The goal of this project is to decipher the quorum sensing (cell-to-cell signaling) abilities of Shewanella oneidensis MR-1, a Gram-negative bacterium well known for its ability to use geologic substrates, such as Fe and Mn oxides, for respiratory purposes. Overall our results show that S. oneidensis cannot utilize either an acyl-homoserine lactone (AHL) or AI-2 quorum sensing signal, despite previous work that indicated that it produced an AHL that would enhance it ability to growth in certain anaerobic environments. Using a variety of quorum sensing signal sensors, no evidence could be found that S. oneidensis has a typical AHL signal. An in silco analysis of the genome also produced little evidence that S. oneidensis has the genes to accept or relay an AHL signal. S. oneidensis can produce a luminescence response in the AI-2 reporter strain, Vibrio harveyi MM32. This luminescence response is abolished upon deletion of luxS, the gene responsible for catalyzing AI-2. Deletion of luxS also affected biofilm formation. Within 16 hours of growth in a biofilm flow-through reactor, the luxS mutant had an inhibited ability to initiate biofilm formation. After 48 hours of growth, the mutant's biofilm had developed similarly to wild-type. The addition of synthetic AI-2 did not restore the mutant's ability to initiation biofilm formation, which led to the conclusion that AI-2 is not likely used as a quorum sensing signal in S. oneidensis for this phenotype. Because of the involvement of LuxS in the activated methyl cycle (AMC) in other organisms, growth on various sulfur sources was examined. A mutation in luxS produced a reduced ability to growth with methionine as the sole sulfur source. Methionine is a key metabolite used in the AMC to produce a methyl source in the cell and homocysteine. This data suggests that LuxS is important in metabolizing methionine and the AMC in S. oneidensis. / Ph. D.

Structural and electronic effects in hydrocarbon reactions over metal oxides have been examined by comparing the reactions of methyl (-CH₃) and methylene (=CH₂) fragments on three different oxide single crystal surfaces: α-Cr₂O₃(101̅2), α-Cr₂O₃(0001), and α-Fe₂O₃(101̅2). The intermediates have been generated through the decomposition of halogenated hydrocarbons. The primary reactions of methyl and methylene over α-Cr₂O₃ are methyl dehydrogenation to methylene, and methylene coupling (C-C bond formation) to ethylene (CH₂=CH₂). The different surface geometric structures of α-Cr₂O₃(101̅2) and (0001) lead to an increase in the activation barrier for methylene surface migration, a critical step in the coupling reaction, of 5.9 kcal/mol over the (0001) surface. For methyl dehydrogenation, differences in the local site pair (cation/anion) geometry and the proximity of surface lattice oxygen to the methyl group do not result in a significant difference in the barrier for dehydrogenation, suggesting that the surface anions play a minor role in the dehydrogenation of methyl on these surfaces. Electronic differences in the Fe³⁺ (𝑑⁵) and Cr³⁺ (𝑑³) cations on structurally-similar α-Cr₂O₃(101̅2) and α-Fe₂O₃(101̅2) surfaces lead to major differences in reaction selectivity. α-Cr₂O₃(101̅2) is nonreducible under the reaction conditions of this study, but α-Fe₂O₃(101̅2) is highly reducible due to the difference in the d electron configuration. Hydrocarbons are formed over α-Cr₂O₃(101̅2), but nonselective oxidation products (CO₂, CO, H₂O) are formed over the stoichiometric α-Fe₂O₃(101̅2) surface along with surface reduction. Reduction of the α-Fe₂O₃(101̅2) leads to a shift in the product selectivity towards formaldehyde (CH₂O) and ethylene. For the limited number of systems examined in this study, examples of geometric structure sensitive (methylene coupling) and structure insensitive (methyl dehydrogenation) reactions have been found on α-Cr₂O₃, and electronic effects are observed for the reactions on α-Cr₂O₃(101̅2) and α-Fe₂O₃. For the structure sensitive reaction, the differences in surface geometry impact the reactions kinetics over Cr₂O₃ but not the types of products formed, while the electronic differences give rise to dramatic changes in the selectivity associated with the very different products formed over α-Cr₂O₃(101̅2) and α-Fe₂O₃(101̅2). / Ph. D.

