Characterization of L-Serine Deaminase and its Potential Role in the Pathogenicity of Pseudomonas aeruginosa

All pathogens require high energetic influxes to counterattack the host immune system and without this energy bacterial infections are easily cleared. This study is an investigation into one highly bioenergetic pathway in Pseudomonas aeruginosa involving the amino acid L-serine and the enzyme L-serine deaminase (L-SD). P. aeruginosa is an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. Recent evidence has linked L-SD directly to the pathogenicity of several organisms including but not limited to Campylobacter jejuni, Mycobacterium bovis, Streptococcus pyogenes, and Yersinia pestis. We hypothesized that P. aeruginosa L-SD is likely to be critical for its virulence. Genome sequence analysis revealed the presence of two L-SD homo logs encoded by sdaA and sdaB. We analyzed the ability of P. aeruginosa to utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains of sdaA (PAOsdaA) and sdaB (PAOsdaB) with their isogenic parent P. aeruginosa P AO 1. We demonstrated that P. aeruginosa is unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine and this glycine-dependent induction of L-SD activity requires the inducer serine. The amino acid leucine was shown to inhibit L-SD activity from both SdaA and SdaB and the net contribution to L-serine deamination by SdaA and SdaB was ascertained at 34% and 66 %, respectively. These results suggest that P. aeruginosa LSD is quite different from the characterized E. coli L-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as a sole carbon source were also isolated and in addition, suicide vectors were constructed which allow for selective mutation of the sdaA and sdaB genes on any P. aeruginosa strain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

Studying Key Amino Acids in the Function of Ras Interference 1 (Rinl) SH2 and Vps9 Domains

Receptor mediated endocytosis effectively removes the "ears" with which a cell would "hear" a signal conveyed by extracellular signaling molecules, but does not necessarily block the signaling pathway in which the endocytosed receptor participates. In the process of signal attenuation, this newly formed vesicle is fused with a phagosome and the receptor molecules are degraded. Receptor mediated endocytosis as a way to attenuate epidermal growth factor (EGF) and insulin signaling will be the focus here. Ras Interference 1 (Rin 1) is a multifunctional protein involved in intracellular membrane trafficking and receptor mediated endocytosis through its Rab5 Guanine Exchange Factor and SH2 domains. The goal of this investigation is to determine the role of key amino acids involved in the interaction of Rinl with Epidermal Growth Factor Receptor and Rab5. To elucidate this role, a number of point mutations have been created and the effects of each mutation on Rin 1 function will be investigated. Key amino acids in the SH2 and Vps9 Domain were identified and effects of mutations on rate of endocytosis were observed.

Diel Patterns of Patch Reef Community Dynamics in a Caribbean Back-Reef System

Back-reef seascapes represent critical habitat for juvenile and adult fishes. Patch reef, seagrass, and mangrove habitats form a heterogeneous mosaic, often linked by species that use reefs as structure during the day and make foraging migrations into soft-bottom habitat at night. Artificial reefs are used to model natural patch reefs, however may not function equivalently as fish habitat. To study the relative value of natural and artificial patch reefs as fish habitat, these communities in the Sea of Abaco, Bahamas were compared using roving diver surveys and time-lapse photography. Diel turnover in fish abundance, recorded with time-lapse photography and illuminated by infrared light, was quantified across midday, dusk, and night periods to explore possible effects of reef type (artificial vs. natural) on these patterns. Diurnal communities on natural reefs exhibited greater fish abundance, species richness, and functional diversity compared to artificial reefs. Furthermore, both types of reef communities exhibited a significant shift across the diel period, characterized by a decline in total fish density at night, especially for grunts (Haemulidae). Cross-habitat foraging migrations by diurnal or nocturnal species, such as haemulids, are likely central drivers of this twilight turnover and can represent important energy and nutrient subsidies. Time-lapse surveys provided more consistent measures of reef fish assemblages for the smaller artificial reef habitats, yet underestimated abundance of certain taxa and species richness on larger patch habitats when compared to the roving diver surveys. Time-lapse photography complemented with infrared light represent a valuable non-invasive approach to studying behavior of focal species and their fine-scale temporal dynamics in shallow-reef communities.

