Bacteria-based cancer therapy (BBCT) holds immense promise in addressing the limitations in treatment of solid tumors. Bacterial strains used for BBCT are engineered to express therapeutics, facilitate precise navigation within the tumor microenvironment by enhancing bacteria's motility, chemotaxis (movement toward or away from specific chemicals), or other mechanisms that aid in reaching and infiltrating the tumor tissue effectively, and complementing traditional chemotherapy and immunotherapies while minimizing side effects. Bacterial motility not only influences the ability of bacteria to navigate within the tumor but also plays a pivotal role in optimizing drug delivery, treatment efficacy, and minimizing potential obstacles associated with the complex microenvironment of human tissues. However, the current understanding of bacterial motility remains limited. In this thesis, we use a reductionist approach and study bacteria motile behavior within human tissue phantoms (collagen and agar) and the bacteria-hydrogel interactions. Apart from motility, it is important to analyze the mechanical properties of the hydrogels the bacteria interact with as they play a vital role in overall behavior and physics of bacteria movement. To that extent, there exists a gap in our understanding of the viscoelastic properties of hydrogels. Lastly, systematic and comprehensive investigation of bacteria behavior in hydrogels requires tracking of thousands of individual cells. Thus, there is an unmet need to develop new automated techniques to reduce the labor-intensive manual tracking of bacteria in low-contrast hydrogel environments, with feature sizes comparable to that of bacteria. To address these gaps, this thesis proposes a trident approach towards mechanistic understanding of bacteria motility in time-invariant agar and temporally evolving collagen hydrogels to bridge critical gaps in understanding bacterial motile behavior in these media, non-destructive microelastography-based mechanical characterization of hydrogels with less than 4.7% error compared with rheology, and the development of deep learning-enabled automated bacteria tracking tools with 77% precision. / Master of Science / There exists a huge scope for improvement in cancer therapies. The gold standard chemotherapy and immunotherapies are responsible for a lot of side effects. Over a century ago, bacteria-based cancer therapy started to develop and over the period, it was discovered that they can be effective when used with traditional therapies improving precision and reducing side effects. The motility of bacteria is shown to improve bacterial distribution in solid tumors. However, the physical underpinnings of bacteria motility in the tumor environment remains understudied. This thesis proposes a trident approach, investigating bacteria motility in tissue-like environments (hydrogels), characterizing the mechanical properties of hydrogels using acoustic waves to capture bacteria-induced alterations, and developing deep-learning-enabled automated bacterial tracking approach for high throughput analysis of experimental data. We report bacteria behavior and motility patterns in hydrogels, the mechanics of these hydrogels with less than 4.7% error compared with standard characterization methods, and automated bacteria tracking with 77% precision to inform the development and advancement of bacteria-based drug delivery systems. In summary, these tools can help improve our understanding of bacteria-hydrogel interactions, allowing us to develop innovative bacteria-based cancer therapies in the long term.

Cytopathology plays a significant role in cancer diagnostics. One of the ancillary methods used for prognostication is immunocytochemistry (ICC). Formalin pre-fixed tissue blocks are often used as validated immunohistochemical controls for cytology samples. However, those are not treated as clinical cytology specimen, which are furthermore commonly fixed in alcohol. As cell structure might be affected by added fixatives and block materials, the aim of this project was to compare the effects of fixation in formalin and PreservCyt®, a methanol-based fixative, and the two cell block materials, plasma-thrombin and agar, on the ICC results. The material used in the study consisted of several cell lines provided by the Technological University Dublin, which were chosen to be representative of the clinical cytology samples. The cells were fixed and put into cell blocks for each cell type or as a mixture of different cell types – a composite cell block. All cell blocks were then stained with suitable antibodies. Composite cell blocks could be tested as simultaneously positive and negative ICC control material. The results showed that in general, PreservCyt®-fixed cell blocks preserved sharper cellular morphology compared to those fixed in formalin. However, PreservCyt® also gave unexpected negative results for some antibodies, including some nuclear markers. Plasma-thrombin cell block method showed to have more advantages than the agar method.This study suggests, that with further optimization and validation of both fixative methods and cell blocks, composite cell block has good potential to be used as source of control blocks for cytology samples.

