Magíster en Ciencias, Mención Computación / Los tipos refinados (Refinement Types) son una técnica efectiva de verificación basada en lenguajes que extienden la expresividad de los sistemas de tipos tradicionales incluyendo la posibilidad de restringir valores usando predicados lógicos. Sin embargo, como cualquier otra disciplina de tipos expresiva, obligan a los programadores a lidiar con las estrictas restricciones impuestas por los tipos. Esto puede resultar ser demasiado tedioso para los programadores, especialmente en etapas tempranas de desarrollo donde el código cambia constantemente. Nosotros conjeturamos que esta rigidez no deseada puede obstaculizar la adopción de los tipos refinados. Los tipos graduales (Gradual Typing) permiten combinar la flexibilidad de los lenguajes dinámicamente tipados con las garantías otorgadas por los lenguajes estáticamente tipados. Usando tipos graduales, los programadores pueden comenzar un desarrollo con código chequeado completamente de forma dinámica y aprovechar la flexibilidad de las construcciones idiomáticas típicas de los lenguajes dinámicos. A medida que el código se vuelve más estable, el programador o la programadora puede escoger verificar estáticamente ciertas porciones del programa, haciendo precisos los tipos en esos puntos. Finalmente, si lo desea, también puede decidir verificar el código completamente de forma estática. Los sistemas de tipos graduales aseguran una transición suave entre estos distintos niveles de precisión. Esta tesis demuestra como los tipos graduales pueden ser extendidos para soportar los tipos refinados, permitiendo una evolución suave además de interoperabilidad entre tipos simples y tipos lógicamente refinados. Al hacer esto, se atacan dos desafíos previamente inexplorados en la literatura sobre tipos graduales: lidiar con información lógica imprecisa y con la presencia de tipos dependientes. El primer desafío condujo a una noción crucial de localidad para fórmulas, mientras que el segundo desafío terminó en la definición de novedosos operadores relacionados con substitución al nivel de tipos y términos, que identifican nuevas oportunidades para errores en tiempo de ejecución en lenguajes graduales con tipos dependientes. / Este trabajo ha sido parcialmente financiado por CONICYT - PCHA Magíster Nacional 2015-22150894

Medical lab work, such as blood testing, will one day be near instantaneous and inexpensive via capabilities enabled by the fast growing world of microtechnology. In this research study, sorting and separation of different ABO blood types have been investigated by applying alternating and direct electric fields using class=SpellE>dielectrophoresis in microdevices. Poly(dimethylsiloxane) (PDMS) microdevices, fabricated by standard photolithography techniques have been used. Embedded perpendicular platinum (Pt) electrodes to generate forces in AC dielectrophoresis were used to successfully distinguish positive ABO blood types, with O+ distinguishable from other blood types at >95% confidence. This is an important foundation for exploring DC dielectrophoretic sorting of blood types. The expansion of red blood cell sorting employing direct current insulative class=SpellE>dielectrophoresis (DC-iDEP) is novel. Here Pt electrodes were remotely situated in the inlet and outlet ports of the microdevice and an insulating obstacle generates the required dielectrophoretic force. The presence of ABO antigens on the red blood cell were found to affect the class=SpellE>dielectrophoretic deflection around the insulating obstacle thus sorting cells by type. To optimize the placement of insulating obstacle in the microchannel, COMSOL Multiphysics® simulations were performed. Microdevice dimensions were optimized by evaluating the behaviors of fluorescent polystyrene particles of three different sizes roughly corresponding to the three main components of blood: platelets (2-4 µm), erythrocytes (6-8 µm) and leukocytes (10-15 µm). This work provided the operating conditions for successfully performing size dependent blood cell insulator based DC dielectrophoresis in PDMS microdevices. In subsequent studies, the optimized microdevice geometry was then used for continuous separation of erythrocytes. The class=SpellE>microdevice design enabled erythrocyte collection into specific channels based on the cell’s deflection from the high field density region of the obstacle. The channel with the highest concentration of cells is indicative of the ABO blood type of the sample. DC resistance measurement system for quantification of erythrocytes was developed with single PDMS class=SpellE>microchannel system to be integrated with the DC- class=SpellE>iDEP device developed in this research. This lab-on-a-chip technology application could be applied to emergency situations and naturalcalamities for accurate, fast, and portable blood typing with minimal error.