Antimicrobial effect of Artemisia species on Pseudomonas aeruginosa

Antibiotic resistance has emerged as a severe problem in hospital-acquired infectious disease. The Gram-negative bacterium Pseudomonas aeruginosa is found to cause secondary infection in immune-compromised patients. Unfortunately, it is resistant to virtually all β-lactam antibiotics such as penicillin, cephalosporin and others. Researchers are seeking for new compounds to treat several antibiotic-resistant bacterial strains. Artemisia plant extracts are commonly used for their therapeutic properties by natives throughout dry regions of North and South America. Here, they are administered as an alternative medicine for stomach problems and other complex health issues. In this study, the antimicrobial effects of plant extracts from several Artemisia species as well as compounds dehydroleucodine and dehydroparishin-B (sesquiterpenes derived specifically from A. douglasiana) were used as treatments against the pathogenicity effects of P. aeruginosa. Results showed that both compounds effectively inhibit the secretion of LasB elastase, biofilm formation and type III secretion, but fail to control LasA protease. This is a significant observation because these virulent factors are crucial in establishing P.aeruginosa infection. The results from this study signify a plausible role for future alternative therapy in the biomedical field, which recommends DhL and DhP can be studied as key compounds against bacterial infections of Pseudomonas aeruginosa.

The Role of Ras Oncogene in Differentiation of 3T3-L1 Preadipocytes

Ras is a proto-oncogene that codes for a small GTPase and is responsible for linking several extracellular signals to intracellular mechanisms that involve cell growth, differentiation and cell-programmed death in normal and diseased cells. In all these processes, Ras has been extensively investigated. However, the role of Ras GTPases is still poorly understood during the differentiation of 3T3-L1 preadipocytes. In this study I investigated the role of the H-Ras defective mutant, Ras:G12V on the differentiation of 3T3-L1 preadipocytes. Preadipocytes were differentiated in vitro to adipocytes (fat cells) by adding an induction medium containing several factors including glucose and insulin. The formation of fat cells evidenced by the visualization of lipid drops as well as by quantifying the accumulation of Oil red O into lipid drops. To examine the role of Ras:G12V mutant, several selective mutations were introduced in order to determine the signaling transduction pathways (i.e., PI3(K)kinase and MAP(K)Kinase) responsible for the Ras-dependent adipogenesis. Cells expressing Ras:G12V mutant stimulated 3T3-L1 preadipocyte differentiation without he need for induction media, suggesting that Ras activation is an essential factor required for 3T3-L1 preadipocyte differentiation. Introduction of a second mutation on Ras:G12V (i.e., Ras:G12V;E37G), which blocks the activation of the MAPKinase pathway, strongly inhibited the 3T3-L1 preadipocyte differentiation. It is also important to note Ras:G12V:E37G double mutant does not inhibit the activation of the PI3kinase pathway. Other Ras double mutants (Ras:G12V;S35T, and V12G;C40Y) showed a modest inhibition of the 3T3-L1 preadipocyte differentiation. Taken together, these observations indicate that Ras plays a selective role in 3T3-L1 preadipocyte differentiation. Thus, understanding which specific pathway Ras employs during preadipocyte differentiation could clarify some of the uncertainties surrounding fat production.

Comparison of community structure of mangrove and riprap rock habitats along urban coastlines of south Florida

Globally, mangrove ecosystems have substantially declined, largely a result of human impacts. Mangroves provide a number of ecosystem services such as shoreline stabilization and nursery habitat for fish species. As declines continue, many of these ecosystem services are lost or altered. The need for shoreline stabilization has become increasingly apparent when chronic erosion wear away coastlines once mangroves are removed. Limestone boulders called riprap have been employed to offset continued erosion associated with mangrove clearing. In urban coastal areas adjacent to Biscayne Bay, Florida, as much as 80 percent of mangroves have been lost. More recently, riprap has been used in conjunction with mangroves to restore wetlands throughout the Bay. This riprap-mangrove habitat provides structure for marine organisms to colonize. However, fish assemblages and benthic composition could vary between this hybridized habitat and natural mangrove systems. Comparisons of fish and benthic community structure were made, to determine if abundance, species richness, and overall diversity differed between the two habitat types. Visual census and benthic quadrat surveys were conducted in vi mangrove and mangrove-riprap sites within two regions of Biscayne Bay. Total fish abundance was greater in mangroves, but the effect of habitat type on species richness varied between regions. The community structure of fishes and benthic composition differed significantly between mangroves and riprap habitats. Because species composition is so distinct, it is likely that the two communities do no function in the same manner. In areas with cleared shorelines, it may be important to consider the function of added anthropogenic structure for ecological communities.

