The Florida manatee (Trichechus manatus latirostris), a subspecies of the West Indian manatee, is one of the most endangered marine mammals in United States waters. The Florida manatee is the only manatee that ranges into subtropical and temperate regions. During the winter months manatees adopt a “refuging strategy” where they aggregate at warm-water sources immediately following decreases in the ambient water temperature to below 20° C (68° F) in order to avoid cold stress syndrome (CSS). During the winter manatees aggregate in warm water refuges, including natural warm water springs and the effluent discharges of power plants. The purpose of this study was to determine the number of manatees that aggregate and utilize the waters of the Florida Power and Light (FPL) Plant in Port Everglades (PPE), Florida, its effluent canal, and the surrounding Intracoastal Waterway during the winter months. This study documents the importance of Port Everglades as a wintering refuge for the Florida manatee. This study analyzed the inverse relationship between the number of manatees present at a warm water effluent and water temperature. In this study data were collected over five manatee winter seasons (between 15 November and 31 March) from 1999-2004, from both boat-based and land-based surveys monitoring the presence of manatees in the effluent canal from the FPL electricity generating facility in Port Everglades, FL and the Intracoastal Waterway. Findings indicated that there was an inverse relationship between the number of manatees present and water temperature, where more manatees were present in cooler months, and fewer in warmer months. This study also analyzed the parameters of Catch per Unit Effort (CPUE) as well as heating degree-days and their effect on and relationship to the number of manatees present. The higher the heating degree-days number, the more severe, or cold, winter this indicates. The year with the highest heating degree-days, 24.98, was the 2002-2003 season, which was also the season with the highest number of manatees observed, 393, and the highest CPUE, 10.62 manatees/day.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Strawberry (Fragaria spp.) is an agricultural crop grown often in temperate regions that has high variability in its susceptibility to freezing injury. To breed cultivars for frost and freezing tolerance, identification of molecular markers associated with low temperature tolerance is advantageous. In this work, I investigated a high-throughput method for protein assays and western blotting. Success in streamlining these processes saves an immense amount of time and allows for the processing of more samples and obtaining larger datasets. Thirty-three octoploid varieties were tested for their accumulation of five different potential biomarkers in response to cold exposure. It was found that total dehydrin content, has the strongest potential to be reliable biomarkers for breeding programs. Previous work identified seven putative dehydrins in Fragaria, where two were purified and positively identified by mass spectrometry and determined to be COR47-like (SKn) and XERO2-like (YnSKn). This work demonstrated that cold tolerance positively correlated with dehydrin protein expression levels. To understand the cold-regulated expression of dehydrins as a function of cold exposure time, the levels of transcripts and corresponding proteins were examined in strongly cold tolerant (Alta) and lesser cold tolerant (FDP817, NCGR1363) Fragaria diploid genotypes. The COR47-like (SKn) and XERO2-like (YnSKn) dehydrins both had higher transcript accumulation and protein levels in the more cold tolerant line in comparison to the two less cold tolerant lines. Lack of correlation between transcript and resulting COR47 protein level in Alta were observed at several different timepoints, where protein accumulation preceded an increase in RNA. This trend was not seen with XERO2. This initiated an investigation to discover at what level COR47 is being regulated. First, the COR47 coding region was sequenced for all the genotypes to confirm against the predicted sequence. In addition, since two isoforms of the COR47 gene exist, and could possibly explain the discrepancy in transcript counts, primers were designed for both isoforms and RT-qPCR was performed to examine the transcripts of COR47 more closely. Through examination of the non-congruence of COR47 transcripts and protein, it was found that transcriptional mechanisms of regulation are not involved, and that post transcriptional and post-RNA splicing mechanisms are likely to be responsible for the observed trend in Alta. Conclusions from this work demonstrate that dehydrin transcripts and dehydrin protein accumulations are strong potential biomarkers for identifying low temperature tolerance in diploid strawberry.