The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields. Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor. Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates. Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline. In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar. These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields. / Ph. D.

In 2007 and 2008 more than 100 million dollars of fresh market tomatoes were grown in Virginia, with the majority of production occurring on the Eastern Shore of Virginia (ESV), according to the National Agricultural Statistics Service. Bacterial wilt of tomato, caused by Ralstonia solanacearum (Smith) and Yabucchi et al., is the most devastating disease of tomato on the ESV. Four 'observational trials' were conducted on the ESV over three growing seasons to determine the temporal and spatial distribution of this disease in commercial tomato fields. Plants were assessed at approximately one-week intervals throughout the growing seasons and the incidence of bacterial wilt for each individual plant was recorded. A steady increase in both disease incidence and clustered distribution of the disease within rows was observed as the growing season progressed. Positive correlations between disease incidence and percentage of rows exhibiting a significant clustered distribution occurred in all trials, which indicated an increase in clustered distribution as disease incidence increased. Research trials were conducted over three years, beginning in the summer of 2007, to investigate the effects of tomato bacterial wilt resistant cultivars on the ESV. In 2008 and 2009, the selective, systemic compound which induces host plant resistance, acibenzolar-S-methyl (ASM) was incorporated into resistant cultivar trials. Results from the 2007 trial revealed significant resistance in some of the breeding lines, CRA 66 and PI 126408. The 2008 and 2009 trials revealed that ASM was not effective at reducing levels of bacterial wilt. Grafted transplants in the spring trials of 2008 and 2009 had varied results in resistance and yield. Results revealed the tomato cultivar BHN 669 was an excellent resistant cultivar with promising yield potential and fruit quality. / Master of Science

Reactivity ratios for the homogeneous free radical initiated copolymerization of acrylonitrile and methyl acrylate were measured by NMR on isolated, low conversion copolymers and by real time in situ FTIR. The system utilized azobisisobutyronitrile (AIBN) initiator in dimethyl formamide (DMF) at 62°. The FTIR technique allowed rapid generation of extensive copolymer compositions, which permitted application of nonlinear least squares methodology for the first time to this copolymer system. Thus, reactivity ratios at the 95% confidence level were determined to be 1.29 ± 0.2 and 0.96 ± 0.2 for acrylonitrile and methyl acrylate, respectively. The results are useful for the development of acrylonitrile (<90%) melt processable copolymer fibers and films, which could include precursors for carbon fibers. / Master of Science

Sinorhizobium meliloti (Ensifer meliloti) is a bacterium that will exist saprotrophically in the soil and rhizosphere or as a differentiated bacteroid inside root nodules of the legume genera Medicago, Melilotus, and Trigonella. It exists in symbiosis when inside a host plant and will fix gaseous N2 into ammonium for the plant. In return, a population of the bacteria is harbored inside the plant where it can proliferate beyond what would be possible in the rhizosphere or bulk soil. This symbiosis is a defining feature of the Fabaceae (legume) family, a clade that diverged approximately 60 million years ago and is now the 5th largest plant family by species count. Each legume species pairs with one or several strains of bacteria, referred to broadly as rhizobia. The rhizobia identify their proper host plant by a cocktail of secondary metabolites called flavonoids released from specific parts of the roots. Initiation of the symbiosis may only occur at the tips of young root hairs. Therefore, the means rhizobia take to localize themselves to these sites must be the inceptive step in the symbiotic interaction. The studies here examine the mechanisms and priorities rhizobia use to achieve this goal. Movement of bacteria is referred to as motility and is achieved via (in rhizobia, multiple) rotating flagella, proteinaceous extracellular appendages that propel the cell through liquid environments. On their own, flagella may only move but not guide the cell. Navigation is achieved through sensors that detect chemical attractant or repellent cues in the environment and an intracellular signaling system that relays information to