Thesis (Ph. D.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Protein-based materials are an important area of research for various reasons. Natural protein materials such as spider silk have mechanical properties which compare favourably to artificial or inorganic materials, and in addition are biodegradable and can be produced from easily available feedstocks. It is also possible to produce materials that incorporate the functionality of a natural protein, such as ligand-binding or catalysis of reactions, thus allowing this functionality to be used in the solid rather than solution phase. Two particularly interesting components for protein-based materials are amyloid fibrils and tandem repeat proteins. Amyloid fibrils are exceptionally strong, tough, highly-ordered structures that self-assemble from a wide range of simple building blocks. Meanwhile, tandem repeat proteins are a class of proteins that act as scaffolds to mediate protein-protein interactions and are known to act as elastic springs. Unlike globular proteins, tandem repeat proteins can be designed to bind specific ligands, and their ligand-binding properties and stability can be tuned separately. This work details the synthesis and characterisation of repeat protein and amyloid fibril components for a “smart” hydrogel, the production of these gels, and their characterisation using a microfluidic method that I developed. Although amyloid fibrils have previously been decorated with functional proteins, hitherto, this has usually been done by assembling the fibrils from already-functionalised components. This approach limits the functionality to species that can survive the harsh conditions of amyloid aggregation and do not disturb fibril assembly. Therefore, a method was developed to produce amyloid fibrils that displayed an alkyne functionality on their surface to allow functional proteins or other species to be attached after assembly. This involved the design and synthesis (using solid-phase peptide chemistry) of a peptide based on the previously known TTR105-115 peptide (derived from the amyloidogenic Transthyretin protein). These fibrils were characterised by AFM and TEM and it was then shown that the assembled fibrils could be functionalised using an azide-alkyne “click” reaction. The reaction was shown to work with a variety of ligands including proteins, which were found to retain their structure and function after crosslinking to the fibril. The fibrils with ligands attached were characterised by a variety of methods including LCMS (liquid chromatography-mass spectrometry) and super-resolution optical microscopy. Next, repeat proteins were produced recombinantly containing non-natural azido amino acids at their termini. Incorporation of non-natural amino acids was carried out using a number of different methods including amber codon suppression and methionine replacement. Micron-sized hydrogels were then formed from microfluidic-generated droplets by covalently crosslinking the alkyne-functionalised fibrils with the azide-functionalised repeat proteins. The initial experiments to show proof of principle were carried out with consensus-designed repeat proteins, but repeat proteins based on natural sequences were also used to make hydrogels that could later be tested for potential uptake of peptides known to bind these proteins. These hydrogels could potentially be used for drug delivery or other applications in which a chemical response to a mechanical stimulus is desired. The mechanical properties of the hydrogels were measured using novel microfluidic devices, which were designed and fabricated using standard PDMS-based soft lithography.

Parkinson's Disease (PD) is a late-onset neurodegenerative disease characterized by progressive motor dysfunction caused by a loss of dopaminergic neurons for which there is no known cure. Among the most common genetic causes of PD are mutations in the leucine-rich repeat kinase 2 gene (LRRK2), encoding a multi-domain protein with kinase activity. The LRRK2 G2019S mutation causes hyperactivity of the kinase domain and is the most frequent LRRK2 mutation in patients with familial PD, though its role in PD pathology remains unclear. Preliminary data from the lab of our collaborator, Dr. David Park, demonstrated through a genetic screen in Drosophila melanogaster that the deletion of rbp9 encoding an RNA-binding protein prevented pathology induced by PD-relevant mutations in the LRRK2 kinase domain. The neuronal homolog of RBP9 in humans is HuD, a member of the Hu family of RNA-binding proteins that regulates the expression of many transcripts involved in neuronal development, plasticity, and survival. In addition, HuD has been shown to modify the age-at-onset or risk of developing PD. Here, we studied the effect of LRRK2 on the post-transcriptional regulation of mRNAs bound by HuD in the context of PD. Our findings showed that HuD is a substrate for LRRK2 phosphorylation in vitro, and that LRRK2 G2019S hyperphosphorylates HuD. We demonstrated that LRRK2 kinase activity is required for the binding of several transcripts by HuD that encode PD-relevant proteins such as α-synuclein and neuronal survival factor BDNF. Our findings in human neuroblastoma cells indicated that LRRK2 regulates the protein levels of HuD mRNA targets α-synuclein and BDNF in a mechanism that can by modified by HuD. Finally, we showed that the combination of HuD knockout with LRRK2 G2019S expression in mice rescues aberrant expression of HuD targets in mice with only the LRRK2 G2019S mutation or the knockout of HuD alone. Together, our findings demonstrate that LRRK2 affects the post-transcriptional regulation of HuD-bound mRNAs, and suggest the use of HuD as a potential therapeutic target in patients with PD caused by the LRRK2 G2019S mutation.

