ENHANCING COMMUNICATION THROUGH SUCCESSFUL REPAIR: EXAMINING FLUENCY AND MECHANISMS FOR SYNTACTIC PROGRESS IN APHASIC AND NON-APHASIC SPEECH

The fields of Rhetoric and Communication usually assume a competent speaker who is able to speak well with conscious intent; however, what happens when intent and comprehension are intact but communicative facilities are impaired (e.g., by stroke or traumatic brain injury)? What might a focus on communicative success be able to tell us in those instances? This project considers this question in examining communication disorders through identifying and analyzing patterns of (dis) fluent speech between 10 aphasic and 10 non-aphasic adults. The analysis in this report is centered on a collection of data provided by the Aphasia Bank database. The database’s collection protocol guides aphasic and non-aphasic participants through a series of language assessments, and for my re-analysis of the database’s transcripts I consider communicative success is and how it is demonstrated during a re-telling of the Cinderella narrative. I conducted a thorough examination of a set of participant transcripts to understand the contexts in which speech errors occur, and how (dis) fluencies may follow from aphasic and non-aphasic participant’s speech patterns. An inductive mixed-methods approach, informed by grounded theory, qualitative, and linguistic analyses of the transcripts functioned as a means to balance the classification of data, providing a foundation for all sampling decisions. A close examination of the transcripts and the codes of the Aphasia Bank database suggest that while the coding is abundant and detailed, that further levels of coding and analysis may be needed to reveal underlying similarities and differences in aphasic vs. non-aphasic linguistic behavior. Through four successive levels of increasingly detailed analysis, I found that patterns of repair by aphasics and non-aphasics differed primarily in degree rather than kind. This finding may have therapeutic impact, in reassuring aphasics that they are on the right track to achieving communicative fluency.

DOUBLE VAULT COMPOSTING LATRINES IN RURAL PARAGUAY : FEASIBLE CONSTRUCTION AND OPTIMAL USE

Water resource depletion and sanitation are growing problems around the world. A solution to both of these problems is the use of composting latrines, as it requires no water and has been recommended by the World Health Organization as an improved sanitation technology. However, little analysis has been done on the decomposition process occurring inside the latrine, including what temperatures are reached and what variables most affect the composting process. Having better knowledge of how outside variables affect composting latrines can aid development workers on the choice of implementing such technology, and to better educate the users on the appropriate methods of maintenance. This report presents a full, detailed construction manual and temperature data analysis of a double vault composting latrine. During the author’s two year Peace Corps service in rural Paraguay he was involved with building twenty one composting latrines, and took detailed temperature readings and visual observations of his personal latrine for ten months. The author also took limited temperature readings of fourteen community member’s latrines over a three month period. These data points were analyzed to find correlations between compost temperatures and several variables. The two main variables found to affect the compost temperatures were the seasonal trends of the outside temperatures, and the mixing and addition of moisture to the compost. Outside seasonal temperature changes were compared to those of the compost and a linear regression was performed resulting in a R2-value of 0.89. Mixing the compost and adding water, or a water/urine mixture, resulted in temperature increases of the compost 100% of the time, with seasonal temperatures determining the rate and duration of the temperature increases. The temperature readings were also used to find events when certain temperatures were held for sufficient amounts of time to reach total pathogen destruction in the compost. Four different events were recorded when a temperature of 122°F (50°C) was held for at least 24 hours, ensuring total pathogen destruction in that area of the compost. One event of 114.8°F (46°C) held for one week was also recorded, again ensuring total pathogen destruction. Through the analysis of the temperature data, however, it was found that the compost only reached total pathogen destruction levels during ten percent of the data points. Because of this the storage time recommendation outlined by the World Health Organization should be complied with. The WHO recommends storing compost for 1.5-2 years in climates with ambient temperatures of 2-20°C (35-68°F), and for at least 1 year with ambient temperatures of 20-35°C (68-95°F). If these storage durations are obtainable the use of the double vault composting latrine is an economical and achievable solution to sanitation while conserving water resources.