Identification of fungal isolates is critical in studying Aspergillus flavus ecology and for developing methods to reduce aflatoxin contamination. In our efforts to track biocontrol applications of the atoxigenic A. flavus K49 (NRRL 30797), we have developed a rapid and accurate classification system for A. flavus based on PCR product melting temperatures (Tm). Using 18 primers and a total of 59 Aspergilli strains, including all 49 representatives of the Georgian peanut Vegetative Compatibility Groups (VCGs), a decision tree Tm flowchart was generated. The decision tree can classify all 59 strains using only 9 of the SSR primers and an average of 3.4 primers for each definitive classification. To confirm the effectiveness of the decision tree for strain identification, unknown samples isolated from experimental fields inoculated with various A. flavus strains in Stoneville, MS were analyzed. Ninety-six percent of the samples could be placed into a VCG using Tm(s) coupled with the decision tree. This dynamic system is an excellent tool for researchers studying biodiversity of A. flavus.

The cryosphere has been shown to be particularly adept as a proxy for climate change by various studies. Accordingly, historical records from the field of ice phenology have been harvested by climate scientists for the express purpose of studying the temporal variation of ice phenomena, namely freeze-up and ice-out. Ice-out records from 20 lakes in Maine, U.S.A. were collected and clustered by z-score for this thesis. Rather than attempt to relate ice-out to spring air temperature or global teleconnections/oscillations, the Spatial Synoptic Classification (SSC) method was used to encapsulate several meteorological variables that could have a bearing on ice-out variation. The balance between occurrence of relatively cool Moist Polar (MP) and relatively warm Dry Moderate (DM) weather-types during the winter-spring "superseason" was found to be a synoptic barometer of whether ice-out would occur seasonably early or late. The significance of this is predicated upon the finding that quantity of DM days has steadily risen at the expense of MP days during the latter-half of the twentieth-century, in accordance with observed climatic warming during the same period. The remaining SSC weather-types, most notably omnipresent Dry Polar (DP), remained generally stable during the historical record in Maine, further undergirding the significance of the DM-MP relationship. / Master of Science

A mastite bovina é considerada a enfermidade de maior impacto na pecuária leiteira mundial. Apesar da adoção de práticas de manejo para minimizar a ocorrência desta, os resultados não são satisfatórios para o controle de Staphylococcus aureus, o principal agente causador. Além da importância veterinária, possui implicações na saúde humana devido ao uso extensivo de antibióticos no tratamento e controle da doença e devido ao potencial de disseminação zoonótica de S. aureus. A principal preocupação em relação à resistência aos antimicrobianos é referente à meticilina, mediada pelo gene mecA. Estudos moleculares são importantes para o conhecimento da diversidade genética dos agentes causadores de mastite. A tipagem por sequenciamento de um único locus, do gene da região X da proteína A de S. aureus (spa Typing), tem sido utilizada com sucesso para investigar estrutura populacional, sendo adequada para estudos epidemiológicos. O presente projeto teve como objetivo pesquisar o perfil de resistência aos antimicrobianos e a presença de genes de resistência à meticilina e de S. aureus isolados de mastite bovina, além de compreender a diversidade genética, identificar possíveis novos perfis e conhecer a distribuição destes isolados. Os resultados encontrados foram a ausência de resistência à meticilina no antibiograma, confirmada pela ausência do gene mecA, e a baixa prevalência de multirresistência aos antimicrobianos testados. Apesar de mais de 58,74% dos isolados (242/412) serem resistentes a pelo menos um antibiótico, 53,64% (221/412) correspondem a resistência à ampicilina. Foram determinados 44 spa tipos diferentes, sendo os três mais frequentes t605 (57,45%), t002 (8,4%) e t127 (8,13%). Não houve associação entre a distribuição dos spa tipos e as variáveis analisadas de ano, estado, município e fazenda, e perfil de resitência aos antibicrobianos. Este é o primeiro estudo que utiliza o spa typing para avaliar uma coleção temporalmente diversa e, portanto, representativa da região estudada ao longo de 22 anos. Logo, espera-se que a descrição dos tipos de S. aureus obtidos contribua para a