<p>This doctoral research comparatively assesses the biogeochemical properties of suspended aquatic flocs through a integrated field-laboratory approach; providing new insight into the linkages among floc associated bacteria, floc-reactive solid phases and trace metal uptake.</p> <p>Results show flocs to possess a distinct geochemistry, microbiology and composition from bed sedimentary materials in close proximity (III-oxyhydroxide minerals (FeOOH); resulting in localized floc-Fe-mineral precipitates and enhanced reactivity. Further, the Fe-enrichment of floc and of floc bio-mineral constituents in turn provides an important and novel lens through which to examine how environmental microbial communities, microbial metabolism and Fe^III/Fe^IIredox transformations interact. The results were the discovery of floc-hosted, Fe^III/II-redox cycling bacterial consortia across diverse oxygenated (O₂^Sat.=1-103%) aquatic systems, which were not predicted to sustain bacterial Fe-metabolism. Both environmental<em> </em>and experimentally-developed consortial aggregates constituted multiple genera of aero-intolerant Fe^III-reducing and Fe^II-oxidizing bacteria together with oxygen consuming organotrophic species. These findings highlight that the implementation of geochemical thermodynamic constraints alone as a guide to investigating and interpreting microbe-geosphere interactions may not accurately capture processes occurring <em>in situ.</em></p> <p><em> </em> Seasonal investigation of microbial Fe^III/II-redox transformations highlighted the interdependence of floc Fe-redox cycling consortia members, revealing that cold conditions and a turnover in putative Fe-reducing community membership extinguishes the potential for coupled Fe-redox cycling by wintertime floc bacteria. Further, the observed summer-winter seasonal turnover of <em>in situ</em> floc community membership corresponded with an overall shift from dominant Fe to S redox cycling bacterial communities. This significantly impacted observable floc Fe and TE (Cd, Pb) geochemistry, resulting in a shift in floc associated Fe-phases from dominantly Fe^(III)_(s) to Fe^(II)_(s), and, in turn, corresponded to a large decrease of TE uptake by flocs under ice.</p> / Doctor of Science (PhD)

Engineered nanomaterials (ENMs) have become ubiquitous in consumer products and industrial applications, and consequently the environment. Much of the environmentally released ENMs are expected to enter terrestrial ecosystems via land application of nano-enriched biosolids to agricultural fields. Among the organisms most likely to encounter nano-enriched biosolids are the key soil bacteria known as plant growth promoting rhizobacteria (PGPR). I reviewed what is known concerning the toxicological effects of ENMs to PGPR and observed the need for high-throughput methods to evaluate lethal and sublethal toxic responses of aerobic microbes. I addressed this issue by developing high-throughput microplate assays which allowed me to normalize oxygen consumption responses to viable cell estimates. Oxygen consumption is a crucial step in cellular respiration which may be examined relatively easily along with viability and may provide insight into the metabolic/physiological response of bacteria to toxic substances. Because many of the most toxic nanomaterials (i.e. metal containing materials) exhibit some level of ionic dissolution, I first developed my methods by examining metal ion responses in the PGPR, Bacillus amyloliquefaciens GB03. I found this bacterium exhibits differential oxygen consumption responses to Ag+, Zn2+, and Ni2+. Exposure to Ag+ elicited pronounced increases in O2 consumption, particularly when few viable cells were observed. Also, while Ni2+ and Zn2+ are generally thought to induce similar toxic responses, I found O2 consumption per viable cell was much more variable during Ni2+ exposure and that Zn2+ induced increased O2 utilization to a lesser extent than Ag+. Additionally, I showed my method is useful for probing toxicity of traditional antibiotics by observing large increases in O2 utilization in response to streptomycin, which was used as a positive control due to its known effects on bacterial respiration. After showing the utility of my method for examining metal ion responses in a single species of PGPR, I investigated the toxicity of silver ENMs (AgENMs) and ions to three PGPR, B. amyloliquefaciens GB03, Sinorhizobium meliloti 2011, and Pseudomonas putida UW4. The ENM exposures consisted of untransformed, polyvinylpyrrolidone coated silver ENMs (PVP-AgENMs) and 100% sulfidized silver ENMs (sAgENMs), which are representative of environmentally transformed AgENMs. I observed species specific O2 consumption responses to silver ions and PVP-AgENMs. Specifically, P. putida exhibited increased O2 consumption across the observed range of viable cells, while B. amyloliquefaciens exhibited responses similar to those found in my first study. Additionally, S. meliloti exhibited more complex responses to Ag+ and PVP-AgENMs, with decreased O2 consumption when cell viability was ~50-75% of no metal controls and increased O2 consumption when cell viability was <50%. I also found the abiotically dissolved fraction of the PVP-AgENMs was likely responsible for most of the toxic response, while abiotic dissolution did not explain the toxicity of sAgENMs. My work has yielded a straightforward, cost-effective, and high-throughput method of evaluating viability and oxygen consumption in aerobic bacteria. I have used this method to test a broad range of toxic substances, including, metal ions, antibiotics, and untransformed and transformed ENMs. I observed species specific toxic responses to Ag+, PVP-AgENMs, and sAgENMs in PGPR. These results not only show the clear utility of the methodology, but also that it will be crucial to continue examining the responses of specific bacterial strains even as nanotoxicology, as a field, must move toward more complex and environmentally relevant systems.

