger abundance of polar compounds in the dry season extract. Furthermore, the antifungal activity observed is probably correlated with the abundance of some secondary metabolites produced by water stress and dry season conditions. Thebioactivityof aqueous extracts of Diospyros cuneata leaves could be stored and released through encapsulation; an effective example of this was tested using alginate-inulin to prepare microbeads by ionic gelation.
Diet of feral cats during the Scopoli’s shearwater breeding season on Linosa Island, Mediterranean Sea

Abstract Feral cats (Felis catus) are considered to be one of main successful invasive species of island ecosystems. The introduced cats often negatively affect seabird populations due to the lack of anti-predatory strategies resulting from the absence of mammalian terrestrial predators during their evolutionary development. Linosa Island hosts the second largest colony of Scopoli’s shearwater (Calonectris diomedea) of the species. In this study, we estimated the number of cats on Linosa Island and we evaluated their diet using a non-invasive method: scat analysis. Scat collection took place in the northern part of the island in the Scopoli’s shearwater colony, where a feral cat colony lives permanently. We analysed a total of 188 cat scats, collected during the different stages of the shearwater breeding period. Introduced mammals were the most frequent preys: wild rabbits and black rats occurred in scats with the same frequency (28.72%). Birds were the second most frequent prey category found, primarily represented by the seabird Scopoli’s shearwater (16.49%). The cats’ diet varied seasonally in relation to prey availability and accessibility: shearwaters were mainly preyed upon during spring when they exhibited the greatest vocal activity and spent the most time out of their nests. Regarding the biomass, mammals were the dominant prey, and wild rabbits were the most consumed species. However, Scopoli’s shearwater represented approximately one-third of the total biomass consumed. Consequently, the management of feral cats living in proximity of the seabird colony is strongly required, and the increase of “trap-neuter-release” (TNR) programmes is necessary in order to avoid uncontrolled gains in the cat population on Linosa Island. Since the cats have a crucial role in controlling the rat population (the main threat for shearwaters), before planning management strategies, the complex inter-relationship that exists between species must be taken into account.
Investigator Group Expedition 2006: Sponges (Porifera)

Abstract The diversity of marine sponges on the rocky reefs surrounding the Investigator Group of islands in South Australia was investigated for the first time. Line intercept transects at Flinders, Pearson and Ward Islands at ~5 m deep, showed the algal dominance of the sub-tidal rocky reefs. Along these transects, quadrat surveys showed low sponge occurrence on upward facing reefs, but in caves and under overhangs sponges and other sessile fauna were dominant. Divers collected 71 sponge species, 10 in the Class Calcarea and 61 in the Class Demospongiae from the reefs of five islands: Flinders, Pearson, Dorothee, Ward and Topgallant. Many of the sponges had evidence of algal or cyanobacterial symbionts, possibly reflecting the clear shallow-water conditions of the sites. There was little overlap between this collection and a previous collection from the Great Australian Bight Marine Park Benthic Protection Zone, with only three sponges in common.
Genetic Engineering of Plant Seeds to Increase Thiamin (Vitamin B1) Content

Thiamine (Vitamin B1) in the form of thiamine pyrophosphate (TPP) is an essential cofactor for the function of numerous enzymes which are involved in central metabolism such as citric acid cycle, pentose phosphate pathway, Calvin cycle, isoprenoid biosynthesis, and branched-chain amino acid biosynthesis. All living organisms need thiamine. However, human and animals can synthesize TPP from thiamine, but they are not able to synthesize thiamine de novo. Therefore, human and animals must obtain thiamine from their diet to maintain a normal metabolism. Severe thiamine deficiency causes the lethal disease beriberi and Wernicke-Korsakoff syndrome in humans. The enzymes involved in thiamine de novo biosynthesis pathway are well known in microorganisms and plants, but little is known regarding the salvage pathways in plants. In order to have better insight about the thiamine salvage pathways in plants, the homologs of bacterial ThiM (thiazole kinase) were analyzed. It has been revealed that this protein in plants has thazole kinase activity which is important for thiamine salvage. In addition, analyzing the TenA_E proteins in plants shows that these proteins have amidohydrolase and aminohydrolase activity to form 4-amino-5-hydroxymethyl-2methylpyrimidine (HMP) from the salvage of thiamine breakdown products. Thiamine plays a vital role in resistance against biotic and abiotic stresses in plants in addition to its role as a cofactor. It has been shown that elevated levels of thiamine content achieved by the seed overexpression of Thi4, ThiC, and ThiE genes can enhance the seed germination and seedlings viability under abiotic stress conditions. Additionally, thiamine and TPP over-producing lines shows altered seed carbon partitioning.
Isolation and expression analysis of differentially expressed genes in stem tissue of the Greek lemon cv. Adamopoulou