No / The isotopic effect of exchanging deuterium with hydrogen on the mechanical and surface properties of agar gel is examined. The elastic modulus of the D2O gels obtained by AFM nanoindentation is significantly higher (factor of 1.5¿2) than the modulus found in H2O agar gels. Furthermore, the modulus is independent of loading rate. Surface imaging reveals that the surface roughness gets progressively smaller with increasing agar concentration. All these data suggest that the isotopic replacement of deuterium enhances the mechanical properties of the agar gel, with significant advantages in its use as a biphasic scaffold. / MRC

Tesis por compendio / [ES] Esta tesis doctoral se centró en la valorización de diferentes fuentes de biomasa marina para desarrollar materiales biopoliméricos e ingredientes destinados a la industria alimentaria. Así pues, en la primera parte de la tesis, se llevó a cabo la valorización de la biomasa de diversas especies de algas con el objetivo de identificar compuestos de interés y evaluar su potencial para la producción de materiales biopoliméricos e ingredientes alimentarios ricos en proteínas. Entre estas especies se encontraba R. okamurae, un alga parda invasora que representa una amenaza para los ecosistemas marinos. No obstante, se demostró que esta especie posee un gran potencial como fuente sostenible de compuestos bioactivos, transformando su papel de amenaza en oportunidad. La biomasa de las algas rojas también destacó por su potencial en diversas aplicaciones, incluyendo el desarrollo de materiales rentables y respetuosos con el medio ambiente obtenidos mediante de un procesado mínimo. En este contexto, la composición y la estructura de las paredes celulares de las algas desempeñaron un papel fundamental para entender cómo se comporta cada especie durante el procesado y en las propiedades de las películas resultantes. Además de la producción de materiales, también se estudiaron las algas rojas como fuente de proteínas para el consumo humano. Aunque las algas nativas presentaron una baja digestibilidad, esta mejoró mediante su procesado. No obstante, a pesar de esta mejora en la digestibilidad, el procesado podía inducir la degradación de los aminoácidos más lábiles, lo que repercutía negativamente en la calidad nutricional de las proteínas. En la segunda parte de esta tesis, se exploraron varias especies de algas rojas para extraer agar de forma más eficiente mediante métodos simplificados, y se evaluó la idoneidad de los extractos obtenidos para desarrollar materiales biopoliméricos e hidrogeles para aplicaciones alimentarias. El grado de purificación del agar tuvo un impacto significativo en las propiedades finales tanto de las películas como de los hidrogeles. En el caso de las películas, las fabricadas con agar puro presentaron propiedades superiores; sin embargo, sufrieron cambios significativos en su estructura semicristalina durante el almacenamiento. Por el contrario, las películas derivadas de extractos de agar menos purificados mostraron una mayor estabilidad en el tiempo, sugiriendo su potencial como aditivo para reducir costes y mejorar la estabilidad de las películas de agar puro. En el caso de los hidrogeles, la purificación del agar condujo a la formación de hidrogeles con una resistencia y dureza más altas. Por otro lado, el protocolo de extracción simplificado produjo fracciones de agar menos purificadas que contenían compuestos adicionales como proteínas o polifenoles. Aunque esto dio lugar a hidrogeles menos rígidos y resistentes, podrían ser de interés para la producción de hidrogeles con propiedades bioactivas. En la tercera y última parte de la tesis, se abordó la valorización de los residuos industriales generados tras la extracción de alginato, con el propósito de obtener fracciones de celulosa mediante métodos simplificados para su aplicación en la producción de materiales biopoliméricos. En este contexto, se observó que los residuos de A. esculenta y S. latissima resultaron adecuados para la extracción de fracciones celulósicas, mientras que A. nodosum podría ser más interesante para la producción de extractos ricos en fucoidano. Las fracciones de celulosa con mayor pureza generaron películas con propiedades mecánicas y aspecto visual más deseables. En cambio, las fracciones menos purificadas presentaron una mayor barrera al vapor de agua. De acuerdo con los resultados, se determinó que la simple aplicación de un tratamiento alcalino puede producir fracciones de celulosa que dan lugar a películas con un equilibrio óptimo entre propiedades funcionales y eficiencia económica y medioambiental. / [CA] Aquesta tesi doctoral es va centrar en la valorització de diferents fonts de biomassa marina para desenvolupar materials biopolimèrics i ingredients destinats a la indústria alimentària. Així doncs, en la primera part de la tesi, es va dur a terme la valorització de la biomassa de diverses espècies d'algues amb l'objectiu d'identificar compostos d'interés i avaluar el seu potencial per a la producció de materials biopolimèrics i ingredients alimentaris rics en proteïnes. Entre aquestes espècies es trobava R. okamurae, una alga marró invasora que representa una amenaça per als ecosistemes marins. No obstant això, es va demostrar que aquesta espècie posseeix un gran