A mesocosm experiment was carried out in a Norwegian fjord near Bergen in May 2006, with the main objective being the study of the effects of increasing concentrations of atmospheric CO2 (and associated effects such as increased acidification) on blooms of natural marine coastal plankton. Three mesocosms were bubbled with CO2(g) to achieve a high (~700ppm) CO2 concentration (pH ~7.8) to simulate predicted future conditions as a result of rising atmospheric CO2 concentrations. Another three mesocosms were treated as controls and bubbled with ambient air to represent a near pre-industrial scenario (atmospheric CO2 concentration ~300ppm, surface seawater pH ~8.15). Blooms in the mesocosms were stimulated by the addition of nutrients at a near-Redfield ratio ([N:P] ≈ [16:1]), and scientific measurements and analyses were carried out over the course of the blooms for approximately one month. Of particular interest in this study were the autotrophic plankton. The diversity and activities of these microorganisms under the two treatments was therefore investigated. By designing and using new degenerate primers specifically targeting ‘Green-type’ (Form IA and IB), ‘Red-type’ (Form IC and ID) and Form II RuBisCO, analysis of primary producers was carried out using PCR and either gDNA or cDNA (mRNA) templates from key time points spanning the complete duration of the blooms throughout the mesocosm experiment. Over 1250 novel RuBisCO large subunit sequences have been fully annotated and deposited in the NCBI GenBank® database. These sequences revealed distinct changes in the diversity of primary producers both over the courses of the blooms and between treatments. Particularly striking was the effect of acidification on the community structure of the eukaryotic picoplankton, Prasinophytes. A clade of prasinophytes closely related to Micromonas pusilla showed a distinct preference for the high CO2 conditions; a laboratory-based experiment confirmed the high tolerance of Micromonas pusilla to lower pH. Conversely, a clade related to Bathycoccus prasinos was almost entirely excluded from the high CO2 treatments. Clades of form II RuBisCO-containing dinoflagellates were also abundant throughout the experiment in both treatments. The high similarity of some of these clades to the toxin-producing species Heterocapsa triquetra and Gonyaulax polyedra, and apparent high tolerance of some clades to high CO2 conditions, is perhaps cause for concern in a high CO2 world and demands further research. In parallel with the RubisCO work, new primers were designed that target the gene encoding the Fe protein of nitrogenase (NifH). 82 Bergen genomic nifH sequences have been annotated and submitted to GenBank®. These sequences include those from organisms related to Alpha, Beta, and Gammaproteobacteria, and Cluster II and Cluster III sequences that align most closely with anaerobic Bacteria, Gram positive, and/or sulphur-reducing Bacteria. The biggest surprise, however, was the apparent abundance and significance of a Rhodobacter sphaeroides-like microorganism throughout the duration of the experiment in both treatments. Whilst this clade was unsurprisingly absent in the RuBisCO cDNA libraries, all but two of 128 nifH cDNA clones analysed were identical to the gene from Rhodobacter sphaeroides. This shows that this clade was potentially fixing N2 throughout the entire experiment, even in the presence of combined N added to both sets of mesocosms at the start of the experiment. A group of Rhodobacter sphaeroides-like microorganisms present at Bergen may therefore have been an unexpected source of new N during the experiment and contributed to the maintenance of the mesocosm communities as nutrients became depleted. One organism dominated the autotrophic communities after the blooms in both treatments. Synechococcus spp. Form IA rbcL clones most closely related to the coastal strain Synechococcus sp. strain CC9902 were recovered throughout the experiment but were particularly numerous toward the end of the experiment and dominated the “Green-type” libraries at this time. Initially, rbcL clones from these