ABSTRACT Lemon (Citrus limon (L.) Burm. f. ) is susceptible to mal secco, a serious vascular disease caused by the fungus Phoma tracheiphila (Petri) Kant. and Gik. , as well as low temperatures. The greek lemon cultivar Adamopoulou, thought to be derived from the Portuguese cultivar Lisbon, exhibits enhanced resistance to mal secco and cold as opposed to cv. Lisbon. Suppression subtractive hybridization (SSH) was employed for the isolation of differentially expressed genes in lemon stem tissue. A subtractive cDNA library was constructed and a total of 296 clones were sequenced. The obtained sequences were edited, resulting in 56 non-redundant ESTs. Sequence analysis revealed homology to previously identified genes involved in defense mechanisms against biotic and abiotic stresses, as well as sequences with no significant similarity in the GenBank. Selected ESTs were analyzed by real-time PCR for confirmation purposes. This analysis revealed significant expression differences between the two cultivars for genes expressing allantoinase, ultraviolet-B-repressible protein, 4-coumarate:CoA ligase and other proteins that are known to be upregulated under biotic and abiotic stress conditions.
Identification of Novel D11 Hepatitis B Surface Antigen Subgenotype in Jeddah, Kingdom of Saudi Arabia

Hepatitis B virus (HBV) infection remains to be a worldwide health problem. In Saudi Arabia, HBV is the most predominant type of hepatitis followed by hepatitis C and hepatitis A while little is known about the molecular epidemiology of the prevalence of HBV genotype/subgenotype particularly in Jeddah. Serum samples were collected from HBV chronic patients and subjected to HBsAg gene amplification. Sequencing and phylogenetic analysis of the entire HBsAg gene sequences revealed that 11 isolates belonged to HBV/D while 4 isolates were associated with HBV/C. Interestingly, HBV/D subgenotypes identified eight HBV/D present isolates belonged to HBV/D1 while three isolates showed a new cluster supporting by a branch with 99% bootstrap value and 4.3-5.8% nucleotide divergence over the entire HBsAg gene from other known subgenotypes D1 to D10, despite they were appearing more related to HBV/D5. The three strains of the new D subgenotype showed unique amino acid sequences consisting of Thr7non, 75Pro in the preS1 gene, 112Ile, 161Gly in the preS2 gene and 196Glu, 197Ala, 238Ser, 259Cys in the S gene. In addition to three amino acid residues in the S gene (373Ile, 374Ala and 381Thr) were specified S118S isolate. Subsequently, it have been verified that HBV/D1 is the most prevalent HBV subgenotype in Jeddah as well as we proposed a novel subgenotype designated HBV/D11. The identification of HBV/D11 novel subgenotypes in the present study suggested that further studies with a large number of subjects in previously examined and unexamined areas may lead to discovering new HBV strain genotypes and/or subgenotypes circulating in Saudi Arabia.
Product Analysis of Starch Active Enzymes by TLC

[Abstract] Thin layer chromatography (TLC) is a useful technique for detecting the presence of monosaccharides through to oligosaccharides, though it needs to be optimized for the specific sugars that are analyzed. Here we present a method for visualizing the reaction product(s) of starch active enzymes, which can contain α-1, 4 linked and α-1, 6 linked glucose. This was first published in Molecular Microbiology (Cockburn et al., 2015). The TLC protocol is an adapted version of that published by Robyt and Mukerjea (Robyt and Mukerjea, 1994). For a summary of the products generated by starch active enzymes see the review by Hii et al. (2012).
Sorus formation on the holdfast haptera of the kelp Ecklonia radicosa (Phaeophyceae, Laminariales)