Enzyme engineering of aminotransferases for improved activity and thermostability

The production by biosynthesis of optically active amino acids and amines satisfies the pharmaceutical industry in its demand for chiral building blocks for the synthesis of various pharmaceuticals. Among several enzymatic methods that allow the synthesis of optically active aminoacids and amines, the use of minotransferase is a promising one due to its broad substrate specificity and no requirement for external cofactor regeneration. The synthesis of chiral compounds by aminotransferases can be done either by asymmetric synthesis starting from keto acids or ketones, and by kinetic resolution starting from racemic aminoacids or amines. The asymmetric synthesis of substituted (S)-aminotetralin, an active pharmaceutical ingredient (API), has shown to have two major factors that contribute to increasing the cost of production. These factors are the raw material cost of biocatalyst used to produce it and product loss during biocatalyst separation. To minimize the cost contribution of biocatalyst and to minimize the loss of product, two routes have been chosen in this research: 1. To engineer the aminotransferase biocatalyst to have greater specific activity, and 2. Improve the engineering of the process by immobilization of biocatalyst in calcium alginate and addition of cosolvents. An (S)-aminotransferase (Mutant CNB03-03) was immobilized, not as purified enzyme but as enzyme within spray dried cells, in calcium alginate beads and used to produce substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the immobilized biocatalyst was recycled. Initial rate of reaction for cycle 1 (6 hr duration) was determined to be 0.258 mM/min, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle 1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1 (immobilized preparation consisted of 50 mg of spray dried cells per gram of calcium alginate). Conversion to product for each cycle decreased as well, from 100% in cycle 1 (About 50 mM), 80% in cycle 2, and 30% after cycle 3. This mutant was determined to be deactivated at elevated temperatures during the reaction cycle and was not stable enough to allow multiple cycles in its immobilized form. A new mutant aminotransferase was isolated by applying error-prone polymerase chain reaction (PCR) on the gene coding for this enzyme and screening/selection: CNB04-01. This mutant showed a significant improvement in thermostability in comparison to CNB03-03. The new mutant was immobilized and tested under similar reaction conditions. Initial rate remained fairly constant (0.2 mM/min) over four cycles (each cycle with a duration of about 20 hours) with the mutant retaining almost 80% of initial rate in the fourth cycle. The final product concentrations after each cycle did not decrease during recycle experiments. Thermostability of CNB04-01 was much improved compared to CNB03-03. Under the same reaction conditions as stated above, the addition of co-solvents was studied in order to increase substituted tetralone solubility. Toluene and sodium dodecylsulfate (SDS) were used. SDS at 0.01% (w/v) allowed four recycles of the immobilized spray dried cells of CNB04-01, always reaching higher product concentration (80-85 mM) than the system with toluene at 3% (v/v) -70 mM-. The long term activity of immobilized CNB04-01 in a system with SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles (20 to 24 hours each one), reaching always final product concentration between 80-85 mM, but dropping precipitously in the fourth cycle to a final product concentration of 50 mM. Although significant improvement of immobilization on productivity and stability were observed using CNB04-01, another observation demonstrated the limitations of an immobilization strategy on reducing process costs. After analyzing the results of this experiment it was seen that a sudden drop occurred on final product concentration after the third recycle. This was due to product accumulation inside the immobilized preparation. In order to improve the economics of the process, research was focused on developing a free enzyme with an even higher activity, thus reducing raw material cost as well as improving biomass separation. A new enzyme was obtained (CNB05-01) using error-prone PCR and screening using as a template the gene derived from the previous improved enzyme. This mutant was determined to have 1.6 times the initial rate of CNB04-01 and had a higher temperature optimum (55°). This new enzyme would allow reducing enzyme loading in the reaction by five-fold compared to CNB03-03, when using it at concentration of one gram of spray dried cells per liter (completing the reaction after 20-24 hours). Also this mutant would allow reducing process time to 7-8 hours when used at a concentration of 5 grams of spray dried cells per liter compared to 24 hours for CNB03-03, assuming that the observations shown before are scalable. It could be possible to improve the economics of the process by either reducing enzyme concentration or reducing process time, since the production cost of the desired product is primarily a function of both enzyme concentration and process time.