cyanobacteria were mostly derived from the ambient CO2 mesocosms but were equally distributed between treatments by the end of the experiment. This suggests that cyanobacteria related to strain CC9902 may be less tolerant of elevated CO2 (which was greatest at the beginning rather than the end of the experiment). However, despite the mesocosms being Pi-limited at the end of the experiment, several Synechococcus species (including those related to strain CC9902 and another coastal strain, CC9311) thrived. Following on from this observation, Pi uptake and assimilation mechanisms in a Synechococcus species were investigated in the laboratory. This led to the sequencing and characterisation of a pstS gene from the marine cyanobacterium Synechococcus sp. WH 8103. Unlike conventional pstS, it was discovered that the pstS II gene in this organism is constitutively expressed and unresponsive to or only weakly regulated by Pi supply. The use of PstS/pstS as a marker for P-limitation in natural samples, therefore, should be interpreted with caution.

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No. of bitstreams: 2 Dissertação - Erika Goulart Rodrigues - 2014.pdf: 2108349 bytes, checksum: 05c7df95766f5d5af12041646500a0a0 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-03-31 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / BACKGROUND: Dressing rooms attend colonized and infected chronic wounds carriers users and the environment may be contaminated by pathogens present in these lesions during the care. The literature shows the surfaces role corroborating the infection epidemiological chain and have identified low standards of environmental hygiene. This theme has been explored with emphasis on the medium and high complexity. However, it is noted that investments directed to the primary health care are rare. OBJECTIVE: To analyze the effect of environmental surfaces and non-critical health products contamination for Staphylococcus spp. for the safety of dressing rooms workers and users in primary health care. METHODOLOGY: Cross-sectional study of microbiological nature, conducted from July to November 2013 in dressing rooms references in the treatment of chronic wounds of primary health care in Goiás, Brazil. Approved by the Federal University of Goiás Research Ethics Committee (protocol 178/2012). It is began collecting direct observation of ambience processing. Then, have been done the interview, guided by semistructured instrument. The specimens collection from environmental surfaces and noncritical health products was performed using a sterile swab, rubbed on the research site and sent to the laboratory for processing. The microbiological procedures were performed according to standard methodologies. Was used SPSS 19.0 (Statistical Package for Social Sciences for Windows®) and the data were analyzed using descriptive statistical procedures. RESULTS: 38 articles were analyzed, including 32 environmental surfaces and six noncritical health products. It was found at the microbiological testing seven (18.4%) environmental surfaces and products were contaminated by Staphylococcus spp. Eight bacteria were isolated, which of seven (87.5%) were identified as coagulase negative Staphylococcus (CoNS) and one (12.5%), as S. aureus. Staphylococcus spp. were resistant to penicillin G (87.5%) and CoNS, presented a constitutive resistance to macrolidelincosamide-streptogramin B group (MLSBc) (28.6%) and resistance to oxacillin (42.9%). All micro-organisms were resistant to at least one antimicrobial agent. Of the three units investigated, the process of cleaning and disinfection was done by 91.7% of the 12 workers in the study. Nonetheless, was at unconformity with current guidelines. CONCLUSION: The results demonstrate the presence of multidrugresistant pathogens in the ambience of the dressing rooms, which denotes failure in articles processing. These failures may be due to lack of supervision and/or the worker unpreparedness to perform the procedure. Also exposes workers and service users at risk for colonization and eventual infection by these bacterias. This situation