Abstract Kelps usually form sori on their vegetative blades or sporophylls, and occasionally on stipes, with a notable exception being the primitive kelp Aureophycus aleuticus (Aureophycaceae), which forms sori on its discoid (haptera-free) holdfast. Here we reveal sorus formation on the holdfast (and blade and stipe) of the warm temperate annual kelp Ecklonia radicosa (Lessoniaceae), whose holdfast is formed by haptera, as is typical of the Laminariales. Surprisingly, hapteral sori continued to release zoospores until December, following blade loss in October. Such production of sori on holdfasts may be advantageous for surviving higher water temperatures and grazing pressure in warm temperate waters.
Water flow impacts group behavior in zebrafish (Danio rerio)

Constant temporal and spatial fluctuations of the physical environment pose a great challenge for individual survival, making plastic behavioral responses an important mechanism for coping with environmental fluctuations. For aquatic animals, water flow is one of the most important factors, imposing additional physiological costs and changing their relationship with the biotic environment including conspecifics, predators, and disease agents. Here, we conducted a controlled laboratory experiment to test the influence of water flow on the behavior of zebrafish (Danio rerio), considering also the impact of obstructions. Using wild-caught zebrafish, we formed groups of 6 fish and tested their behavior in 4 treatments that varied in water flow and obstructions. We used automated tracking software to estimate shoal cohesion, aggression, and activity level. Zebrafish strongly responded to the presence of even a weak flow by forming less cohesive, more aggressive, and more active groups. The effect of flow was not exaggerated by turbulence generated from obstructions. Zebrafish were also more active and more aggressive when in a structurally complex context. These findings highlight the plasticity of zebrafish social behavior and provide insight to understand the impact of water flow on behavioral plasticity of social groups.
Modeling ice storm damage to a mature, mixed-species hardwood forest in eastern Ontario

Abstract In January 1998, the worst ice storm of the last century hit regions of southeastern Ontario, Québec, New Brunswick, and the northeastern United States. Using standard multiple regression and classification tree models, we examined the ice damage suffered by trees in a mature, deciduous forest in eastern Ontario at two scales: plot (5 m radius) and individual tree. Canopy trees were damaged significantly more than mid-story trees and there were significant differences among tree species in damage susceptibility. At the plot scale, the best predictors of damage were average tree size and plot species evenness. Plots dominated by large trees were damaged more than those dominated by small trees and plots with higher species evenness suffered higher levels of damage than did less even plots. Models incorporating damage to neighbouring plots explained more variance than did models without the neighbour information. At the individual tree scale, damage suffered by the dominant canopy tree species, sugar maple, was best predicted by tree size. Damage suffered by the dominant mid-story tree species, ironwood, was best predicted by neighbour information and tree size. Disturbances that differentially affect canopy and mid-story layers have been shown to accelerate forest succession by creating light gaps. However, given the species composition and structure of our study forest, we feel that this disturbance will not overly influence forest succession in mature, deciduous forests in eastern Ontario.
Characterization of Pratylenchus crenatus and P. neglectus (Nematoda: Pratylenchidae) associated with wheat crop

Abstract The distribution of Pratylenchus species associated with wheat crops was investigated in Bohemian region of the Czech Republic. In total twelve localities were sampled. The populations were identified based on morphology and morphometrics, and further characterised based on sequences of the rDNA D2/D3 region and 18S gene. Pratylenchus crenatus was present in two localities and P. neglectus in five localities. At one locality both species were detected. Sequence analysis of 18S and D2/D3 region of three populations of P. crenatus reveal no variation while five populations of P. neglectus differ by 0 to 0.14 % (18S) and 0.17 to 0.50 % (D2/D3).
Isolation and characterization of Vibrio alginolyticus from cultured amphioxus Branchiostoma belcheri tsingtauense

