One-dimensional and two-dimensional polyacrylamide gel electrophoresis: a tool for protein characterisation in aquatic samples

Dissolved organic nitrogen (DON) represents the least understood part of the nitrogen cycle. Due to recent methodological developments, proteins now represent a potentially characterisable fraction of DON at the macromolecular level. We have applied polyacrylamide gel electrophoresis to characterise proteins in samples from a range of aquatic environments in the Everglades National Park, Florida, USA. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that each sample has a complex and characteristic protein distribution. Some proteins appeared to be common to more than one site, and these might derive from dominant higher plant vegetation. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) provided better resolution; however, strong background hindered interpretation. Our results suggest that the two techniques can be used in parallel as a tool for protein characterisation: SDS-PAGE to provide a sample-specific fingerprint and 2D-PAGE to focus on the characterisation of individual protein molecules.

The applied computer science of economic analyses: Internet prioritization and Internet pricing, and software engineering and software pricing for the mass market

This research examines evolving issues in applied computer science and applies economic and business analyses as well. There are two main areas. The first is internetwork communications as embodied by the Internet. The goal of the research is to devise an efficient pricing, prioritization, and incentivization plan that could be realistically implemented on the existing infrastructure. Criteria include practical and economic efficiency, and proper incentives for both users and providers. Background information on the evolution and functional operation of the Internet is given, and relevant literature is surveyed and analyzed. Economic analysis is performed on the incentive implications of the current pricing structure and organization. The problems are identified, and minimally disruptive solutions are proposed for all levels of implementation to the lowest level protocol. Practical issues are considered and performance analyses are done. The second area of research is mass market software engineering, and how this differs from classical software engineering. Software life-cycle revenues are analyzed and software pricing and timing implications are derived. A profit maximizing methodology is developed to select or defer the development of software features for inclusion in a given release. An iterative model of the stages of the software development process is developed, taking into account new communications capabilities as well as profitability. ^

An aquatic risk assessment of ethion and bromacil inputs to the C-25 Canal and Indian River Lagoon

Pesticide monitoring in St. Lucie County by various local, state and federal agencies has indicated consistent residues of several pesticides, including ethion and bromacil. Although pesticides have long been known to pose a threat to non-target species and much background monitoring has been done, no pesticide aquatic risk assessment has been done in this geographical area. Several recognized United States Environmental Protection Agency (USEPA) methods of quantifying risk are employed here to include hazard quotients (HQ) and probabilistic modeling with sensitivity analysis. These methods are employed to characterize potential impacts to aquatic biota of the C-25 Canal and the Indian River Lagoon (in St. Lucie County, Florida) based on current agricultural pesticide use and drainage patterns. The model used in the analysis incorporates available physical-chemical property data, local hydrology, ecosystem information, and pesticide use practices. HQ's, probabilistic distributions, and field sample analyses resulted in high levels of concern (LOCs), which usually indicates a need for regulatory action, including restrictions on use, or cancellation. ^

The conflict resolution behaviors of occupational therapy students as a function of the personality types, demographic characteristics and contextual factors

Interpersonal conflicts have the potential for detrimental consequences if not managed successfully. Understanding the factors that contribute to conflict resolution has implications for interpersonal relationships and the workplace. Researchers have suggested that personality plays an important and predictable role in conflict resolution behaviors (Chanin & Schneer, 1984; Kilmann & Thomas, 1975; Mills, Robey & Smith, 1985). However, other investigators have contended that contextual factors are important contributors in triggering the behavioral responses (Shoda & Mischel, 2000; Mischel & Shoda, 1995). The purpose of this study was to investigate the relationships among personality types, demographic characteristics and contextual factors on the conflict resolution behaviors reported by graduate occupational therapy students (n = 125). ^ The study design was correlational. The Myers Briggs Type Indicator (MBTI) and the Thomas-Kilmann (MODE) Instrument were used to establish the personality types and the context independent conflict resolution behaviors respectively. The effects of contextual factors of task vs. relationship and power were measured with the Conflict Case Scenarios Questionnaire (CCSQ). One-way ANOVA and linear regression procedures were used to test the relationships between personality types and demographic characteristics with the context independent conflict behaviors. Chi-Square procedures of the personality types by contextual conditions ascertained the effects of contexts in modifying the resolution modes. Descriptive statistics established a profile of the sample. ^ The results of the hypotheses tests revealed significant relationships between the personality types of feeling-thinking and sensing-intuition with the conflict resolution behaviors. The contextual attributes of task vs. relationship orientation and of peer vs. supervisor relationships were shown to modify the conflict behaviors. Furthermore, demographic characteristics of age, gender, GPA and educational background were shown to have an effect on the conflict resolution behaviors. The knowledge gained has implications for students' training, specifically understanding their styles and use of effective conflict resolution strategies. It also contributes to the knowledge on management approaches and interpersonal competencies and how this might facilitate the students' transition to the clinical role. ^