benefits cross-contamination and spread of multiresistant microorganisms in primary health care. / INTRODUCCIÓN: Salas de curativo asisten a usuarios portadores de heridas crónicas colonizadas e infectadas y el ambiente puede contaminarse por agentes patógenos presentes en estas lesiones durante la asistencia. La literatura envidencia el papel de las superficies que corroboran la cadena epidemiológica de la infección y se han identificado bajos niveles de higiene ambiental. Este tema ha sido explorado con énfasis en media y alta complejidad. Sin embargo, se percibe que las embestidas dirigidas hacia la atención primaria de salud son escasas. OBJETIVO: Analizar el efecto de contaminación de superficies ambientales y productos de salud no críticos por Staphylococcus spp. para seguridad de trabajadores y usuarios de salas de curativo de la atención primaria de salud. METODOLOGÍA: Estudio transversal de naturaleza microbiológica, realizado entre julio y noviembre de 2013, en salas de curativo referencias en el tratamiento de heridas crónicas de la atención primaria de salud en Goiás, Brasil. Aprobado por el Comité de Ética en Investigación de la Universidad Federal de Goiás (protocolo 178/2012). Comenzó la obtención de datos por observación directa del procesamiento de ambiente. A continuación, se ha hecho la entrevista, guiada por instrumento semi-estructurado. La recolección de espécimen de superficies ambientales y productos para salud no críticos se realizó con una torunda estéril, frotado en el sitio de investigación y enviado al laboratorio para su procesamiento. Los procedimientos microbiológicos se realizaron de acuerdo con metodologías estandarizadas. Se utilizó el programa SPSS 19.0 (Statistical Package for Social Sciences para Windows®) y los datos se analizaron mediante procedimientos estadísticos descriptivos. RESULTADOS: Se analizaron 38 artefactos, incluyendo 32 superficies ambientales y seis productos para salud no críticos. Se encontró, en las pruebas microbiológicas, siete (18,4%) de las superficies ambientales y de los productos estaban contaminados por Staphylococcus spp. Se aislaron ocho bacterias, de cuales siete (87,5%) fueron identificadas como Staphylococcus coagulasa negativo (CoNS) y uno (12,5%) como S. aureus. Staphylococcus spp. eran resistentes a penicilina G (87,5%) y los CoNS, presentarón resistencia constitutiva a el grupo macrólidos, lincosamidas, estreptogramina B (MLSBc) (28,6%) y resistencia a oxacilina (42,9%). De las tres unidades investigadas, el proceso de limpieza y desinfección se realizaba por 91,7% de los 12 trabajadores en el estudio. Sin embargo, estaba en desacuerdo con las directrices actuales. CONCLUSIÓN: Los resultados demuestran la presencia de patógenos resistentes a múltiples fármacos en el ambiente de salas de curativo, que denota el fracaso en el procesamiento de los artefactos. Estas fallas pueden deberse a falta de supervisión y falta de preparación del trabajador para realizar el procedimiento. Además expone a los trabajadores y usuarios del servicio en situación de riesgo para la colonización y la infección posterior por estas bacterias. Esta situación promueve la contaminación cruzada y propagación de microorganismos multirresistentes en la atención primaria de salud. / INTRODUÇÃO: Salas de curativo atendem usuários portadores de feridas crônicas colonizadas e infectadas e a ambiência pode ser contaminada por patógenos presentes nessas lesões durante a assistência. A literatura evidencia o papel das superfícies corroborando a cadeia epidemiológica da infecção e tem identificado baixos padrões de higiene ambiental. Essa temática tem sido explorada com maior ênfase nos serviços de média e alta complexidade. Entretanto, percebe-se que os investimentos direcionados para a atenção primária à saúde são escassos. OBJETIVO: Analisar as repercussões da contaminação de superfícies ambientais e produtos para saúde não críticos por Staphylococcus spp. para a segurança de trabalhadores e usuários de salas de curativo da atenção primária à saúde. MÉTODOLOGIA: Estudo do tipo transversal de natureza microbiológica, realizado de julho