Miracle-dz/UniFAR-FTUs huggingface dataset:
The Systemic Response to Fire Damage in Tomato Plants: A Case Study in the Development of Methods for Gene Expression Analysis Using Sequence Data

COKER, JEFFREY SCOTT. The systemic response to fire damage in tomato plants: A case study in the development of methods for gene expression analysis using sequence data. (Under the direction of Dr. Eric Davies.) Fire is a natural component of most terrestrial ecosystems and can act as a local wound stimulus to plants. The ultimate goal of this work was to characterize the array of transcripts which systemically accumulate in plants after fire damage. Before this could be accomplished, substantial development of methods for gene expression analysis using sequence data was necessary. This involved developing methods for identifying contamination in DNA sequence data (Chapter 2), identifying over 78,000 false sequences in GenBank and several thousand more in the indica rice genome (Chapter 2), developing a novel method for identifying housekeeping controls using sequence data (Chapter 3), performing relative expression analyses for 127 potential housekeeping control transcripts (Chapter 3), and characterizing 23 transcripts which encode all 13 subunits of vacuolar HATPases in tomato plants (Chapter 4). A subtractive cDNA library served as a starting point to identify and characterize 9 novel tomato transcripts systemically up-regulated in leaves in the first hour after a distant leaf is flame wounded (Chapters 5). Real-time RT-PCR using leaf RNA isolated at different times after flaming showed that the most common pattern of transcript accumulation was an increase within 30 to 60 minutes, followed by a return to basal levels within 3 hours. Expression analyses also showed that most up-regulated transcripts were already present in unwounded tissues. A total of 46 different transcripts were identified from the subtractive cDNA library (Chapters 6). Compared with the entire tomato transcriptome, these 46 transcripts are very highly conserved in plants. The vast majority fell into 5 classes: enzymes of general metabolism; protein synthesis, modification, and transport; transcription; membrane transport; and photosynthesis and respiration. At least half of the transcripts have been previously associated with wounding or stress, suggesting that the systemic response to fire damage has components similar to those of other wound and stress responses. On the other hand, 30% of transcripts were associated with photosynthesis and respiration, suggesting that part of the response to fire damage is notably different from other wound and stress responses. Conclusions and future directions are included in Chapter 7. THE SYSTEMIC RESPONSE TO FIRE DAMAGE IN TOMATO PLANTS: A CASE STUDY IN THE DEVELOPMENT OF METHODS FOR GENE EXPRESSION ANALYSIS USING SEQUENCE DATA by JEFFREY SCOTT COKER A dissertation submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy DEPARTMENT OF BOTANY Raleigh 2004
Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh, India to Explore Bacterial Diversity

ABSTRACT Extraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.
Diversity and dynamics of microbial communities in natural and mixed starter cultures

In spite of their many drawbacks, the production of many traditional cheeses in Europe still relies on natural mixed strain starters, reproduced each day at the cheese plant. Cultivation-dependent and independent molecular methods have provided insight on the composition of natural starters for many cheese varieties. Because of the selective conditions used in their production they are almost invariably dominated by thermophilic homofermentative lactic acid bacteria (Lactobacillus helveticus, Lb. delbmeckii subsp. lactis, Streptococcus thennophilus). Natural starters are often dominated by a limited number of strains but subdominant species and strains may become dominant in the early phases of cheese making. Recent studies have proven that most of the diversity of the microflora of the cheese varieties for which natural starters are used originates from the use of raw milk rather than from the starter. Studies on the existence of a link between the composition of the starters and the area of the origin of the cheese are often inconclusive. However, because of their complexity, natural starters are still an important source of strains with novel properties for the cheese industry and mainly for historical and cultural reasons are unlikely to be replaced by defined strain starters in the production of traditional cheeses.
On the regulation of centriole duplication in human cells : exploring the interactions of polo-like kinase 4 with the centrosomal proteins Cep192 and STIL