compreensão dos aspectos epidemiológicos envolvidos na transmissão do patógeno e do papel dos hospedeiros como reservatórios para linhagens resistentes. Tal compreensão é imprescindível para a prevenção, controle e tratamento da mastite bovina. / Bovine mastitis is considered the disease of greatest impact in global dairy industry. Despite the adoption of management practices to minimize its occurrence, the results are not satisfactory for the control of Staphylococcus aureus, the major causative agent. Besides veterinary importance, there are also implications for human health due to the extensive use of antibiotics in treatment and control of the disease and due to the potential for zoonotic spread of S. aureus. The main concern regarding antimicrobial resistance is methicillin resistance mediated by the mecA gene. Molecular epidemiology studies are important for the understanding of the genetic diversity of the causative agents of mastitis. Typing by sequencing of a single locus of the X region gene of the S. aureus protein A (spa Typing) has been successfully used to investigate population structure. This project aims to investigate the antimicrobial susceptibility profile and the presence of methicillin resistance genes in S. aureus isolated from bovine mastitis, in addition to understanding the genetic diversity, identifying possible new profiles and knowing the distribution of these isolates. The results obtained to the present were the absence of methicillin resistance in the antibiogram, confirmed by the absence of mecA gene, and the low prevalence of multidrug resistance to antimicrobials. Even though resistance to at least one antibiotic was present in more than 58,74% of the isolates (242/412), ampicillin resistance corresponds to 53.64% (221/412). Different spa types were determined, the three most frequent being t605 (57.45%), t002 (8.4%) and t127 (8.13%). There was no association between the spa type distribution and the variables of year, state, municipality, farm, and antibiotic resistance profile analyzed. This is the first study that uses spa Typing to evaluate a collection as temporarily diverse and representative of the region studied in 22 years. Therefore, it is expected that the results of this study, by allowing comparison of the profile of S. aureus from different sources (animals and property), will contribute to the understanding of the epidemiological aspects involved in the transmission of the pathogen and the role of hosts as reservoirs for pathogenic strains. Such understanding is essential for the prevention, control and treatment of bovine mastitis

A mastite bovina é considerada a enfermidade de maior impacto na pecuária leiteira mundial. Apesar da adoção de práticas de manejo para minimizar a ocorrência desta, os resultados não são satisfatórios para o controle de Staphylococcus aureus, o principal agente causador. Além da importância veterinária, possui implicações na saúde humana devido ao uso extensivo de antibióticos no tratamento e controle da doença e devido ao potencial de disseminação zoonótica de S. aureus. A principal preocupação em relação à resistência aos antimicrobianos é referente à meticilina, mediada pelo gene mecA. Estudos moleculares são importantes para o conhecimento da diversidade genética dos agentes causadores de mastite. A tipagem por sequenciamento de um único locus, do gene da região X da proteína A de S. aureus (spa Typing), tem sido utilizada com sucesso para investigar estrutura populacional, sendo adequada para estudos epidemiológicos. O presente projeto teve como objetivo pesquisar o perfil de resistência aos antimicrobianos e a presença de genes de resistência à meticilina e de S. aureus isolados de mastite bovina, além de compreender a diversidade genética, identificar possíveis novos perfis e conhecer a distribuição destes isolados. Os resultados encontrados foram a ausência de resistência à meticilina no antibiograma, confirmada pela ausência do gene mecA, e a baixa prevalência de multirresistência aos antimicrobianos testados. Apesar de mais de 58,74% dos isolados (242/412) serem resistentes a pelo menos um antibiótico, 53,64% (221/412) correspondem a resistência à ampicilina. Foram determinados 44 spa tipos diferentes, sendo os três mais frequentes t605 (57,45%), t002 (8,4%) e t127 (8,13%). Não houve associação entre a distribuição dos spa tipos e as variáveis analisadas de ano, estado, município e fazenda, e perfil de resitência aos antibicrobianos. Este é o primeiro estudo que utiliza o spa typing para avaliar uma coleção temporalmente diversa e, portanto, representativa da