a novembro de 2013 em salas de curativo referências em tratamento de feridas crônicas da atenção primária à saúde em Goiás, Brasil. Aprovado em Comitê de Ética em Pesquisa da Universidade Federal de Goiás (protocolo 178/2012). Iniciou-se a coleta com observação direta do processamento da ambiência. Em seguida, realizou-se a entrevista, norteada por formulário semiestruturado. A coleta dos espécimes de superfícies ambientais e de produtos para saúde não críticos foi realizada por meio swab estéril, friccionado sobre o sítio de investigação e enviado ao laboratório para processamento. Os procedimentos microbiológicos foram realizados conforme metodologias padronizadas. Utilizou-se o SPSS 19.0 (Statistical Package for the Social Sciences for Windows®) e os dados foram analisados mediante procedimentos de estatística descritiva. RESULTADOS: Foram analisados 38 artefatos, sendo 32 superfícies ambientais e seis produtos para saúde não críticos. Verificou-se, à análise microbiológica, que sete (18,4%) das superfícies ambientais e dos produtos estavam contaminadas por Staphylococcus spp. Foram isoladas oito bactérias, das quais sete (87,5%) foram identificadas como Staphylococcus coagulase negativos (CoNS) e uma (12,5%), como S. aureus. Os Staphylococcus spp. apresentaram resistência à penicilina G (87,5%) e os CoNS, resistência constitutiva ao grupo macrolídeo, lincosamida, estreptogramina B (MLSBc) (28,6%) e à oxacilina (42,9%). Todos os micro-organismos apresentaram resistência a, pelo menos, um antimicrobiano. Das três unidades pesquisadas, o processo de limpeza e desinfecção era realizado por 91,7% dos 12 trabalhadores do estudo. No entanto, estava em desacordo com as diretrizes vigentes. CONCLUSÃO: Os resultados demonstram presença de patógenos multirresistentes na ambiência das salas de curativo, o que denota falhas no processamento dos artefatos. Essas falhas podem ser decorrentes da falta de supervisão e/ou despreparo do trabalhador para executar o procedimento. Igualmente, expõe trabalhadores e usuários do serviço aos riscos de colonização e eventual infecção por essas bactérias. Essa situação beneficia a contaminação cruzada e disseminação de micro-organismos multirresistentes na atenção primária à saúde.

The teratogenic potential of commercial formulations of atrazine (40.8%) and 2,4-D was evaluated using FETAX (frog embryo teratogenic assay--Xenopus). Because these herbicides have been detected in ground and surface water, this study was designed to determine the adverse effects in buffer and natural water for both herbicides. All treatments showed a significant concentration-response effect on exposed embryos, except for the 2,4-D natural water sample. Atrazine (solubility of the commercial formula used 70 mg/L at 20 degrees C), compared to 2,4-D (solubility = 311 mg/L at pH = 1 and 25 degrees C), had a significantly greater teratogenic effect in both the buffer (atrazine EC50 = 33 mg/L, LC50 = 100 mg/L, TI = 3.03; 2,4-D EC50 = 245 mg/L, LC50 = 254 mg/L, TI = 1.04) and natural water samples (atrazine EC50 < 8 mg/L, LC50 = 126 mg/L; 2,4-D EC50 and LC50 > 270 mg/L). The 2,4-D EC50 and LC50 values for the buffer were similar at 245 mg/L and 254 mg/L. These similar values and the teratogenic index (TI) of 1.04 suggested that 2,4-D was more embryotoxic than teratogenic to frog embryos at high concentrations. Atrazine in natural water demonstrated a significantly greater EC50 (100% abnormality at 8 mg/L, the lowest test concentration) to frog embryos than the buffer experiment (EC50 = 33 mg/L). The extrapolated lowest observable adverse effect concentration (LOAEC) for the natural water experiment was 1.1 mg/L. These results suggest that atrazine toxicity is enhanced by the synergistic or additive effects of some component of the water or atrazine was already present in the sample. In contrast to atrazine, 2,4-D was less toxic in natural water than buffer. These results suggest that both atrazine and 2,4-D pose little threat, since their embryotoxicity and teratogenicity to frog embryos occur at high concentrations approaching their maximum solubility levels in water.