Centrioles duplicate once in each cell cycle to give rise to two centrosomes that form the spindle poles during mitosis. Aberrant centriole duplication can result in the formation of supernumerary centrosomes, leading to incorrect spindle assembly and chromosome segregation errors, thereby possibly contributing to carcinogenesis (Ganem et al., 2009; Nigg, 2002; Zyss and Gergely, 2008). Thus, to ensure genome stability, centriole duplication has to be precisely regulated. Polo-like kinase 4 (PLK4) is a key regulator of centriole duplication (Bettencourt-Dias et al., 2005; Habedanck et al., 2005). PLK4 is characterized by an N-terminal Ser/Thr kinase domain and three C-terminal Polo-boxes (PB1-PB3) (Slevin et al., 2012). The PB1-PB2 domain is required for PLK4's centrosomal localization and binding to Cep152 (Cizmecioglu et al., 2010; Hatch et al., 2010; Slevin et al., 2012). In contrast to PB1-PB2, no binding partners have been described for PB3. Here, we identify Cep192 and STIL as novel interaction partners of PLK4-PB1-PB2 and PLK4-PB3, respectively. In the first part of this study, we reveal that Cep192 directly binds PB1-PB2 via a short region within its N-terminus, which contains conserved patches of acidic residues. We show that also in the case of Cep152 a short N-terminal acidic region is critical for the binding to PB1-PB2. These acidic regions of Cep192 and Cep152 enable electrostatic interactions with positively charged residues of the PB1-PB2 domain in order to promote PLK4 centriolar recruitment (Sonnen et al., 2013). In the second part of this study, we identify STIL as the first known binding partner of PLK4-PB3. We show that the coiled-coil motif of STIL (STIL-CC) is necessary and sufficient for this interaction and thus important for centriole duplication. Based on a collaboration for crystallographic and NMR analyses, we furthermore demonstrate that PB3 adopts a canonical PB fold, and that the PLK4-PB3/STIL-CC binding mimics coiled-coil formation. Analysis of structure-guided STIL mutants suggests a dual binding mode of STIL-CC to PB3 and L1 of PLK4 (linker between the catalytic domain and the PB domains). Taken together, we propose a speculative model for the initial steps of procentriole assembly according to which PLK4 is recruited to centrioles by electrostatic interactions between PB1-PB2 and Cep192/Cep152, and thereafter is stabilized and activated via STIL-CC binding to PB3 and L1.
Development of a RAPD-based SCAR marker for sex identification in Momordica dioica Roxb

Momordica dioica Roxb. , commonly known as spine gourd, is a perennial, rhizomatous, distinctly dioecious climber belonging to the family Cucurbitaceae. There is an urgent need for sex identification at pre-flowering stage to support selection and breeding of this plant because of its sex-specific economic values. Since there are no morphological markers to identify sex, an easy, rapid and reliable molecular method for sex identification at the pre-flowering stage in M. dioica is reported in this paper. Both Random amplification of polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) markers were used to search for specific markers linked to the sex locus. A total of 50 random decamer primers were used for screening of specific RAPD markers in male and female populations. Only one primer, OPA-15, amplified genomic DNA in different patterns in the male versus female genotypes. This sex-specific band OPA-151500 was cloned and sequenced. Based on the RAPD sequence, a pair of SCAR primers .
Investigating the role of Epstein-Barr virus in the pathogenesis of Multiple Sclerosis