região estudada ao longo de 22 anos. Logo, espera-se que a descrição dos tipos de S. aureus obtidos contribua para a compreensão dos aspectos epidemiológicos envolvidos na transmissão do patógeno e do papel dos hospedeiros como reservatórios para linhagens resistentes. Tal compreensão é imprescindível para a prevenção, controle e tratamento da mastite bovina. / Bovine mastitis is considered the disease of greatest impact in global dairy industry. Despite the adoption of management practices to minimize its occurrence, the results are not satisfactory for the control of Staphylococcus aureus, the major causative agent. Besides veterinary importance, there are also implications for human health due to the extensive use of antibiotics in treatment and control of the disease and due to the potential for zoonotic spread of S. aureus. The main concern regarding antimicrobial resistance is methicillin resistance mediated by the mecA gene. Molecular epidemiology studies are important for the understanding of the genetic diversity of the causative agents of mastitis. Typing by sequencing of a single locus of the X region gene of the S. aureus protein A (spa Typing) has been successfully used to investigate population structure. This project aims to investigate the antimicrobial susceptibility profile and the presence of methicillin resistance genes in S. aureus isolated from bovine mastitis, in addition to understanding the genetic diversity, identifying possible new profiles and knowing the distribution of these isolates. The results obtained to the present were the absence of methicillin resistance in the antibiogram, confirmed by the absence of mecA gene, and the low prevalence of multidrug resistance to antimicrobials. Even though resistance to at least one antibiotic was present in more than 58,74% of the isolates (242/412), ampicillin resistance corresponds to 53.64% (221/412). Different spa types were determined, the three most frequent being t605 (57.45%), t002 (8.4%) and t127 (8.13%). There was no association between the spa type distribution and the variables of year, state, municipality, farm, and antibiotic resistance profile analyzed. This is the first study that uses spa Typing to evaluate a collection as temporarily diverse and representative of the region studied in 22 years. Therefore, it is expected that the results of this study, by allowing comparison of the profile of S. aureus from different sources (animals and property), will contribute to the understanding of the epidemiological aspects involved in the transmission of the pathogen and the role of hosts as reservoirs for pathogenic strains. Such understanding is essential for the prevention, control and treatment of bovine mastitis

published_or_final_version / Physiology / Master / Master of Philosophy

Giardia lamblia is a eukaryotic parasite that causes diarrhea in humans worldwide. Diarrheal diseases cause stunting and mental retardation in children in developing nations, therefore it is important to understand the molecular epidemiology of G. lamblia. Compounding this, it is not clear if companion animals such as dogs contribute to infections in humans through zoonotic transmission. The genotypes of G. lamblia that have been found in humans are A1, A2 and B, while those in dogs have been on rare occasions all three human genotypes, but largely C and D, which have only been reported in dogs and appear to be species-specific. The molecular epidemiology of G. lamblia in humans and dogs was assessed in an endemic region of Lima, Peru. With one exception, dogs were found to harbor the C and D dog genotypes of G. lamblia. A single family dog was found to harbor a human genotype of G. lamblia. A2 and B genotypes of G. lamblia, but not A1, were found in humans in the endemic region. Previous literature reported that A2 and B typing within genotype tools were available, however the A2 samples from the endemic region could not be distinguished from one another through nucleotide polymorphism sequence analysis. A molecular typing technique was developed to type A2 samples. The extensive sequence analysis performed on two chromosomes of G. lamblia, yielded different phylogenetic tree groupings for the same samples. This lead to algorithmic analysis, which demonstrated a significantly high probability that meiotic recombination is occurring in the A2 samples of G. lamblia. As G. lamblia is largely believed to be asexual, the conclusion of doctoral research performed in this study yielded controversial, yet significant evidence that sex in G. lamblia A2 genotype samples is indeed occurring.