Financial aid and the persistence of associate of arts graduates transferring to a senior university

This study examined the effects of financial aid on the persistence of associate of arts graduates transferring to a senior university in one of four consecutive fall semesters (1998-2001). Situated in an international metropolitan area in the southeastern United States, the institution where the study was conducted is a large public research university identified as a Hispanic Serving Institution. Archival databases served as the source of information on the academic and social background of the 4,669 participants in the study. Data from institutional financial aid records were pooled with the data in the student administrative system.^ For purposes of this study, persistence was defined as ongoing progress until completing the baccalaureate degree. Student social background variables used in the study were gender, ethnicity, age, and income, with GPA and part-time or full-time enrollment status being the academic variables. Amount and type of aid, including grants, loans, scholarships, and work study were incorporated in the models to determine the effect of financial aid on the persistence of these transfer students. Because the dependent variable persistence had three possible outcomes (graduated, still enrolled, dropped out) multinomial logistic regression was the appropriate technique for analyzing the data; four multinomial models were employed in the analysis.^ Findings suggest that grants awarded based on the financial need of students and loans were effective in encouraging the persistence of students, but scholarships and work study were not effective.^

A micro-opto-electro-mechanical system (MOEMS) for microstructure manipulation

Microstructure manipulation is a fundamental process to the study of biology and medicine, as well as to advance micro- and nano-system applications. Manipulation of microstructures has been achieved through various microgripper devices developed recently, which lead to advances in micromachine assembly, and single cell manipulation, among others. Only two kinds of integrated feedback have been demonstrated so far, force sensing and optical binary feedback. As a result, the physical, mechanical, optical, and chemical information about the microstructure under study must be extracted from macroscopic instrumentation, such as confocal fluorescence microscopy and Raman spectroscopy. In this research work, novel Micro-Opto-Electro-Mechanical-System (MOEMS) microgrippers are presented. These devices utilize flexible optical waveguides as gripping arms, which provide the physical means for grasping a microobject, while simultaneously enabling light to be delivered and collected. This unique capability allows extensive optical characterization of the structure being held such as transmission, reflection, or fluorescence. The microgrippers require external actuation which was accomplished by two methods: initially with a micrometer screw, and later with a piezoelectric actuator. Thanks to a novel actuation mechanism, the "fishbone", the gripping facets remain parallel within 1 degree. The design, simulation, fabrication, and characterization are systematically presented. The devices mechanical operation was verified by means of 3D finite element analysis simulations. Also, the optical performance and losses were simulated by the 3D-to-2D effective index (finite difference time domain FDTD) method as well as 3D Beam Propagation Method (3D-BPM). The microgrippers were designed to manipulate structures from submicron dimensions up to approximately 100 μm. The devices were implemented in SU-8 due to its suitable optical and mechanical properties. This work demonstrates two practical applications: the manipulation of single SKOV-3 human ovarian carcinoma cells, and the detection and identification of microparts tagged with a fluorescent "barcode" implemented with quantum dots. The novel devices presented open up new possibilities in the field of micromanipulation at the microscale, scalable to the nano-domain.