Do long-tailed macaques (Macaca fascicularis) know what others can and cannot hear?

Theory of mind has been defined as the ability to attribute mental sates such as perceptions, knowledge, and belief to others. Studies examining theory of mind in primates have been the center of intense controversy. Much of the research on this subject has focused on designing methodologies to test a primate’s ability to discern the perceptions of others. Namely, many studies have examined an individual’s knowledge of what others can and cannot see. However, other sensory modalities have not undergone as much extensive research. This study aimed to replicate the methodology of a previous experiment with the addition of two novel experimental conditions. Individual long-tailed macaques (Macaca fascicularis) were allowed to approach one of two identical, lidded, clear boxes which had jingle bells attached to them. One of the boxes had the metal bits removed from inside of the jingle bells attached to it, thus creating one “silent” box and leaving the remaining one “noisy”. The experimenter either looked directly at the subject, down at the ground between their knees, or in the novel conditions, turned their back to the subject, or wore a welder’s mask while facing the subject after demonstrating each box’s auditory properties. It was predicted that subjects would choose to approach the silent container in the latter three conditions. The results indicated that subjects selected boxes at random in all conditions. Additionally, in order to explore the possibility of perspective-taking representing a derived trait in the genus Macaca, a phylogeny of the genus was created and annotated to display the presence of perspective-taking as a phenotypic trait in extant species. Three likely evolutionary scenarios leading to the current distribution of perspective-taking are postulated and analyzed for parsimony through the number of assumed gains and losses. The most parsimonious tree suggests that perspective taking could be a conserved trait among the order, giving credence to the argument that some other variable was responsible the negative results in this experiment. It is suggested that the results of the present study represent an artifact of the social environment of the subject population. Moreover, arguments are made for the development of more naturalistic studies for examining mental state attribution in primates.

Achievement motivation attributes of international students attending a *postsecondary institution

The purpose of this study was to identify and compare the causal attributes of achievement motivation for academic success possessed by international students to those causal attributes of achievement motivation by other legal immigrant student groups attending a postsecondary institution. In this study, F-1 international students have obtained permission from the federal Department of Immigration and Naturalization Services to attend Miami-Dade Community College in Miami, Florida. There were two additional groups of students selected to participate in this study: Permanent Resident students and Others comprised of asylees, refugees and paroles, which are specific categories of legal immigrant students.^ From the observations of the investigator, international students were more successful at the institution as compared to the other two student groups as measured by such variables as Grade Point Average, Number of Credits Earned and Honors Day awards. By identifying these attributes of achievement motivation, additional curricula, support services and activities can be developed to meet the needs of these highly motivated students.^ Incorporating the theories of McClelland and Weiner in the theoretical framework of the study, both quantitative and qualitative data were collected from 150 subjects using the Measurement of Achievement Motivation Questionnaire and the Guided Biography Interview as data collection instruments. Statistical analyses on the data collected from the three groups of foreign born students at the institution indicated differences in these identified attributes and their effects on academic success.^ Results indicated that F-1 international students at Miami-Dade Community College were highly motivated to achieve, possessed different causal attributes of achievement motivation and displayed their need for achievement in different activities and in different ways as compared to the Permanent Resident and Other student groups. Further investigation on the importance of residency status as an influencing factor of achievement motivation is suggested. ^

Factors positively influencing academically successful, highly motivated African -American high school students