appropriately control locomotion. This sensing is called chemotaxis. A research focus is directed on the sensing aspect of chemotaxis to understand which chemical compounds are the preferred attractants for S. meliloti. An emphasis is placed on those compounds released from germinating host seeds. Chapter 2 spearheads our research goals by examining the chemotactic potential of host-derived flavonoids, the compounds that induce the symbiotic signaling in the rhizobial symbiont. While a logical place to start, this study reveals that our strain of rhizobia is not attracted to flavonoids. We determined that the best chemoattractants are hydrophilic in nature and that hydrophobic compounds, such as flavonoids, are not effective chemoattractants. In addition, we discuss the nature of chemotactic agents and symbiosis inducers to fortify our understanding of how classes of compounds contribute to the rhizobia-plant interaction. In chapter 3, we characterize the sensor protein, McpV, and its ligand profile for carboxylates. The protein is first screened using a high-throughput assay to test numerous possible ligands simultaneously. We confirm positive reactions using direct binding studies and quantify dissociation constants. Then, the phenotypic response to these ligands is measured using capillary chemotaxis assays, and the role mcpV plays in this response is confirmed using deletion mutants. Last, the symbiotic context is addressed by quantifying these ligands in exudates of the host alfalfa. These experiments show that McpV is a chemotactic sensor dedicated to detecting 2 – 4 C monocarboxylates. Only one of the compounds found in the ligand profile, glycolate, was detected in seed exudates, so the contribution of McpV to host sensing is yet to be expounded. Chapter 4 follows the model of chapter 2 but is complicated when the ligand screen used previously gives ambiguous results. Using direct binding studies, we were able to confirm the true ligand amidst numerous false positives. Analytical gel filtration suggests that McpT exists as a dimer regardless of ligand binding. Capillary chemotaxis assays quantified the responses mediated by McpT to di- and tri-carboxylates, which were slightly weaker, but still on-par with the responses to McpV ligands. Strains with mcpT deletions showed strongly reduced, but in some cases, not abolished, chemotaxis to carboxylates. Chapter 5 examines McpX – the chemoreceptor already known to be a sensor of quaternary ammonium compounds. This is a structural investigation into the binding of McpX to its ligands. A crystal structure of the ligand binding region of the protein is resolved to understand how ligands fit into the binding pocket of McpX and what determines its structurally diverse ligand profile. The contribution of certain residues to ligand binding are further probed using direct binding studies on single point variants of McpX. The analysis of chemoreceptor functions hint at what kinds of molecules are most important to bacterial survival and reproduction. Knowing what the bacterium is tuned to seek out grants understanding of what niches they prefer, and how they thrive in those niches. For S. meliloti and other rhizobia, the preeminent niche is one in symbiosis with a host plant. The sum of this knowledge we have accrued with S. meliloti lends itself to agricultural goals of soil enrichment, legume inoculation, nutrient cycling, and environmentally safe and efficient crop fertilization. / Doctor of Philosophy / Sinorhizobium meliloti and other soil-dwelling bacteria termed rhizobia are crucial to the cultivation of leguminous crops such as alfalfa, soy, pea, lentil, peanut, and many more. The bacterium can be internalized by the plant host's roots where it will supply the plant with nitrogen. This is a great boon to crops when they need to accumulate more protein in seed stores, or for plants that survive in nutrient depleted soils. The bacterium must begin seeking out the host plant by sensing chemical cues. It can navigate to the proper location by using a process called chemotaxis. This process is centered around chemoreceptors that can be likened to the nose of the bacterium. Using these chemoreceptors, the bacterium will seek out compounds that benefits it – these are usually food sources. Identifying what each individual chemoreceptor senses allows us to understand what the bacterium needs to seek out to survive. We correlate this information with compounds that the plant secretes and find that many chemoreceptors have evolved to sense signals that will lead the bacterium to a plant root. This interaction is a key part of how the symbiosis is propagated and ultimately benefits the agriculture of leguminous plants.