Resumo: A variabilidade entre progênies é criada pela segregação cromossômica independente dos genes e pela recombinação genética intracromossomal durante a meiose. O objetivo deste estudo foi analisar a variabilidade derivada de crossing-overs em cruzamentos biparentais (G2 e J2), quádruplos (G4 e J4) e óctuplos (G8 e J8), avaliados em populações segregantes derivadas de parentais contrastantes para resistência ao nematóide de cisto da soja (raça 3) - NCS - e ao oídio - O. A análise foi realizada em populações F2, através de marcadores SSR (single sequence repeat) concentrados em uma região de 55 cM ao redor do gene rmd (resistência ao oídio) e rhg1 (resistência ao NCS). Após o teste dos marcadores, quanto ao polimorfismo, apenas marcadores polimórficos foram utilizados para detectar crossing-over. Todos os marcadores analisados foram não significativos pelo teste de qui-quadrado (P > 0,05), indicando que os valores observados se ajustam à proporção genotípica esperada em F2 (1:2:1). As maiores médias de crossing-over por genótipo foram obtidas para G4 (4,00), no grupo G, e J8 (2,91), no grupo J. Por outro lado, as maiores médias de crossing-over considerando o número de gerações para formar cada população, foram para G2 (2,02) e J8 (0,97). A recombinação entre alelos ocorreu em algumas populações, entretanto para G4 e J8 em 1,89% dos genótipos não ocorreram. Em geral, nos cruzamentos com maior número de parentais envolvidos a ocorrência de crossingover foi maior, sendo satisfatórios na criação de variabilidade. O progresso no melhoramento de soja tem sido alcançado em partes pela criação de novas combinações alélicas dentro dos cromossomos. / Abstract: The variability among the progenies is created by chromosome segregation, independent assortment of genes, and intra-chromosomal genetic recombination during meiosis. The objective of this study was to analyze the variability derived from crossovers in soybean biparental (G2 and J2), quadruple (G4 and J4) and octuple (G8 and J8) crosses, measured in segregant population derived from contrasting parental regarding their resistance to cist nematode (race 3) - SCN and powdery mildew - PM. The analyses were made in F2 population through SSR (single sequence repeat) markers located in a 55CM region around Rmd (powdery mildew) and Rhg1 (cist nematode) resistance genes. After screening makers for their polymorphism, only polymorphic markers were used to detect crossovers. All markers were not significant by chi-square test (P > 0.05), showing that observed values corroborates to genotypic inheritance ratio expected in F2 population (1:2:1). Thus, the higher average of crossovers for some populations were observed for G4 (4.00), at linkage group G and J8 (2.91), at linkage group J. On the other hand, the higher average of crossovers considering the generation number to form each population, was found for G2 (2.02) and J8 (0.97). The recombination between alleles occurred in some populations, however, to G4 and J8, in 1.89% of the genotypes not showing crossover. In general, the crosses with larger numbers of parents showed higher number of crossovers, being very satisfactory for the creation of genetic variability. Soybean breeding progress has been accomplished in part by creating on new within_chromossome allele combinations. / Orientador: Antonio Orlando Di Mauro / Coorientador: Todd Pfeiffer / Banca: Natal Antonio Vello / Banca: Osvaldo Toshiyuki Hamawak / Banca: José Roberto Môro / Banca: Janete Apparecida Desidério Sena / Doutor

abstract: Worldwide, rivers and streams make up dense, interconnected conveyor belts of sediment– removing carved away earth and transporting it downstream. The propensity of alluvial river beds to self-organize into complex trains of bedforms (i.e. ripples and dunes) suggests that the associated fluid and sediment dynamics over individual bedforms are an integral component of bedload transport (sediment rolled or bounced along the river bed) over larger scales. Generally speaking, asymmetric bedforms (such as alluvial ripples and dunes) migrate downstream via erosion on the stoss side of the bedform and deposition on the lee side of the bedform. Thus, the migration of bedforms is intrinsically linked to the downstream flux of bedload sediment. Accurate quantification of bedload transport is important for the management of waters, civil engineering, and river restoration efforts. Although important, accurate qualification of bedload transport is a difficult task that