Multiple Sclerosis (MS), a neuronal demyelination disease of the central nervous system, is the most common neurological disease in young adults worldwide, with an unknown cause. It affects over 23,000 people in Australia, and despite effective immune-based treatments, clinical deterioration and disability still occur. Epstein-Barr Virus (EBV), a human herpesvirus, which infects early in life and remains dormant within immune cells, is common in the unaffected population but almost universal in MS patients. Studies have found that MS patients maintain elevated EBV-specific antibody levels, most notably against latent Epstein-Barr Nuclear Antigen-1 (EBNA-1) proteins. The aims of this research were to investigate the significance of EBV immune responses, including those targeting the novel epitope EBNA-1(398-413), previously associated with MS risk in disease-discordant identical twins, and to understand the relationship between EBV-specific serological responses and genetic risk factors in an established MS cohort (n=426) and healthy controls (n=186). This study also investigated the influence of arginine modification (citrullination) on EBNA-1(398-413)-specific antibody responses, as well as the role of EBNA-1-specific IgG subclass bias. Novel in-house assays were compared with commercial ELISAs for EBV viral capsid antigen (VCA) and EBNA-1. MS patients had significantly higher antibodies against all EBV targets. Inclusion of EBNA-1(398-413)-specific antibody levels further discriminated cases and controls in risk analysis, in addition to genetic risk factors. Citrullinated and IgG1-specific EBNA-1 antibody levels were also elevated in MS cases compared to controls, although they did not improve case-control classification in combined statistical models. EBNA-1(398-413)-specific IgG from acute patients showed different protein reactivity from whole serum, as well as differences between progressive MS serum and healthy control plasma. This project contributes to the importance of EBV in the pathogenesis of the complex disease MS, and has identified novel and statistically powerful relationships between environmental and genetic MS risk factors.
The complete mitochondrial genome sequence of the giant mudskipper, Periophthalmodon schlosseri (Perciformes: gobiidae)

Abstract The complete mitochondrial genome sequence of the giant mudskipper, Periophthalmodon schlosseri, was first reported in this study. The circle genome is 16 470 bp in length, with the base composition of 28.4% T, 15.4% G, 29.2% A and 27.0% C. The mitogenome includes 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one D-loop region. Only the NADH dehydrogenase subunit 6 (NAD6) and eight tRNA genes are encoded on the light strand. The mitochondrial gene arrangement of P. schlosseri is similar to those of most other gobies. The phylogenetic analysis using the neighbor-joining method showed that the kinship between Periophthalmodon and Periophthalmus is closer than those between Periophthalmodon and other selected genera. Our complete mitogenome data are going to provide the basis for taxonomic and phylogenetic research of amphibious mudskippers.
Brain size and the risk of getting shot

Hunting kills hundreds of millions of animals annually, potentially constituting an important selection pressure on hunted species. We hypothesized that hunted individuals differing from survivors by having better ability to distinguish between dangerous humans and other human beings would be at a selective advantage. We tested whether shot individual birds had smaller brains than survivors, under the assumption that individuals with larger brains had superior escape ability. We used a large database on birds from Denmark to test whether getting shot was predicted by brain mass, while controlling statistically for the potentially confounding effects of age, sex, body mass and body condition. Analyses based on all species, or only species that were hunted, while controlling for differences in sampling effort in random effects models, showed consistently that shot individuals had smaller brains than survivors.
Analysis of Species Variety and Physiological Characteristics of Denitrifying Oligotrophic Bacteria Isolated from the Specific Environment in Korea

In an effort to isolate novel bacteria for the bioremediation of over-fertilized soils, we identified 135 denitrifying cells out of 3,471 oligotrophic bacteria pools (3.9%) using a denitrification medium supplemented with potassium nitrate as the sole nitrogen source. Soil samples were taken from ecologically well-conserved areas, including a mountain swamp around the demilitarized zone (Yongneup), two ecoparks (Upo and the Mujechi bog), and ten representative islands around the Korean peninsula (Jejudo, Daecheongdo, Socheongdo, Baekryeongdo, Ulrungdo, Dokdo, Geomundo, Hongdo, Huksando and Yeonpyeongdo). All of the 135 bacteria produced nitrogen gas from the denitrification medium, and were proved to be nitrate reductase positive by API-BioLog tests. Phylogenetic analysis using 16S rDNA sequences revealed that the 135 bacteria consisted of 44 different genera. Along with the most prominent, Proteobacteria (87.4%), we identified denitrifying bacteria from Firmicutes (9.4%), Actinobacteria (2.4%), and Bacteroidetes (0.8%). Physiological analyses of the 44 representative denitrifying bacteria, under various pH levels, growth temperatures and salt stresses, revealed 12 favorable denitrifying strains for soil bioremediation.
Biochemical and biophysical characterization of a peroxidase isolated from Euphorbia tirucalli with antifungal activity