Yes / The production of methyl decanoate (MeDC) through esterification of decanoic acid (DeC) with methanol by reactive distillation is operationally challenging and energy-intensive due to the complicated behaviour of the reaction system and the difficulty of retaining the reactants together in the reaction region. Methanol being the lightest component in the mixture can separate itself from the reactant DeC as the distillation proceeds which will cause a massive reduction in the conversion of DeC utilizing either a batch or continuous distillation process. Aiming to overcome this type of the potential problem, novel integrated divided-wall batch reactive distillation configuration (i-DWBD) with recycling from the distillate tank is established in this study and is examined in detail. This study has clearly demonstrated that the integrated divided-wall batch reactive distillation column (i-DWBD) is superior to the traditional conventional batch distillation (CBD) and both the divided-wall (DWBD), and split reflux divided-wall (sr-DWBD) batch reactive distillation configurations in terms of maximum achievable purity of MeDC and higher conversion of DeC into MeDC. In addition, significant batch time and energy savings are possible when the i-DWBD is operated in multi-reflux mode.

Yes / Although batch reactive distillation process outperforms traditional reactor-distillation processes due to simultaneous reaction and separation of products for many reaction systems, synthesis of Methyl lactate (ML) through esterification of lactic acid (LA) with methanol in such process is very challenging due to difficulty of keeping the reactants together when one of the reactants (in this case methanol) has the lowest boiling point than the reaction products. To overcome this challenge, two novel reactive distillation column configurations are proposed in this work and are investigated in detail. These are: (1) integrated conventional batch distillation column (i-CBD) with recycled methanol and (2) integrated semi-batch and conventional batch distillation columns (i-SBD) with methanol recovery and recycle. Performances of each of these configurations are evaluated in terms of profitability for a defined separation task. In i-SBD column, an additional constraint is included to avoid overflow of the reboiler due to continuous feeding of methanol into the reboiler as the reboiler is initially charged to its maximum capacity. This study clearly indicates that both integrated column configurations outperform the traditional column configurations (batch or semi-batch) in terms of batch time, energy consumption, conversion of LA to ML, and the achievable profit.

Yes / Methyl Decanoate (MeDC) is a Fatty Acid Methyl Ester (FAME) and is an important chemical compound with global production of 31 million tons per year. However, synthesis of methyl decanoate (MeDC) via esterification of Decanoic Acid (DeC) with methanol by reactive distillation is operationally challenging due to difficulty of keeping the reactants together in the reaction zone as methanol being the lightest component in the mixture can separate itself easily form the other reactant deteriorating significantly the conversion of DeC using either conventional batch or continuous distillation column. This is probably the main reason for not applying the conventional route for MeDC synthesis. Whether Semi-batch Distillation column (SBD) and the recently developed Integrated Conventional Batch Distillation column (i-CBD) offer the possibility of revisiting such chemical reactions for the synthesis of MeDC is the focus of this paper. The minimum energy consumption (Qtot) as the performance measure is used to evaluate the performances of each of these reactive column configurations for different range of methyl decanoate purity and the amount of product. It is observed that the use of i-CBD column provides much better performance than SBD column in terms of the production time and the maximum energy savings when excess methanol is used in the feed. However, the SBD column is found to perform better than the i-CBD column when both reactants in the feed are in equal amount. Also, the optimization results for a given separation task show that the performance of two-reflux intervals strategy is superior to the single-reflux interval in terms of operating batch time, and energy usage rate in the SBD process at equimolar ratio.