continues t elude researchers. This dissertation focuses on improving our understanding and quantification of bedload transport on the two spatial scales: the bedform scale and the reach (~100m) scale. Despite a breadth of work investigating the spatiotemporal details of fluid dynamics over bedforms and bedload transport dynamics over flat beds, there remains a relative dearth of investigations into the spatiotemporal details of bedload transport over bedforms and on a sub-bedform scale. To address this, we conducted two sets of flume experiments focused on the two fundamental regions of flow associated with bedforms: flow separation/reattachment on the lee side of the bedform (Chapter 1; backward facing-step) and flow reacceleration up the stoss side of the next bedform (Chapter 2; two-dimensional bedform). Using Laser and Acoustic Doppler Velocimetry to record fluid turbulent events and manual particle tracking of high-speed imagery to record bedload transport dynamics, we identified the existence and importance of “permeable splat events” in the region proximal to flow reattachment. These coupled turbulent and sediment transport events are integral to the spatiotemporal pattern of bedload transport over bedforms. Splat events are localized, high magnitude, intermittent flow features in which fluid impinges on the bed, infiltrates the top portion of bed, and then exfiltrates in all directions surrounding the point of impingement. This initiates bedload transport in a radial pattern. These turbulent structures are primarily associated with quadrant 1 and 4 turbulent structures (i.e. instantaneous fluid fluctuations in the streamwise direction that bring fluid down into the bed in the case of quadrant 1 events, or up away from the bed in the case of quadrant 4 events) and generate a distinct pattern of bedload transport compared to transport dynamics distal to flow reattachment. Distal to flow reattachment, bedload transport is characterized by relatively unidirectional transport. The dynamics of splat events, specifically their potential for inducing significant magnitudes of cross-stream transport, has important implications for the evolution of bedforms from simple, two dimensional features to complex, three-dimensional features. New advancements in sonar technology have enabled more detailed quantification of bedload transport on the reach scale, a process paramount to the effective management of rivers with sand or gravel-dominated bed material. However, a practical and scalable field methodology for reliably estimating bedload remains elusive. A popular approach involves calculating transport from the geometry and celerity of migrating bedforms, extracted from time-series of bed elevation profiles (BEPs) acquired using echosounders. Using two sets of repeat multibeam sonar surveys from the Diamond Creek USGS gage station in Grand Canyon National Park with large spatio-temporal resolution and coverage, we compute bedload using three field techniques for acquiring BEPs: repeat multi-, single-, and multiple single-beam sonar. Significant differences in flux arise between repeat multibeam and single beam sonar. Mulitbeam and multiple single beam sonar systems can potentially yield comparable results, but the latter relies on knowledge of bedform geometries and flow that collectively inform optimal beam spacing and sampling rate. These results serve to guide design of optimal sampling, and for comparing transport estimates from different sonar configurations. / Dissertation/Thesis / Doctoral Dissertation Geological Sciences 2018

Sammanfattning: De ökande testdatavolymerna som krävs för att testa moderna System-On-Chip (SOC) bidrar i hög grad till den ökande produktionskostnaden. De stora testdatavolymerna kräver stora och dyra Automatic Test Equipment-minnen (ATE-minnen). För att minska behovet av dessa minnen så har olika komprimeringsmetoder utvecklats. Denna rapport beskriver arbetet med att implementera en given komprimeringsmetod för testdata till SOC. Den metod som används heter vector repeat och den implementeras genom skapandet av ett för ändamålet avsett dataprogrogram. För att vector repeat ska fungera effektivt så förutsätts det att in- och utgångarna på den ATE som används kan delas in i olika portar. Portarna används för att korta ner testdatats vektorer, vilket möjliggör en bättre komprimering. Resultaten av implementationen har verifierats med hjälp av experiment där testdata från en benchmark SOC använts och jämförts med