potencial com a font sostenible de compostos bioactius, transformant el seu paper d'amenaça en oportunitat. La biomassa de les algues roges també va destacar pel seu potencial en diverses aplicacions, incloent-hi el desenvolupament de materials rendibles i respectuosos amb el medi ambient obtinguts mitjançant d'un processament mínim. En aquest context, la composició i l'estructura de les parets cel·lulars de les algues van exercir un paper fonamental per a entendre el comportament cada espècie durant el processament i en les propietats de les pel·lícules resultants. A més, també es van estudiar les algues roges com a font de proteïnes per al consum humà. Encara que les algues natives van presentar una baixa digestibilitat, aquesta va millorar mitjançant el seu processament. No obstant això, malgrat aquesta millora en la digestibilitat, el processament podia induir la degradació dels aminoàcids més làbils, la qual cosa repercutia negativament en la qualitat nutricional de les proteïnes. En la segona part d'aquesta tesi, es van explorar diverses espècies d'algues roges per a extraure agar de forma més eficient mitjançant mètodes simplificats, i es va avaluar la idoneïtat dels extractes obtinguts per a desenvolupar materials biopolimèrics i hidrogels per a aplicacions alimentàries. El grau de purificació de l'agar va tindre un impacte significatiu en les propietats finals tant de les pel·lícules com dels hidrogels. En el cas de les pel·lícules, les fabricades amb agar pur van presentar propietats superiors; no obstant això, van patir canvis significatius en la seua estructura semicristal·lina durant l'emmagatzematge. Per contra, les pel·lícules derivades d'extractes d'agar menys purificats van mostrar una major estabilitat en el temps, suggerint el seu potencial com a additiu per a reduir costos i millorar l'estabilitat de les pel·lícules d'agar pur. En el cas dels hidrogels, la purificació de l'agar va conduir a la formació d'hidrogels amb una resistència i duresa més altes. D'altra banda, el protocol d'extracció simplificat va produir fraccions d'agar menys purificades que contenien compostos addicionals com a proteïnes o polifenols. Encara que això va donar lloc a hidrogels menys rígids i resistents, podrien ser d'interés per a la producció d'hidrogels amb propietats bioactius. En la tercera i última part de la tesi, es va abordar la valorització dels residus industrials generats després de l'extracció d'alginat, amb el propòsit d'obtindre fraccions de cel·lulosa mitjançant mètodes simplificats per a la seua aplicació en la producció de materials biopolimèrics. En aquest context, es va observar que els residus de A. esculenta i S. latissima van resultar adequats per a l'extracció de fraccions cel·lulòsiques, mentre que A. nodosum podria ser més interessant per a la producció d'extractes rics en fucoidans. Les fraccions de cel·lulosa amb major puresa van generar pel·lícules amb propietats mecàniques i aspecte visual més desitjables. En canvi , les fraccions menys purificades van presentar una major barrera al vapor d'aigua. D'acord amb els resultats, es va determinar que la simple aplicació d'un tractament alcalí pot produir fraccions de cel·lulosa que donen lloc a pel·lícules amb un equilibri òptim entre propietats funcionals i eficiència econòmica i mediambiental. / [EN] This doctoral thesis focused on the valorization of different sources of marine biomass with the main purpose of developing biopolymeric materials and ingredients for the food industry. Thus, in the first part of the thesis, the valorization of biomass from different algae species was carried out to identify compounds of interest and assess their potential to produce biopolymeric materials and protein-rich food ingredients. Among these species was Ruguloperyx okamurae, an invasive brown seaweed that poses a threat to marine ecosystems. However, this species was shown to have significant potential as a sustainable source of bioactive compounds, transforming its role as a threat into a valuable opportunity. Red seaweed biomass was also highlighted for its potential in a variety of applications, including the development of cost-effective and environmentally friendly materials for food applications obtained through minimal processing. In this context, the composition and structure of the algal cell walls played a crucial role in understanding how each species behaves during processing and in the properties of the resulting films. In addition to the production of biopolymeric materials, red seaweeds were also studied as a source of proteins for human consumption. Although native seaweeds had low digestibility, this was improved through processing. However, it is important to note that, despite the improvement in digestibility, processing could induce the degradation of more labile amino acids, having a negative impact on the nutritional quality of the proteins. In the second part of this thesis, several species of red seaweeds were explored to extract agar more efficiently by simplified methods, and the suitability of the extracts obtained to develop biopolymeric materials and hydrogels for food applications was evaluated. It could be observed that the degree of agar purification had a significant impact on the final properties of both films and hydrogels. In the case