O gênero Erythroxylum P. Browne pertence a família Erythroxylaceae Kunth e é principalmente conhecido devido as espécies E. coca e E. novogranatense, as “plantas da coca”. O gênero é a única fonte natural da cocaína e de outros alcaloides com núcleo ecgonina, principais responsáveis pelos efeitos tóxicos e biológicos originados pelo consumo de suas espécies. A presença da cocaína é amplamente aceita para as plantas de coca, porém a sua biossíntese por outras espécies apresenta divergência, o que pode estar relacionada ao período de coleta. Além destas particularidades fitoquímicas, as espécies do gênero também apresentam importantes atividades como antioxidante e antimicrobiana, além de toxicidade a humanos e a ovinos. O Brasil é o centro da diversidade e do endemismo do gênero e no sul do país, nos estados do Rio Grande do Sul e de Santa Catarina, as carcaterísticas fitoquímicas e potencialidades tóxicas e farmacológicas das espécies apresentam um campo aberto para estudos. Desta forma, foram avaliadas características fitoquímicas e potencialidades tóxicas e farmacológicas de espécies nativas dos estados do Rio Grande do Sul e de Santa Catarina, coletadas em dois períodos climáticos distintos, verão e inverno, além de revisão bibliográfica sobre as espécies produtoras de cocaína e as implicações segundo a legislação brasileira vigente. Foram utilizadas metodologia de planária, disco-difusão em ágar, microdiluição em caldo, medida da extinção da absorção do cátion 2,2-difenil-1-picrilhidrazil e procedimentos preconizados pelas Nações Unidas para a investigação fitoquímica de espécies produtoras de cocaína. Foi verificado que 24 espécies apresentam o aparato enzimático para a produção da cocaína. Embora E. novogranatense origina rendimentos apropriados ao tráfico, segundo a legislação Brasileira vigente, apenas E. coca encontra-se proscrita, o que torna necessário a inclusão de E. novogranatense na lista de plantas proscritas. Para o estudo toxicológico desenvolvido com E. deciduum sobre modelo de planária, foi necessário adaptar a metodologia existente. O extrato da espécie ocasionou aumento significativo da velocidade de locomoção (p= 0.016) e comportamentos estereotipados padronizados de posição tipo C e hipercinesia tipo parafuso, eventos característicos da neurotransmissão dopaminérgica em planárias. Os resultados, além de ampliar as possibilidades do uso destes vermes na busca por extratos ativos, demonstram que E. deciduum biossintetiza metabólitos ativos sobre este neurotransmissor, os quais podem estar relacionados com os efeitos ocasionados pelo consumo da espécie vegetal. A influência do período de coleta sobre as potencialidades farmacológicas e as características fitoquímicas foi investigada e os resultados demonstram esta infuência. Diferentes extratos de E. argentinum e E. deciduum, coletadas durante o verão, apresentaram atividade antimicrobiana superiores a verificada para os extratos obtidos de coletas realizadas no inverno. O extrato etanólico de E. argentinum, coleta verão, pode ser considerado um verdadeiro antimicrobiano, MIC= 0,78mg/ mL, e desta forma torna-se uma fonte promissora para a descoberta de novas moléculas antimicrobianas. Os extratos das espécies E. argentinum e E. deciduum demonstram pertinentes resultados sobre a atividade antioxidante. A influência do período de coleta, verão/ inverno, sobre a atividade foi identificada. A influência do período de coleta sobre as atividades tóxico-farmacológicas é corroborada pelo perfil alcaloídico obtido de noves espécies do gênero, coletadas durante o verão e o inverno, nos estados do Rio Grande do Sul e de Santa Catarina. O perfil alcaloídico das espécies E. amplifolium, E. argentinum, E. cuneifolium, E. cuspidifolium, E. deciduum, E. microphyllum, E. myrsinites, E. pelleterianum e E. vacciniifolium demonstra a presença dos alcaloides: éster de metecgonidina, éster de metilecgonina, cuscohigrina e tropacocaína e dos intermediários da biossíntese: higrina, tropinona e tropanol. A identificação dos metabólitos éster de metilecgonina e éster de metilecgonidina também demonstra que as espécies do gênero Erythroxylum presentes no Sul do Brasil apresentam potencialidade econômicas, pois podem ser empregadas na produção de padrões toxicológicos. Os objetivos propostos foram cumpridos e permitem concluir que espécies do gênero Erythroxylum dos estados do Rio Grande do Sul e de Santa Catarina, Sul do Brasil, demonstram importância tóxica, farmacológica e química, sendo parte destas influenciada pelo