Cyanobacteria are a group of photo-oxygenic bacteria found in nearly every ecosystem, but much cyanobacterial diversity, in various habitats, has yet to be explored. Cyanobacteria are often conspicuous components of photosynthetic flora, providing significant carbon and nitrogen inputs to surrounding systems. As possible primary colonizers of stone substrates not native to this region, cyanobacteria isolated from headstones may provide biogeographically informative data. An exploratory study of lichen-dominated microbial consortia, growing on headstones, was conducted to isolate and identify novel microaerophytic cyanobacteria, and resulted in the establishment of four novel cyanobacterial taxa. Phylogenetic analyses of photobionts in one tripartite lichen revealed two novel taxa: Brasilonema lichenoidesand Chroococcidiopsis lichenoides. Using a total evidence approach, analyzing ecology, morphology, ITS structure, and molecular data two additional taxa were described: Brasilonema geniculosusand Calothrix dumas. Analysis of secondary structures of the Internal Transcribed Spacer (ITS) regions of the 16S-23S operon in cyanobacteria are commonly used in cyanobacterial taxonomy studies and were applied to the identification of the new taxa in this study. However, the relationship between ITS structures, hairpin loops (helices) in a region of non-coding DNA, has not been thoroughly evaluated. The 16S-23S operon is one of many in prokaryotes with multiple copies and there is evidence that operons may vary due to differential selective pressures or drift. A study was undertaken analyzing ITS operons from 224 previously published cyanobacterial taxa for domain inclusion and exclusion, intragenomic heterogeneity of ITS operons, and the possible relevance of variable selective pressures affecting individual domains. Analysis revealed highly variable ITS domain inclusion even in complete sequences, as well as high variation between domains containing two or no tRNA sequences. Recommendations were made to standardize ITS analysis in the future to account for this possible variation. Further study is required to statistically demonstrate to what extent ITS secondary structures correlate with taxonomy.

Breast cancer mortality is mainly due to distant recurrence of the disease arising from dormant tumor cells established by cancer therapies. Patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy. Although immunotherapy of breast cancer by means of anti-HER2 antibodies prolongs survival of breast cancer patients, disease recurrence remains a major challenge. On the other hand administration of human vaccines against infectious disease in a preventive setting or during latency/dormancy has been successful in offering a cure. Here, we sought to use adoptive immunotherapy (AIT) at the time of tumor dormancy in order to prevent progression of breast cancer. We used a low dose immunogenic chemotherapy by means of 5-FU, Adriamycin, and Cyclophosphamide (FAC) in order to stabilize tumor progression prior to AIT using autologous tumor-reactive lymphocytes. Low dose FAC established local tumor dormancy, inhibited distant tumor dormancy occurring long before distant metastasis, and induced predominate a Ki67- quiescent type of tumor dormancy, which is less susceptible to tumor immunoediting. Dormant tumor cells expressed the cell survival pathways, including the endothelin receptor/ligand (ETRA, ETRB and ET-1) and PD-L1, thereby protecting them from elimination by AIT. In addition, tumor-reactive CD8+ T cells also produced ET-1 as a survival ligand for ETRA positive tumor cells. A combination of AIT with the blockade of tumor cell survival pathways resulted in a significant improvement of AIT against tumor dormancy. We also showed that the inhibition Bcl-xL downstream of the tumor cell survival pathways is specifically effective against dormant tumor cells, suggesting a combination of AIT with small molecules inhibitors of Bcl-xL. Altogether, we showed that distant tumor dormancy is established long before distant recurrence of breast cancer, and that the expression of several tumor cell survival pathways in dormant cells protects them from immunotherapy. Our results suggest that immunotherapeutic targeting of tumor dormancy combined with the blockade of a common downstream cell survival pathway could prevent tumor progression and recurrence of the disease.

Black band disease (BBD) of corals is a temperature dependent, highly virulent, polymicrobial disease affecting reef-building corals globally. The microbial consortium of BBD is primarily comprised of functional physiological groups that include photosynthetic cyanobacteria, sulfate reducers, sulfide oxidizers and a vast repertoire of heterotrophic bacteria. Quorum sensing (QS), the cell-density dependent communication phenomenon in bacteria, is known to induce expression of genes for a variety of virulence factors in diseases worldwide. Microbes capable of QS release signals such as acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), which coordinate microbial interaction. The focus of the present study was to investigate the presence and potential role of QS in BBD pathogenicity, utilizing culture dependent and independent methodologies. Isolates across coral health states including BBD, were screened for production of QS signals, and AHL and AI-2 production capabilities were analyzed via LC-MS/MS. The effect of temperature on AHLs was also examined. Additionally, antimicrobial production capabilities of isolates were tested. BBD metagenomes were utilized to screen for sequences related to QS, antimicrobial synthesis, and antimicrobial resistance genes. BBD isolates represented a significantly higher proportion of isolates capable of producing QS signals in comparison to healthy coral isolates. Several AHLs produced by coral derived bacterial cultures were identified, and three AHLs, specifically 3OHC4, 3OHC5 and 3OHC6, showed a significant increase in production at an elevated temperature of 30 °C, which correlates with increased BBD incidence on reefs with increasing water temperature. Most of the BBD cultured isolates were identified as vibrios. Several sequences related to QS, antimicrobial synthesis and resistance genes were detected in the BBD metagenomes. Based on the findings of this study, a model for potential microbial interactions amongst BBD heterotrophs, centered around QS, is proposed. Taken together, the findings from this study provide a clearer understanding of the potential role of QS in BBD, and serve as the basis for further studies aimed at elucidating the pathogenesis of an intricate coral disease.