Explaining the effects of class size on faculty and students

This study explored the effects of class size on faculty and students. Specifically, it examined the relationship of class size and students' participation in class, faculty interactive styles, and academic environment and how these behaviors affected student achievement (percentage of students passing). The sample was composed of 629 students in 30 sections of Algebra I at a large, urban community college. A survey was administered to the students to solicit their perceptions on their participation in class, their faculty interaction style, and the academic environment in their classes. Selected classes were observed to triangulate the findings. The relationship of class size to student participation, faculty interactive styles, and academic environment was determined by using hierarchical linear modeling (HLM). A significant difference was found on the participation of students related to class size. Students in smaller classes participated more and were more engaged than students in larger classes. Regression analysis using the same variables in small and large classes showed that faculty interactive styles significantly predicted student achievement. Stepwise regression analyses of student and faculty background variables showed that (a) students' estimate of GPA was significantly related to their achievement (r = .63); (b) older students reported more participation than did younger ones, (c) students in classes taught by female, Hispanic faculty earned higher passing grades, and (d) students' participation was greater with adjunct professors. Class observations corroborated these findings. The analysis and observational data provided sufficient evidence to warrant the conclusion that small classes were not always most effective in promoting achievement. It was found that small classes may be an artifact of ineffectual teaching, actual or by reputation. While students in small classes participate and are more engaged than students in larger classes, the class-size effect is essentially due to what happens in instruction to promote learning. The interaction of the faculty with students significantly predicted students' achievement regardless of class size. Since college students select their own classes, students do not register for classes taught by faculty with poor teaching reputation, thereby leading to small classes. Further studies are suggested to determine reasons why classes differ in size.

Theoretical investigations of intercellular communication in the microcirculation: Conducted responses, myoendothelial projections and endothelium derived hyperpolarizing factor

The contractile state of microcirculatory vessels is a major determinant of the blood pressure of the whole systemic circulation. Continuous bi-directional communication exists between the endothelial cells (ECs) and smooth muscle cells (SMCs) that regulates calcium (Ca2+) dynamics in these cells. This study presents theoretical approaches to understand some of the important and currently unresolved microcirculatory phenomena. ^ Agonist induced events at local sites have been shown to spread long distances in the microcirculation. We have developed a multicellular computational model by integrating detailed single EC and SMC models with gap junction and nitric oxide (NO) coupling to understand the mechanisms behind this effect. Simulations suggest that spreading vasodilation mainly occurs through Ca 2+ independent passive conduction of hyperpolarization in RMAs. Model predicts a superior role for intercellular diffusion of inositol (1,4,5)-trisphosphate (IP3) than Ca2+ in modulating the spreading response. ^ Endothelial derived signals are initiated even during vasoconstriction of stimulated SMCs by the movement of Ca2+ and/or IP3 into the EC which provide hyperpolarizing feedback to SMCs to counter the ongoing constriction. Myoendothelial projections (MPs) present in the ECs have been recently proposed to play a role in myoendothelial feedback. We have developed two models using compartmental and 2D finite element methods to examine the role of these MPs by adding a sub compartment in the EC to simulate MP with localization of intermediate conductance calcium activated potassium channels (IKCa) and IP3 receptors (IP 3R). Both models predicted IP3 mediated high Ca2+ gradients in the MP after SMC stimulation with limited global spread. This Ca 2+ transient generated a hyperpolarizing feedback of ∼ 2–3mV. ^ Endothelium derived hyperpolarizing factor (EDHF) is the dominant form of endothelial control of SMC constriction in the microcirculation. A number of factors have been proposed for the role of EDHF but no single pathway is agreed upon. We have examined the potential of myoendothelial gap junctions (MEGJs) and potassium (K+) accumulation as EDHF using two models (compartmental and 2D finite element). An extra compartment is added in SMC to simulate micro domains (MD) which have NaKα2 isoform sodium potassium pumps. Simulations predict that MEGJ coupling is much stronger in producing EDHF than alone K+ accumulation. On the contrary, K+ accumulation can alter other important parameters (EC V m, IKCa current) and inhibit its own release as well as EDHF conduction via MEGJs. The models developed in this study are essential building blocks for future models and provide important insights to the current understanding of myoendothelial feedback and EDHF.^

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Responsiveness of voltage-gated calcium channels in SH-SY5Y human neuroblastoma cells on quasi-three-dimensional micropatterns formed with poly (l-lactic acid)