No description available.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-06-22T12:29:24Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_marcelo_nogueira_do_amaral.pdf: 2321866 bytes, checksum: 813c57b5a855e4f2470433f907cc1a61 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-22T20:37:11Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_marcelo_nogueira_do_amaral.pdf: 2321866 bytes, checksum: 813c57b5a855e4f2470433f907cc1a61 (MD5) / Made available in DSpace on 2017-06-22T20:37:11Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_marcelo_nogueira_do_amaral.pdf: 2321866 bytes, checksum: 813c57b5a855e4f2470433f907cc1a61 (MD5) Previous issue date: 2015-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O arroz (Oryza sativa L.) é o segundo cereal mais cultivado no mundo, sendo o Brasil o 9º maior produtor. Apesar dos bons níveis de produtividade, acredita-se que estes números sejam insuficientes para suprir o requerimento populacional no mundo, sendo necessário um aumento na produção. Entretanto, diversos estresses abióticos, como salinidade, toxidez por ferro e baixa temperatura, limitam a produtividade do arroz mundialmente. A resposta das plantas às condições estressantes é um fenômeno complexo, envolvendo alterações morfológicas, fisiológicas, bioquímicas e moleculares. Assim, o conhecimento do transcriptoma e análise fotossintética de plantas de arroz submetidas a estes estresses, pode auxiliar a elucidar quais vias metabólicas são alteradas e quais são as principais respostas bioquímicas e fisiológicas das plantas em tais condições. Desta forma, o objetivo desse trabalho foi analisar os genes diferencialmente expressos (DEGs), através da metodologia de RNA-Seq e quantificar as trocas gasosas em folhas de arroz (cv. BRS Querência), no estádio V3, submetidas aos estresses por frio, ferro e sal durante o período de 24 horas. Um intervalo entre 41 - 51 milhões de reads foram submetidas ao alinhamento, sendo que deste total um intervalo entre 88,47 - 89,21% foram mapeadas no genoma de referência. Foi observado 5.506 genes diferencialmente expressos para o estresse por frio, 1.808 para o sal e 630 para o ferro, sendo que 330 DEGs foram comuns aos três estresses. A anotação funcional através do software MapMan demonstrou que o estresse por frio promoveu maiores alterações no metabolismo em geral. O estresse salino apresentou uma rede de interação de termos de Ontologia Gênica (GOs) sobre-representados mais complexa que os demais estresses. Nos parâmetros de trocas gasosas, a taxa assimilatória liquida foi a única que apresentou diferença significativa, com o estresse por frio apresentando a menor média. A partir dos resultados obtidos foi possível concluir que o estresse por baixa temperatura apresenta um maior número de genes diferencialmente expressos e que há uma maior relação entre o estresse salino e por ferro. Além disso, o estresse por frio é o que afeta mais drasticamente a fotossíntese, tanto em nível molecular quanto fisiológico, e apesar de reduções na taxa assimilatória liquida, a cultivar BRS Querência demonstrou menores alterações nos estresses salino e por excesso de ferro. / Rice (Oryza sativa L.) is the second most cultivated cereal in the world; Brazil is the ninth biggest producer. Despite the high levels of productivity, it is believed that these numbers are insufficient to meet the population requirement in the world, demanding an increase in its production. However, various abiotic stresses, such as salinity, toxicity by iron and low temperature, limit the productivity of rice worldwide. The response of plants to stressful conditions is a complex phenomenon involving morphological, physiological, biochemical and molecular changes. Thus, the knowledge of the transcriptome and photosynthetic analysis of rice plants subjected to these stresses can help to elucidate which metabolic pathways are changed and what are the main biochemical and physiological responses of plants under such conditions. Thus, the objective of this study was to analyze the differentially expressed genes (DEGs), through the methodology of RNA-Seq and quantify gas exchanges in rice leaves (cv. BRS Querência) in V3 stage, under cold, iron and salt stress during 24 hours. A range from 41 to 51 million reads was submitted to alignment, in which a range from 88.47 to 89.21% has been mapped in the reference genome. 5,506 differentially expressed genes under cold stress were observed, as well as, 1,808 for salt and 630 for iron stress; 330 of them were similar to the three DEGs stresses. Functional annotation by MapMan software showed that, usually, cold stress promoted major changes in the metabolism. The saline stress presented a network of interaction of Gene Ontology terms (GOs) over-represented are more complex than the other stresses. In the parameters of gas exchange, the net assimilation rate was the only one significant difference, with stress by cold presenting the lowest average. From the results obtained it was possible to conclude that the low temperature stress has a greater number of differentially expressed genes and that there is a greater relationship between salt and iron stress. In addition, the cold stress is affecting more drastically photosynthesis, either in molecular-level and physiological, and despite reductions in net assimilation rate, BRS Querência showed minor changes in the salt stress and excess iron.

Indiana University-Purdue University Indianapolis (IUPUI) / Drought and cold conditions are among the major factors affecting plant growth and crop production globally. Dehydrins are group II late embryogenesis abundant (LEA) proteins characterized by a conserved K-region (EKKGIMDKIKEKLPG consensus sequence) that accumulate in many plants during drought, low temperature, and high salinity to confer stress tolerance. While it has been demonstrated that overexpression of dehydrins improves cold tolerance in various crop plants, the mechanism leading to cold tolerance is still unclear. Previous studies reported phosphorylation of AtERD14 dehydrin by casein kinase II (CKII) led to an increase in calcium binding activity. Mass spectroscopy analysis determined that the phosphorylation was localized to a poly-serine (S) region. To further characterize the S-region, GST fused ERD14 mutants were created via site-directed mutagenesis and deletion of either the amino or carboxyl ends of ERD14 via the QuickChange® Multi Site-Directed Mutagenesis Kit. Phosphorylation of purified mutant proteins by CKII was analyzed via gel shift and direct phosphorylation assays. The effect of phosphorylation on calcium binding activity was also analyzed. Results showed the serine (S) residue at position 83 was crucial to phosphorylation-dependent molecular mass shift and Ca2+-binding activities followed by the serine residue at position 85 in importance. Mutation of serines at positions 83, 84, and 85 completely eliminated the phosphorylation-dependent gel shift and calcium binding. Examination of truncation mutants determined the N-terminal was an important region for protein structure modification and phosphorylation ability leading to Ca2+ activation. Calcium binding activity of the truncated mutants indicated the calcium binding site was localized in the region between the S-region and the K-region near the C-terminal end. To characterize the acidic dehydrins contribution to cold tolerance in vivo, three single (erd10, erd14, cor47) knockouts (KOs) were characterized. Single KOs produced no cold sensitive phenotype indicating the need for multiple dehydrin KOs in Arabidopsis in order to potentially produce a cold sensitive phenotype.

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.

The study of cis-regulatory elements (CREs) will allow for increased understanding of regulation and lead to insight regarding the mechanisms governing growth, development, health, and disease. The aim of this study was to characterize the de novo in silico predictions of Arabidopsis CREs. Eight synthetic and 30 native promoter-constructs containing an eGFP/GUS reporter protein were generated for cold, genotoxic, heat, osmotic, and salt stress; the circadian clock; ABA signaling; root and epidermis tissue. Constructs were stably transformed into A. thaliana Col-0 and the effects of the CREs were evaluated by in planta stress or tissue assays using GUS expression levels. Results reveal a novel genotoxic element that specifically directs GUS expression in rosette leaves during genotoxic stress. Results also look promising for novel epidermis and root-specific elements. Results of these assays validate the de novo prediction pipeline's ability to identify novel and known CREs related to abiotic stress.