okomprimerat testdata samt ytterliggare en komprimeringsmetod som heter 9C. Resultaten visar att vector repeat är en effektiv komprimeringsmetod om antalet portar är tillräckligt stort. Experiment har även genomförts som visar hur mycket komprimeringen förbättras då antalet portar som används ökas. Dessa resultat kan användas i framtida arbeten, varav ett exempel är, där kostnaden för ökat antal portar tas i beaktande. / Abstract: The increased volume of test data which is required in testing of modern System-On-Chip (SOC) are a high contributor to the increased production costs. The large volumes of test data requires large and expensive Automatic Test Equipment memories. (ATE memories). To decrease the need of these memories, different compression methods have been developed. This report describes the work with implementing a given compression method for test data to SOC. The method which is used is called vector repeat and it is implemented through the creation of a, for the task designated, computer program. If vector repeat should work efficiently it is assumed that the entrances and the exits on the ATE can be divided into different ports. The ports are being used to shorten the vectors of the test data, which enables a better compression. The result of the implementation has been verified through experiments where the test data from a benchmark SOC is used and compared with uncompressed data and another compression method which is called 9C. The result shows that vector repeat is an effective compression method if the number of ports is large enough. Experiments has also been done which show how much the compression is improved when the number of ports in use are increasing. These results can be used in future works, where one example is, where the cost of the increasing number of ports is considered.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-04-08T12:53:20Z No. of bitstreams: 2 Dissertação Nathália Leite.pdf: 1163803 bytes, checksum: ed3fcb6e6b34f0c3e3452518a29c3263 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-08T12:53:20Z (GMT). No. of bitstreams: 2 Dissertação Nathália Leite.pdf: 1163803 bytes, checksum: ed3fcb6e6b34f0c3e3452518a29c3263 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012-02-29 / CNPq / O presente estudo foi realizado com os objetivos de identificar acessos de feijão-caupi tolerantes ao caruncho (C. maculatus) e caracterizar esses acessos a nível molecular, em conjunto a acessos de V. unguiculata ssp. cylindrica e V. radiata, por meio de marcadores DAF (DNA Amplification Fingerprinting) e ISSR (Inter Simple Sequence Repeat), visando identificar parentais promissores para futuros trabalhos de melhoramento, bem como para mapeamento, pela associação com o nível de resposta ao caruncho. Para identificação da tolerância ao caruncho em feijão-caupi, 27 acessos foram avaliados em ensaios de laboratório, utilizando-se o Delineamento Experimental Inteiramente Casualizado. Para cada acesso, uma amostra com 30 grãos de feijão-caupi foi infestada com cinco casais de caruncho com até 24 h de idade, sendo os resultados obtidos submetidos à análise estatística e suas médias comparadas pelo teste de Tukey a 5% de probabilidade. Para as duas variáveis estudadas, o acesso INHUMA comportou-se como suscetível, enquanto os acessos PATATIVA e MNC99-537F-4 apresentaram-se tolerantes para ambas as variáveis. Os demais acessos testados apresentaram comportamento intermediário, o que não os diferencia em relação à INHUMA, PATATIVA e MNC99-537F-4. Na caracterização molecular dos acessos, 25 primers geraram um total de 239 amplicons, dos quais 163 foram polimórficos. Os fragmentos amplificados foram incluídos em uma matriz de dados para análise pelo método de Neighbor-Joining. No fenograma obtido, V. radiata e V. unguiculata ssp. cylindrica assumiram uma posição basal isolada dos subgrupos formados, enquanto que os acessos de V. unguiculata subdividiram-se em dois grupos, nos quais os acessos contrastantes quanto à tolerância ao caruncho se distribuíram em subgrupos distintos, o que evidencia a existência de diferenças genéticas significativas entre alguns candidatos. Nesse sentido, os resultados obtidos demonstraram que os ensaios de tolerância ao caruncho associados à aplicação de marcadores moleculares foram capazes de identificar genótipos contrastantes de feijão-caupi promissores para aplicação em trabalhos de melhoramento vegetal, bem como para a geração de populações segregantes adequadas ao mapeamento genético.