of films, those made of pure agar exhibited superior properties; however, they underwent significant changes in their semicrystalline structure during storage. In contrast, those films derived from less purified agar extracts showed greater stability over time, suggesting the potential of less purified agars as additives to reduce costs and improve the stability of pure agar films. Regarding the hydrogels, agar purification led to the formation of hydrogels with significantly higher strength and hardness. On the other hand, the simplified extraction protocol produced less purified agar fractions containing other compounds such as proteins, polyphenols, and minerals. Although this resulted in less rigid and resistant hydrogels, this feature could be advantageous for the production of hydrogels with bioactive properties. In the third and last part of the thesis, the valorization of industrial wastes generated after alginate extraction was addressed, with the purpose of obtaining cellulosic fractions by simplified methods for their application in the production of biopolymeric materials. In this context, it was observed that the residues of Alaria esculenta and Saccharina latissima were suitable for the extraction of cellulosic fractions, while Ascophyllum nodosum might be more interesting for the production of fucoidan-rich extracts. Cellulosic fractions with higher purity produced films with more desirable mechanical properties and visual appearance. Conversely, less purified fractions presented a greater barrier to water vapor. According to the results, it was determined that the simple application of an alkaline treatment could produce cellulosic fractions that resulted in films with an optimal balance between functional properties and economic and environmental efficiency in biopolymeric material production. / This work was financially supported by the “Agencia Estatal de Investigación” (PCI2018-092886 Grant) and co-funded by the European Union’s Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2) and by the grant TED2021-129711B-I00, funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. This work was done within the framework of a collaboration with HISPANAGAR S.L (Burgos, Spain). The research was funded by the Spanish Ministry of Science, Innovation and Universities through projects PID2020-114821RB-I00, PID2020-117744RJ-I00, as well as CEX2021-001189-S, funded by MCIN/AEI/10.13039/501100011033. The authors also acknowledge funding through project CIRCALGAE (Horizon Europe) under grant agreement 101060607. In all projects, funding from “ERDF A way of making Europe”, the “European Union” or the “European Union NextGenerationEU/PRTR” is also acknowledged. / Cebrián Lloret, V. (2024). Valorization of Marine Biomass for the Development of Biopolymeric Materials and Additives with Functional Properties [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202961 / Compendio

Master of Science / Food Science Institute- Animal Sciences and Industry / Kelly J. K. Getty / The Food and Drug Administration’s Bacteriological Analytical Manual recommends two enumeration methods for Bacillus spp.: 1) standard plating method using mannitol-egg yolk-polymyxin (MYP) agar and 2) most probable number (MPN) method with tryptic soy broth supplemented with 0.1% polymyxin sulfate. Preliminary research evaluated three inoculum preparation methods using EZ-Spore™ B. cereus pellets. Two methods involved EZ-Spore™ B. cereus pellets that were dissolved in deionized (DI) water, grown in brain heart infusion broth with manganese sulfate, and then heated to produce spores. The third inoculum preparation method of dissolving EZ-Spore™ pellets only in DI water was the most efficient due to 100% spores being present in the inoculum. Preliminary research also determined that MPN method recovered greater (p<0.05) B. cereus populations than MYP method in inoculated ultra-high temperature pasteurized skim and 2% milk. The objective of the main study was to compare the MYP and MPN method for detection and enumeration of B. cereus in raw and high-temperature-short-time pasteurized skim, 2%, and whole milk at 4 °C for 96 h. Milk samples were inoculated with B. cereus EZ-Spores™ dissolved in DI water and sampled at 0, 48, and 96 h after inoculation. No differences (p>0.05) were observed among sampling times so data was pooled for overall mean values for each treatment. The overall B. cereus population mean of pooled sampling times for MPN method (2.59 log CFU/mL) was greater (p<0.05) than MYP plating method (1.89 log CFU/mL). B. cereus populations ranged from 3.40 log CFU/mL to 2.40 log CFU/mL for inoculated milk treatments for MYP and MPN methods, which is well below the necessary level for toxin production. Even though MPN method enumerated more B. cereus, the MYP method should be used by industry for enumeration of B. cereus due to its ease of use and rapid turnover time (2 d compared to 5 d with MPN). However, MPN method should be used for validation research due to its greater populations recovered. EZ-Spore™ B. cereus pellets were found to be an acceptable spore inoculum for validation research because the inoculum consists of 100% spores and does not contain vegetative cells.