Abstract Amphioxus is an intermediary species bridging invertebrates and vertebrates used to study the development and evolution of species and genes. In this study, the dominant bacterial strain was isolated from tail lesions of cultured amphioxus and designated WW1. Challenge by intramuscular microinjection revealed the 50% lethal dose of WW1 to be 9 × 102 bacteria/g amphioxus body weight. Based on 16S rRNA gene sequence, physiological and biochemical characteristics, and species-specific collagenase gene marker, WW1 was identified as Vibrio alginolyticus. To our knowledge, this is the first report on the isolation of V. alginolyticus as pathogen from amphioxus. This study extends the known host range of V. alginolyticus and may provide information helpful in preventing and managing its occurrence in cultured animals.
Adenoviral Gene Therapy for the Stimulation of Neovessel Growth

In this thesis, stimulation of the growth of both lymphatic and blood vessels was studied in large animal models using adenovirally (Ad) delivered vascular endothelial growth factors (VEGFs) and hypoxia-inducible factors (HIFs), which transcriptionally regulate a plethora of factors involved in angiogenesis, including VEGF-A isoforms. Two recognized lymphangiogenic growth factors, VEGF-C and VEGF-D, were employed to induce lymphatic vessel growth in a new large animal model of lymphedema. Lymphedema, which often develops as a consequence of the surgical removal of cancer or radiation therapy, lacks curative treatments, thus making the application of gene therapy to repair lymphatic damage an attractive new treatment option for the patients suffering from this condition. Both AdVEGF-C and AdVEGF-D were shown to induce lymphatic vessel regrowth and, consequently, improve lymphatic flow in the pig model of lymphedema, where lymphatic damage was first caused by excising all of the lymphatic vessels around an inguinal lymph node. Interestingly, AdVEGF-D was found to evoke more pronounced blood vascular effects than AdVEGF-C. Therefore, VEGF-C gene therapy was selected for further evaluation and optimization in the same large animal model of lymphedema. A subsequent comparison was performed of VEGF-C gene transfer into the lymph node versus into the fat surrounding the lymph node in terms of efficacy and adverse effects. The regeneration of lymphatic vessels was equally effective regardless of the gene transfer site. However, gene transfer into the fat surrounding the lymph node was found to have less adverse effects on the node. In the final part of this thesis, HIF-1α and HIF-2α, which are upstream regulators of VEGF-A expression, were adenovirally delivered to mouse myocardium and ischemic rabbit hindlimbs to evaluate their capability to induce blood vessel growth. Patients with cardiovascular disease are becoming increasingly older and suffering additional comorbid conditions, i.e. they may not withstand the current invasive procedures but would be suitable for these emerging alternative treatment options. Both of the transcription factors increased blood capillary size and perfusion in skeletal muscles. Additionally, HIF-1α gene transfer enhanced the recovery of ischemic muscles from energy depletion induced by electrical stimulation. In conclusion, adenovirally delivered VEGF family members VEGF-C and VEGF-D efficiently induce lymphangiogenesis, which results in improved lymphatic flow, whereas AdHIF-1α triggers angiogenesis, which is sufficient to improve energy metabolism of ischemic muscles. National Library of Medicine Classification: QU 107, QU 560, QZ 170, WG 500, WH 700 Medical Subject Headings: Lymphangiogenesis; Blood Vessels/growth & development; Angiogenesis Inducing Agents; Genetic Therapy; Adenoviridae; Gene Transfer Techniques; Vascular Endothelial Growth Factors; Basic Helix-Loop-Helix Transcription Factors; Lymphedema/therapy; Ischemia/therapy; Therapies, Investigational; Disease Models, Animal
Targeted Therapeutics against Biliary Cancer Elicit Concurrent Tumor Extrinsic Effects on Immune Suppression and Cancer Cachexia

ii Acknowledgments. v Vita . vi List of Abbreviations . viii List of Tables . xvi List of Figures . xvii Chapter 1: Introduction to Biliary Tract Cancer . 1 1.1 Clinical Features of Biliary Tract Cancer . 2 1.1.1 Incidence, Risk Factors, and Clinical Presentation of Biliary Tract Cancer . 3 1.1.2 Pathogenesis and Disease Progression . 5 1.1. 3 Cancer Cachexia and Biliary Tract Cancer . 8 1.2 Therapeutic Options . 10 1.2. 1 Surgical Resection . 10 1.2. 2 Chemotherapeutic Options . 10