Abstract A novel peroxidase from the latex of medicinal plant Euphorbia tirucalli (Pencil tree) belonging to the Euphorbiaceae family is purified to homogeneity using cation exchange chromatography. The enzyme, named Euphorbia tirucalli peroxidase (ETP) has a molecular mass of 38.8 kDa. The isoelectric point of the enzyme is pH 7.5 with optimum pH and temperature of pH 6.0 and 50 °C respectively. The extinction coefficient (ɛ280 nm1%) of the enzyme is 20.52 and the molecular structure consists of 13 tryptophan, nine tyrosine, and eight cysteine residues forming four disulfide bridges. Three peptide sequences, ALVHKECGPVVSCSDIVAIAARDSVVLTGGPKYDV, YYVDLMNRQGLFTSDQDLYT DKR, and MGQLEVVTGNQGEIR are obtained by MS/MS analysis which confirms the novelty of the enzyme. ETP belongs to α/β class of proteins with secondary structural features of approximately 10% α-helix, 29% β-sheet, and 61% random coil. ETP exhibits antifungal activity against Aspergillus niger and Candida albicans which shows its role in defense mechanism of plants. The enzyme is stable and retains its activity over a broad range of pH and temperature or prolonged storage at 4 °C. Simple purification, high yield, and stability enable exploration of the peroxidase for structure–function relationship studies as well as other biotechnological applications.
Caracterização de variantes virais de HIV-1 em indivíduos soropositivos com perfil de controle da progressão para a AIDS e da replicação viralavaliação da ocorrência de variantes de escape da resposta imune

The HIV infection is characterized by the existence of a clinical latency period (os asymptomatic period) between the acute infection and the AIDS phase, whose duration is extremely variable, generating distinct progression profiles between Hiv infected individuals. A small fraction of infected individuals (5-10%), named long term non progressors (LTNP), remains clinically asymptomatic and with high TCD4+ levels (>500cells/ul) during more than 8 years of infection in absence of treatment. Some of these individuals can even block almost all the viral replication and evolution, generating an evolutionary latency. The objective of this study was to analyze the mutational profile of HIV-1 and the emergency and maintenance dynamic of immune response escape mutants in HIV-1 positive patients classified as LTNPs and with different levels of viral replication control. For this end, a group of 12 LTNP patients, classified as viremic controllers (VC) and Elite Controllers with (ECB) and without Blips (EC) according to their level of viremia control, was used. For each individual, DNA samples were extracted from PBMCs obtained during the earliest (V1) and most recent visits (VX) available. The extracted DNA was used to amplification and sequencing, through a NGS methodology, of the Gag and Nef genes. The sequences obtained allowed to map the whole Nef gene (medium coverage of 286.463 reads/pb) and the first 1000bps of Gag (medium coverage of 212.674 reads/pb) The consensus sequences of each mapping were used to divergence analyses between V1 and VX and variants with frequencies higher than 0,5% in each mapped bp were considered real. The evolution analyses showed a lower degree of diversity and divergence of Gag and Nef genes between controllers. EC individuals presented lower mutational profile in both genes when compared with ECB and VC patients. While Gag presented higher conservation than Nef in VCs, both genes had showed similar variability in ECs, indicating that a higher viremia control limit the evolution in both regions. In some ECB individuals, evidence of superinfection and APOBEC3g mediated Hypermutation was observed. The analyses of mutation along regions of CTL-restricted epitopes had showed the presence of mutations arised between V1 and VX, which were indicative of imune response escape, in most individuals. Non synonymous mutations had been found in lower frequencies in all individuals, including ECs, indicating that those escape variantes arise, but most don\2019t manage to obtain evolutionary success
Morphological variations of the Solidago virgaurea L. complex along an elevational gradient on Mt Norikura, central Japan