Coral reefs are one of the most diverse ecosystems on the planet due partially to the habitat structure provided by corals. Corals are long lived organisms that can live for hundreds of years and as a result growth of many species is very slow. As a result of this, recovery of corals from disease outbreaks is very slow and difficult and therefore the ecosystem is deteriorating rapidly. Due to this increase in disease and its detrimental effect on coral reefs, it has become imperative to study how corals respond to disease outbreaks. The response of the coral to pathogens is believed to be controlled by the innate immune system. However, the immune pathways and components of these pathways used by cnidarians to combat pathogens are still rudimentary. This work showed that C3 and heat shock protein 70 are components of the coral immune system that positively respond to disease occurrence. As disease out breaks become more frequent, the question has arisen as to whether cnidarians have homologs to of the adaptive immune system that allow them to respond more rapidly to subsequent encounters with the same bacterium. In the cnidarian model system Exaiptasia pallida, immune priming occurs up to one month after the initial sub lethal exposure to the pathogen. This transient form of priming could be the result of host energy allocation in place of establishing long term immune priming which could be too energetically costly. Cnidarians may only activate priming during summer months, when ocean temperatures and bacterial load are high. Specificity of immune priming in E. pallida requires further investigation with more bacterial pathogens. In this dissertation, one bacterial strain shows specificity while the other does not. Furthermore, the priming response involves many pathways which include pathogen recognition, inflammation, and activation of NF-κB. The discovery of immune priming in a sea anemone shows that this phenomenon evolved earlier in the tree of life than previously thought. Additionally, identification of priming in E. pallida is suggestive of its presence in corals which would allow for potential vaccinations of vulnerable corals.

Diverse and ecologically important microbial communities (microbiomes) are symbiotic within marine sponges. In this study, the microbiome of Amphimedon compressa from three sample locations (Broward and Dade Counties, Southeast Florida, USA and the Southern Caribbean, Bocas del Toro, Panama) is characterized using 16S rRNA Illumina sequencing. The predominant taxa are Proteobacteria and Cyanobacteria, as expected for Low Microbial Abundance sponges, accounting for over 53% of the total microbiome community. The numbers of Operational Taxonomic Units (OTUs) decrease from Broward County (2,900) to Dade County (2,300) and then Bocas del Toro (1,200). The correlates to a decreasing north-south gradient of sponge microbiome richness and diversity. Sponge microbiome richness and Alpha diversity are nearly identical from the two closest locations (37 km), both in Southeast Florida (Tukey HSD/ANOVA; p=0.999). However Panama sponge microbiome richness and Alpha diversity are distinctly lower, with the primary driver being distance, ~1,850 km from Southeast Florida. Abiotic factors driving this trend of decreased richness and diversity include increased temperature, and deceased salinity in relation to precipitation-based seasons. Sponge microbiome Beta diversity as determined by Bray-Curtis Dissimilarity and Non-Metric Multidimensional Scaling documents the clustering of Panama samples as distinct from the Broward and Dade County samples. In a seasonal comparison, Broward County sponge microbiome richness (p=0.026, r2=0.92) and Alpha diversity (p=0.007, r2=0.98) are significantly different, documenting robust effects of temperature. This comparison confirms lowest microbiome OTU diversity in the season with highest precipitation and highest temperatures of 29.8 °C. These results are consistent with prior studies that report decreasing microbiome OTU richness and diversity under conditions of environmental stress such as decreased salinity and increased temperatures.