In dieser Arbeit wurde überprüft, inwieweit sich verschiedene Lagerungsbedingungen von Zähnen auf die Verwertbarkeit von DNA im Zahninneren zur Gewinnung eines genetischen Fingerabdrucks auswirken. Die Untersuchungen an frisch extrahierten Zähnen ließen erkennen, dass die in dieser Arbeit verwendeten Methoden und DNA-Kits für die weitere Analyse der den unterschiedlichen Bedingungen ausgesetzten Zähne geeignet sind. Asensible Zähne weisen bereits bei der Zahnextraktion Zeichen von Degradation auf. Dagegen ist der kariöse Zerstörungsgrad an sich noch kein Ausschlusskriterium. Bei wurzelkanalbehandelten Zähnen konnten nur Systeme mit sehr kurzen Amplifikationsprodukten typisiert werden. Sie waren für eine genetische Identifizierung kaum geeignet. Bei der Analyse im Erdreich vergrabener Zähne nahm die Anzahl der typisierbaren Systeme nach einem Vierteljahr Liegezeit kontinuierlich ab, bis schließlich nach einem Jahr nur noch vereinzelt kleine Systeme detektiert werden konnten. Ein anderes Bild zeigte sich bei den in Wasser gelagerten Zähnen. Bis zu einem halben Jahr nach Versuchsbeginn konnte eine Typisierung aller Systeme durchgeführt werden. Erst nach einem Jahr war die Degradation so weit vorangeschritten, dass große Systeme keine Typisierung mehr erlaubten. Bei Zähnen, die der Sonnenstrahlung einen Monat ausgesetzt waren, war es dagegen problemlos möglich, einen vollständigen genetischen Fingerabdruck zu erfassen. Nach drei Monaten konnten allerdings nur noch vereinzelt sehr kleine Systeme gewonnen werden. Nach einem halben Jahr ergab lediglich der TPOX-vs eine erfolgreiche Typisierung. UV-Strahlung kann Zahnschmelz und –dentin durchdringen und DNA in recht kurzer Zeit degradieren. Die Zähne aus dem Sektionsgut des Instituts für Rechtsmedizin Würzburg lieferten unterschiedliche Ergebnisse, sodass hierzu keine generelle Aussage gemacht werden kann. Bei der Behandlung der Zähne mit Hitze von 200 °C konnte die DNA nur begrenzte Zeit vor Degradation geschützt werden. Bereits nach einer halben Stunde waren große Teile der Multiplex-Kits nicht mehr für eine Typisierung verwertbar. Das STR-System SE33 konnte allerdings noch ermittelt werden. Die Entnahmen zu späteren Zeitpunkten (60 min, 90 min und 120 min) lieferten fast nur noch einzelne STRSysteme. Eine knapp 30-jährige trockene Lagerung von Zähnen in einem Schrank mit Schutz vor Sonneneinstrahlung schadete der DNA-Analysierbarkeit kaum. Ein vollständiger Ausfall aller Systeme musste bei den Zahnproben von der Ausgrabung in Katzwang verzeichnet werden. Die Zähne dieser 380 bis 550 Jahre alten Schädel lieferten kein auswertbares DNA-Material mehr. Ebenso konnte mit diesen Untersuchungsmethoden aus den 2300 bis 2800 Jahre alten Zähnen aus der Dietersberghöhle kein analysierbares DNA-Material gewonnen werden. Es zeigte sich, dass Lagerbedingungen einen entscheidenden Einfluss auf die DNA-Qualität ausüben. Als DNA-schädigende Einflüsse können neben Mikroorganismen und Feuchtigkeit insbesondere UV-Strahlung und Hitze gelten. Der Faktor Zeit spielt bei günstigen Lagerbedingungen eine untergeordnete Rolle. / In this dissertation the possibility of winning a genetic fingerprint out of teeth that have been rested under different conditions was analysed. The examinations of recently extracted teeth showed that the methods and DNA-Kits used in this study are suitable for further analyses of teeth that have been exposed to different conditions. Asensible teeth already showed at time of extraction signs of degradation. However it was possible to get a genetic DNA-typing out of teeth which were destroyed by caries. Teeth with a root canal filling only showed systems with short amplification products. So they were not suitable for a genetic identification. The analysis of teeth which were buried in soil resulted that the number of detectable systems decreased continually after four month. After one year only a few systems were detected. The teeth which were stored in water gave a positive result in all systems after half a year. When the teeth have laid one year in water the degradation had gone so far that the big systems could not be typified anymore. Teeth that were exposed to sunshine for one month gave a complete detection of all analysed systems. Two month later only a few small systems were detected. After six months only the system TPOX-vs was successfully typed. UV-radiation gets through the dentin and the enamel and destroys the DNA very quickly. Two teeth out of the section good of the institute for forensic medicine showed completely different results. In one case the DNA-typing was successful, in the other one it wasn´t. Teeth that were treated in a 200 °C hot oven could protect the DNA for only a few minutes. After 30 minutes half of the analysed systems already were destroyed. After one hour and more only some sporadic systems were detected. The DNA-typing of a genetic fingerprint out of 30 years old teeth was successful. The complete failure of all systems showed examples of 380 to 550 years old teeth from a dig in Katzwang such as examples of 2.300 to 2.800 years old teeth out of the Dietersberg cave. For the DNA-quality it is very important to which conditions the teeth are exposed. DNA-damaging influences among other things are microorganisms, humidity, UV-radiation and heat. The factor time is not so important if the surrounding conditions are optimal.