Morphological variations in vegetative and reproductive organs in the Solidago virgaurea complex were examined for eight elevations between 1600 and 2400 m a.s. l. in Japan. The rosette diameter and stem height were lower at higher elevations. The stem diameter at any stem height was greater at higher elevations, suggesting that the S. virgaurea complex increases mechanical stiffness against strong wind at high elevations. The number of flower heads at any stem height was less at high elevations (2000–2400 m a.s.l.) than at low elevations (1600–1900 m a.s.l.). Leaf nitrogen concentration did not change along the elevational gradient. Leaf mass per area (LMA) tended to decrease with increasing elevation, except for 2400 m a.s. l. The decrease of LMA would contribute to maintaining a positive carbon balance at high elevations. The number of involucral rays of flower heads was mostly four at low elevations (1600–1900 m a.s.l.) and three at high elevations (2000–2400 m a.s.l.). The number of involucral scales and diameter of flower heads were greater at high elevations (2000–2400 m a.s.l.) than at low elevations (1600–1900 m a.s.l.). Therefore, the S . virgaurea complex is suggested to adapt to high elevations that have cool temperature, a short growing season and strong wind conditions by changing its vegetative and reproductive traits.
Elevated activity in adult mountain gorillas is related to consumption of bamboo shoots

Eastern gorillas (Gorilla beringei) are among the few mammal species that seasonally consume large quantities of young bamboo shoots, which are a rich source of energy. Here, we document how the consumption of bamboo shoots coincides with changes in behavior of adult mountain gorillas (Gorilla beringei beringei) monitored by the Dian Fossey Gorilla Fund's Karisoke Research Center in Volcanoes National Park, Rwanda. We offer a preliminary analysis of possible mechanisms underlying this behavioral change by measuring energy intake rates and the presence of cyanide and alcohol—ingredients potentially associated with increased activity levels—in fresh bamboo shoots. The percentage of bamboo shoots in the diet of gorillas was correlated positively with the rate of play behavior shown by adults in 2 of the 3 study groups. Play behavior was not the result of better weather conditions and also did not reflect the availability of spare time. Rather, there is some, but not consistent, evidence for a link between energy intake rates and play behavior of adults. Cyanide was not detected in young bamboo shoots, and the presence of alcohol remains inconclusive, albeit unlikely. In sum, our results show that consumption of a high-quality food can have a direct influence on the activity budget (and by extrapolation energy expenditure) of mountain gorillas through increased rates of play behavior. However, the physiological aspects underlying this elevated activity warrant further investigation.
Hümik Madde İçeren Yüzeysel Suların UV/H2O2 ile Dezenfeksiyonu

UV/H 2 O 2 disinfection of surface water containing humic substances was investigated. UV radiation was applied at doses of 68, 136, 204, 272, 341, 409, 545, 681 mWs/cm 2 to water samples containing various concentrations of humic acid [0-10 mg Dissolved Organic Carbon (DOC)/L] and 0.125 mg/L hydrogen peroxide. Total coliform numbers were determined after UV radiation and dark incubation of the samples. Results showed that the efficiency of bacterial reduction was reduced significantly with increasing concentrations of humic acid in water. Inactivation coefficients (k) for waters containing 0, 1, 5 ve 10 mg DOC/L humic acid were determined to be 0.0232, 0.0162, 0.0151 ve 0.0135, respectively according to Chick-Watson Model. Results of regrowth experiments indicated that free oxygen radicals produced by H 2 O 2 decomposition were also effective for the inactivation of bacteria after the completion of disinfection process.