período de coleta, verão/ inverno. / “Chemical and Biological Study of Erythroxylum P. Browne Species Collected in Southern Brazil”. Erythroxylum P. Browne genus belongs to the Erythroxylaceae Kunth family and is mainly known due to E. coca and E. novogranatense species, the "coca plants". The genus is the only natural source of cocaine and other alkaloids with ecgonine nucleus, mainly responsible for the toxic and biological effects caused by the consumption of their species. The presence of cocaine is widely accepted for coca plants, but its biosynthesis by other species shows divergence, which may be related to the collect period. Besides these phytochemical peculiarities, the species of the genus also present important activities such as antioxidant and antimicrobial, besides toxicity to humans and sheep. Brazil is the center of diversity and endemism of the genus and in the south of the country, in the states of Rio Grande do Sul and Santa Catarina, the phytochemical characteristics, toxic and pharmacological potentials of the species present an open field for studies. Thus, phytochemical characteristics, toxic and pharmacological potentials of native species of the states of Rio Grande do Sul and Santa Catarina were assessed, collected in two distinct climatic periods, summer and winter, as well as a bibliographical review on cocaine producing species and implications under current Brazilian law. Some methodologies were used such as planaria, disc-diffusion in agar, microdilution in broth, extinction measurement of the absorption of cation 2,2-diphenyl-1-picrylhydrazyl and procedures recommended by the United Nations for the phytochemical investigation of cocaine producing species. It was verified that twenty-four species present the enzymatic apparatus for the production of cocaine. Although E. novogranatense originate income appropriate to traffic according to current Brazilian legislation, only E. coca is outlawed, which makes it necessary to include E. novogranatense in the list of proscribed plants. For the toxicological study developed with E. deciduum on planaria model, it was necessary to adapt the existing methodology. The extract of the species caused a significant increase in locomotion velocity (p= 0.016) and standardized stereotyped behaviors of type C position and screw- type hyperkinesia, characteristic events of dopaminergic neurotransmission in planarians. The results, besides expanding the possibilities of the use of these worms in search for active extracts, demonstrate that E. deciduum biosynthesizes active metabolites on this neurotransmitter, which may be related to the effects caused by the consumption of the plant species. The influence of the collect period on pharmacological potentials and phytochemical characteristics was investigated and the results demonstrate this influence. Different extracts of E. argentinum and E. deciduum collected during summer showed higher antimicrobial activity than the extracts obtained from winter collect. The ethanolic extract of E. argentinum, summer collect, may be considered a true antimicrobial, MIC= 0.78mg/ mL, on Staphylococcus aureus, and in this way it becomes a promising source for the discovery of new antimicrobial molecules. The extracts of E. argentinum and E. deciduum species demonstrate relevant results on antioxidant activity. The influence of the collecting period, summer/ winter, on the activity was verified. The influence of collect period on toxic- pharmacological activities is corroborated by the alkaloid profile obtained from nine species of the genus, collected during summer and winter in the states of Rio Grande do Sul and Santa Catarina. The alkaloid profile of E. amplifolium, E. argentinum, E. cuneifolium, E. cuspidifolium, E. deciduum, E. microphyllum, E. myrsinites, E. pelleterianum and E. vacciniifolium species shows the presence of alkaloids: ecgonidine methyl ester, ecgonine methyl ester, cuscohygrine and tropacocaine and the biosynthesis intermediates: hygrine, tropinone and tropanol. The identification of the ecgonine methyl ester and ecgonidine methyl ester also shows that Erythroxylum genus species present in southern Brazil have economic potential because they can be used in the production of toxicological standards. The aims were met and allow the conclusion that Erythroxylum genus of Rio Grande do Sul and Santa Catarina states, south of Brazil, show toxic, pharmacological and chemical importance, part of these being influenced by the collect period, summer/ winter.