Introduction: In this study, quasi-three-dimensional (3D) microwell patterns were fabricated with poly (l-lactic acid) for the development of cell-based assays, targeting voltage-gated calcium channels (VGCCs). Methods and materials: SH-SY5Y human neuroblastoma cells were interfaced with the microwell patterns and found to grow as two dimensional (2D), 3D, and near two dimensional (N2D), categorized on the basis of the cells’ location in the pattern. The capability of the microwell patterns to support 3D cell growth was evaluated in terms of the percentage of the cells in each growth category. Cell spreading was analyzed in terms of projection areas under light microscopy. SH-SY5Y cells’ VGCC responsiveness was evaluated with confocal microscopy and a calcium fluorescent indicator, Calcium GreenTM-1. The expression of L-type calcium channels was evaluated using immunofluorescence staining with DM-BODIPY. Results: It was found that cells within the microwells, either N2D or 3D, showed more rounded shapes and less projection areas than 2D cells on flat poly (l-lactic acid) substrates. Also, cells in microwells showed a significantly lower VGCC responsiveness than cells on flat substrates, in terms of both response magnitudes and percentages of responsive cells, upon depolarization with 50 mM K+. This lower VGCC responsiveness could not be explained by the difference in L-type calcium channel expression. For the two patterns addressed in this study, N2D cells consistently exhibited an intermediate value of either projection areas or VGCC responsiveness between those for 2D and 3D cells, suggesting a correlative relation between cell morphology and VGCC responsiveness. Conclusion: These results suggest that the pattern structure and therefore the cell growth characteristics were critical factors in determining cell VGCC responsiveness and thus provide an approach for engineering cell functionality in cell-based assay systems and tissue engineering scaffolds.

The effects of student -centered and traditional models of teaching on reading skills

A pre-test, post-test, quasi-experimental design was used to examine the effects of student-centered and traditional models of reading instruction on outcomes of literal comprehension and critical thinking skills. The sample for this study consisted of 101 adult students enrolled in a high-level developmental reading course at a large, urban community college in the Southeastern United States. The experimental group consisted of 48 students, and the control group consisted of 53 students. Students in the experimental group were limited in the time spent reading a course text of basic skills, with instructors using supplemental materials such as poems, news articles, and novels. Discussions, the reading-writing connection, and student choice in material selection were also part of the student-centered curriculum. Students in the control group relied heavily on a course text and vocabulary text for reading material, with great focus placed on basic skills. Activities consisted primarily of multiple-choice questioning and quizzes. The instrument used to collect pre-test data was Descriptive Tests of Language Skills in Reading Comprehension; post-test data were taken from the Florida College Basic Skills Exit Test. A MANCOVA was used as the statistical method to determine if either model of instruction led to significantly higher gains in literal comprehension skills or critical thinking skills. A paired samples t-test was also used to compare pre-test and post-test means. The results of the MANCOVA indicated no significant difference between instructional models on scores of literal comprehension and critical thinking. Neither was there any significant difference in scores between subgroups of age (under 25 and 25 and older) and language background (native English speaker and second-language learner). The results of the t-test indicated, however, that students taught under both instructional models made significant gains in on both literal comprehension and critical thinking skills from pre-test to post-test.

A Micro-Opto-Electro-Mechanical System (MOEMS) for Microstructure Manipulation

Microstructure manipulation is a fundamental process to the study of biology and medicine, as well as to advance micro- and nano-system applications. Manipulation of microstructures has been achieved through various microgripper devices developed recently, which lead to advances in micromachine assembly, and single cell manipulation, among others. Only two kinds of integrated feedback have been demonstrated so far, force sensing and optical binary feedback. As a result, the physical, mechanical, optical, and chemical information about the microstructure under study must be extracted from macroscopic instrumentation, such as confocal fluorescence microscopy and Raman spectroscopy. In this research work, novel Micro-Opto-Electro-Mechanical-System (MOEMS) microgrippers are presented. These devices utilize flexible optical waveguides as gripping arms, which provide the physical means for grasping a microobject, while simultaneously enabling light to be delivered and collected. This unique capability allows extensive optical characterization of the structure being held such as transmission, reflection, or fluorescence. The microgrippers require external actuation which was accomplished by two methods: initially with a micrometer screw, and later with a piezoelectric actuator. Thanks to a novel actuation mechanism, the “fishbone”, the gripping facets remain parallel within 1 degree. The design, simulation, fabrication, and characterization are systematically presented. The devices mechanical operation was verified by means of 3D finite element analysis simulations. Also, the optical performance and losses were simulated by the 3D-to-2D effective index (finite difference time domain FDTD) method as well as 3D Beam Propagation Method (3D-BPM). The microgrippers were designed to manipulate structures from submicron dimensions up to approximately 100 µm. The devices were implemented in SU-8 due to its suitable optical and mechanical properties. This work demonstrates two practical applications: the manipulation of single SKOV-3 human ovarian carcinoma cells, and the detection and identification of microparts tagged with a fluorescent “barcode” implemented with quantum dots. The novel devices presented open up new possibilities in the field of micromanipulation at the microscale, scalable to the nano-domain.