Au cours de notre programme de thèse, nous avons isolé et caractérisé des molécules protéiques à activité antivirale intracellulaire dirigée contre le VIH-1. Ces protéines, appelées aRep, ont été obtenues par criblage d'une banque de protéines artificielles de nouvelle génération, construites de façon combinatoire à partir de protéines naturelles constituées de motifs alpha-hélicoidaux répétés. La cible virale, ou "appât", utilisé pour ce criblage a été une région de la polyprotéine Gag du VIH-1 identifiée comme une cible privilégiée de nouvelles thérapeutiques antivirales, car essentielle à l'assemblage viral, l'empaquetage du génome viral et le clivage de maturation de Gag aboutissant à la formation de virions infectieux. Deux molécules d'aRep à affinité élevée pour la cible virale, l'aRep4E3 (32 kDa; 7 motifs répétés) et l'aRep9A8 (28 kDa; 6 motifs répétés) ont ainsi été identifiées, isolées et clonées. L'étude de l'activité anti-VIH intracellulaire de ces aRep a été réalisée dans différents systèmes d'expression cellulaire, nécessitant la construction de lignées stables de cellules d'insecte et de cellules épithéliales humaines, et leur infection par différents types de vecteurs viraux recombinants, baculovirus ou lentivirus, porteurs du gène rapporteur luciférase. Mais surtout, cette étude a été menée sur des cellules lymphocytaires-T (SupT1), cibles naturelles du virus, exprimant ces aRep et infectées par du VIH-1 naturel infectieux. Nos résultats ont montré que l'aRep4E3 et l'aRep9A8 ont toutes deux un effet négatif significatif sur le cycle réplicatif du VIH-1, mais ciblent des fonctions virales différentes. L'aRep4E3 bloque l'empaquetage du génome viral, tandis que l'aRep9A8 inhibe la maturation et diminue l'infectivité virale. De plus, l'aRep9A8, exprimée de façon constitutive dans les cellules SupT1, leur confère une résistance au VIH: une lignée de SupT1 chroniquement infectée par le VIH-1 a pu être ainsi isolée et maintenue en culture pendant plusieurs semaines, sans effet cytopathique viro-induit apparent. Ces nouvelles données auront des implications non-négligeables dans le choix et la conduite de futures stratégies de thérapie cellulaire anti-VIH / HIV-1 infection is a long-term disease which required a long-life treatment. Besides the standard HAART regiment, HIV gene therapy is a promising alternative strategy which give rise to hope for the better HIV-1 treatment. Protein therapeutics is one another technique that represent high impact results in curing various types of disease. It is already become a significant part of current medical treatments. In this study we first designed aRep, a non-immunoglobulin scaffold protein which target two domains of HIV-1 Pr55Gag, SP1-NC and investigated their roles as an intracellular therapeutic agents. Phage display technology was used for the specific isolation of aRep against a critical C-terminal region of the HIV-1 Pr55Gag precursor from a large and diverse library. The antiviral activity of these two Pr55Gag binders was investigated using different cell systems. Two aRep scaffolds aRep4E3 and aRep9A8 were isolated and characterized. aRep4E3 contains 7 repeat motifs (32 kDa), meanwhile aRep9A8 has 6 repeat motifs (28 kDa). These two scaffold molecules found to be able to display antiviral effects on the late stage of HIV-1 replication, by reducing and delaying the viral progeny production. The difference in the molecular mechanism was observed between these two aRep proteins: aRep4E3 mainly interferes with the packaging of the viral genome, meanwhile aRep9A8 interferes with the proteolytic processing of Gag, and performs as a protease inhibitor to prevent the PR cleavage required for the production of newly infectious mature virus. Interestingly, aRep9A8 is able to survive in the chronical HIV-1 infected cells up to D38 pi with the low level of HIV-1 replication. Taken together, results suggested that aRep, a new type of scaffold protein could serve as a promising alternative agent in protein therapy, not only the HIV-1 infection but also the others pathogens or disorders

Magister Scientiae - MSc / The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex. / South Africa

Magister Artium - MA / Globally, teenage pregnancy remains a disturbing phenomenon which impacts on the lives of teenagers, their families and society as a whole. Numerous attempts at addressing the problem have seen a decline in fertility rates but agreement still exists that the incidence of young girls bearing children is unacceptably high. Studies conducted over the years have emphasised both the causes and consequences of teenage births. Many studies too have explored the benefits of preventative strategies. Yet, despite all this, teenage pregnancy remains a cause for concern with many teenage girls remaining sexually active after a first pregnancy, and exposing themselves to subsequent pregnancies and births. This study was focused on teenage girls who had experienced unintended repeat pregnancies. Through the research a deeper understanding of the meanings that female teenagers ascribe to repeat pregnancies, were sought. A sample group of teenage mothers were allowed to take a step back from their experience of the repeat pregnancy; to think deeply about the experience, and to reflect on what they had learnt and how it has impacted on their current lives. The researcher employed a qualitative approach with a descriptive, explorative design in order to obtain a rich description of the experiences of teenage mothers who have been through a repeat pregnancy. The goal of the study was to explore and describe the reflections of these teenage mothers who had experienced unintended, repeat pregnancies. Data was obtained through semi-structured individual interviews where an interview guide was used. The data was analysed according to the steps outlined by Creswell (2009). Findings were noted and recommendations made. These recommendations are designed for role-players involved with teenagers and youth in general. Emphasis was placed on recommendations to professionals, like educators, healthcare workers and social workers who are at the coalface of dealing with teenagers who engage in sexual activity. Finally, recommendations for further research were made.