A dissertation submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Medicine. / The emergence of strains of S. pneumoniae resistant to penicillin and to other antibiotics, and the spread of that resistance over the world, have become major concerns and increase the need for epidemiological surveillance. The following typing methods have been used to detect strain variability in pneumococci: Serotyping, antibiotic susceptibility profiles, multilocus enzyme electrophoresis (MLEE), penicillin-binding protein (PBP) profiles, pulse-field gel electrophoresis (pFGE), and ribotyping. Serotyping, antibiograms, and MLEE only detect phenotypic variability. PBP gene profiles, PFGE, and ribotyping detect genotypic differences but these techniques are labour intensive and time consuming. Random amplified polymorphic DNA (RAPD) is a new technique that bas proved useful for typing bacteria, fungi, and parasites, but has not been. studied using pneumococci. Unlike conventional polymerase chain reaction (peR), RAPD utilizes single, short primers, usually 10 oligonucleotides in length. As the primer is short and low astringency annealing temperatures are used, there will be many complimentary sites scattered randomly throughout a bacterium's genome, When such sites occur a few hundred base pairs away from each other and on opposite DNA strands, the enclosed region can be amplified by peR This results in numerous discrete target fragments which can be separated by agarose-gel electrophoresis and ethidium bromide staining. RAPD requires no sequence information and it scans the whole genome rather than relying on hypervariability within one specific gene. The aims of this study were: to determine strain variability using RAPD, to determine the reproducibility ofRAPD, and to demonstrate intercontinental spread of a multiresistant pneumococcal strain. The following strains were evaluated: a) 10 strains from a day-care centre (DCC), the index case being a 3 year old girl 'with otitis media. An aunt from Spain had recently been staying with the family. The other strains were isolated from class mates and siblings of the index case.; b) 18 clinical isolates from Seoul, Korea; and c) 11 epidemiologically unrelated isolates from South Africa, including the reference strain, R6. Two DNA extraction methods were used. The first involving lysis with sodmm-dodecyl-sulphaze and proteinase K. Proteins were removed with phenol-chloroform, and the DNA precipitated with ethanol. The second method involved incubating the cells at 95 0C for 15 microlitres, followed by centrifugation. 2 microlitres of the supernatant was then used for each PCR reaction, Three primers were evaluated. After 01uimisation of the RAPDreaction for pneumococci, the final peR mixtures per 50 ul was: primer (4 plY1), template (0.5 ng), nuc1eotides (300 pMeach), magnesium (4 mM~, and Taq polymerase (2 U). 35 cycles were used with an annealing temperature of 35'C. Both DNA extraction methods: gave reproducible results but were not comparable to each other. All 10 strains from the DCC gave the same banding pattern as the Spanish done for all 3 primers. 7 of the Korean strains gave the same banding pattern as the Spanish clone using the first two primers, however one strain showed an additional band using the third primer. Of the remaining 22 strains, 21 different banding patterns were obtained. This study has shown that RAPD is a simple and rapid technique that can distinguish strain variation among pneumococci. The reproducibility is excellent within the same laboratory. Finally using RAPD. this study identified a Spanish multiresistant 23F clone in South Africa and Korea. / Andrew Chakane 2018