The purpose of this study is two-fold: (1) to investigate the common factors positively influencing academically successful, highly motivated African-American high school students in their educational pursuits; and (2) to determine ways in which educators can facilitate an increased positive impact on minority students' achievement motivation and transition to higher education. ^ Three sources of data inform the qualitative study. Short autobiographical essays address the topic of antecedent factors influencing the students' selection of an academic program of study. Individual interviews using a semi-structured format elicit discussion of prior experiences and influences related to the research sub-questions. Focus group interviews elicit discussion of emergent patterns perceived by the researcher from the autobiographical essays and individual interviews. ^ Results indicate a generic model for success for high achieving African-American students. Students prioritize education as a means to success in life. They receive strong support for educational aspirations from at least one adult within the home. The students are focused on a professional career in the future. The students realize and desire the financial benefits of academic success. The students desire academic challenge. Students have feelings of control over their academic choices; however, they frequently seek the counsel of family members. Prior experience in honors, gifted, or magnet programs did not meet needs. ^ Recommendations for improved educational experiences include: Increase teacher and counselor responsiveness to students. Provide classroom activities which afford more interaction among the students and teacher and relate curriculum to topics of interest. Increase involvement of parents and Black community members in establishing goals and visions of opportunity. Recognize and revere diversity among students within the classroom environment. Set a climate that assumes more individual student responsibility and sets higher academic expectations. ^ The factors influencing academically successful, highly motivated African-American high school students echo the patterns of experiences of many groups. Pride in achievement, emotional support for efforts, expectations of success, independence in thought, and consternation with mediocrity and malaise drive the students to excel academically just like all others who succeed. ^

Sugar Feeding Affects the Male Contribution in Reproduction in Aedes Aegypti

Juvenile hormone (JH) is crucial for the stimulation and progression of oogenesis from emergence to the previtellogenic resting stage in female Aedes aegypti mosquitoes. Juvenile hormone has been suggested to be among the many substances transferred form the male accessory glands to the female during copulation but no evidence for this has previously been provided. Quantification of JH III in the accessory glands of males and in the bursae copulatrix and spermathecae of mated females was performed using HPLC-FD. These amounts were measured in relation to the quality of adult sugar feeding in the male. The effect of this variable transfer was measured on two fecundity markers that occur during the previtellogenic stage of oogenesis, specifically follicular resorption and ovarian lipids. Male mosquitoes provided with 20% sucrose contained ~ 60% greater amount of JH in the accessory glands and transferred 4 fmol more JH during copulation than males provided with 3% sucrose. These differences resulted in a nearly 40% reduction in follicular resorption and an approximate 3-fold increase in lipid content in the ovaries of mated females during the previtellogenic stage. These results suggest that the contribution of JH from the male is dependent on the quality of nutrition obtained during adult sugar feeding. Female fecundity is likely responsive to these variable previtellogenic effects, possibly resulting in a difference in the number of eggs laid. Improvements in female reproductive output may have wider implications in the transmission of diseases attributed to this important arbovirus vector.

Multiplex PCR for Assessment of Tetracycline Resistance (tet) Genes in Antibiotic-Resistant Soil Bacteria and the Ecological Implications

Antibiotics are becoming increasingly prevalent in bacterial communities due to clinical and agricultural misuse and overuse in their environment. As exposure increases, so does the incidence of microbial resistance. Such is the case with bacterial resistance to tetracyclines, a phenotype often acquired through the horizontal gene transfer of tet genes between bacteria. The objective of this project was to analyze the bacterial diversity of tet resistance genes in soil from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine each bacterium’s degree of resistance. The 16S rRNA gene from antibiotic-resistant isolates was amplified by PCR and sequenced to identify the isolates. All isolates’ tet genes were amplified by multiplex PCR, sequenced, and compared. Among eight isolates, three distinct species were positively identified based on their 16S rRNA sequences and four distinct tet genes were identified, though all tested susceptible to tetracycline via the Kirby-Bauer test. This project clarifies some aspects of the ecology of antibiotic resistance genes, their natural ecological function and the potential for the expansion of intrinsic multi-antibiotic resistance into new ecosystems and/or hosts.