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-27T17:03:09Z No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-27T17:03:21Z (GMT) No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-27T17:03:21Z (GMT). No. of bitstreams: 2 Dissertação - Glennyha Fernandes Duarte - 2014.pdf: 1764909 bytes, checksum: fa26338a2793bde550bdf59fd0986bc1 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-07-17 / Biomphalaria glabrata snails are aquatic and inhabit often ephemeral basins. They have high reproductive capacity, and egg masses are laid on submerged substrates on the banks of breeding sites exposed to desiccation and pathogens like fungi. Virtually nothing is known about the viability of egg masses in breeding and transient sites and their pathogens. This information is important to control this intermediate host of Schistosoma mansoni, which causes schistosomiasis. This study describes the feasibility of egg masses in water films and susceptibility infection with to Beauveria bassiana and Metarhizium ansiopliae. Egg masses were exposed to water film formed in two different devices, one with a stone clay in a cup with water and another one with water agar at different concentrations of agar (0.3% - 3%) in Petri dishes in different relative humidity (RH > 98% and 75%). Conidia or blastospores of B. bassiana (ARSEF 9588) or M. anisopliae (IP 46) (2 x 107 conidia or blastospores/egg mass) were inoculated in egg masses exposed in the water film in stone clay or submerged (2 x 107 conidia/ml or blastospores/ml). Egg masses exposed in the water film in stone clay or in water agar (agar < 1%) at RH > 98% were viable. The eclosion and the displacement of juveniles in the films were observed regardless of the tested device (RH > 98%, agar < 1%), except at 75% RH and glass without water film. Mycelium and conidia developed on egg masses treated with conidia or blastospores of M. anisopliae in the water film in stone clay (RH > 98%), and there was no eclosion of juveniles from egg masses treated with blastospores of M. anisopliae. In egg masses submerged treated with blastospores of M. anisopliae or B. bassiana no eclosion of juveniles and egg masses treated with conidia accumulated average onset was < 52.5%. The feasibility of egg masses and the displacement of juveniles in both devices on RH > 98% was due to the formation of stable water film. Blastospores may have released toxic metabolites that inhibited the eclosion of juvenile B. glabrata. Both devices are promising for testing egg masses in the water film. The feasibility of egg masses in water under laboratory conditions film should be considered to control B. glabrata. Blastospores of M. anisopliae and B. bassiana are promising against B. glabrata. / Biomphalaria glabrata são caramujos aquáticos e habitam coleções hídricas frequentemente efêmeras. Possuem alta capacidade reprodutiva, e massas ovígeras são postas em substratos submersos nas margens dos criadouros expostas ao ressecamento e a patógenos como fungos. Praticamente nada é sabido sobre a viabilidade de massas ovígeras em criadouros transitórios e a sua susceptibilidade a fungos. Estas informações são importantes para o controle desse caramujo hospedeiro intermediário de Schistosoma mansoni, causador de esquistossomose. O presente trabalho relata a viabilidade de massas ovígeras em filme de água e a susceptibilidade a Metarhizium ansiopliae e Beauveria bassiana. Massas ovígeras foram expostas em filme de água formado em dois dispositivos diferentes confeccionados, um com pedra de argila em copo com água e outro com meio ágar água com diferentes concentrações de ágar (0,3% - 3%) em placas de Petri em diferentes umidades relativas (UR > 98% e 75%). Conídios ou blastosporos de B. bassiana (ARSEF 9588) ou M. anisopliae (IP 46) (2 x 107 conídios ou blastosporos/massa ovígera) foram inoculados em massas ovígeras expostas em filme de água em pedra de argila ou submersas (2 x 107 conídios/ml ou blastosporos/ml). As massas ovígeras expostas em filme de água em pedra de argila ou em meio ágar água (ágar < 1%) em UR > 98% foram viáveis. A eclosão e o deslocamento de juvenis nos filmes foram observados independentemente do dispositivo testado (UR > 98%, ágar < 1%), exceto em UR 75% e em vidro sem filme de água. Micélio e conídios desenvolveram sobre massas ovígeras tratadas com conídios ou blastosporos de M. anisopliae em filme de água em pedra de argila (UR > 98%), e não houve eclosão de juvenis a partir de massas ovígeras tratadas com blastosporos de M. anisopliae. Em massas ovígeras submersas tratadas com blastosporos de M. anisopliae ou B. bassiana não houve eclosão de juvenis e em massas ovígeras tratadas com conídios a eclosão média acumulada foi < 52,5%. A viabilidade de massas ovígeras e o deslocamento de juvenis em ambos os dispositivos em UR > 98% foi devido à formação de filme de água estável. Blastosporos podem ter liberado metabólitos tóxicos que inibiram a eclosão de juvenis de B. glabrata. Ambos os dispositivos são promissores para testes com massas ovígeras em filme de água. A viabilidade de massas ovígeras em filme de água em condições laboratoriais deve ser considerada no controle de B. glabrata. Blastosporos de M. anisopliae e B. bassiana são promissores contra B. glabrata.

Nas últimas décadas as espécies de Campylobacter spp. são reconhecidas como importantes agentes de gastroenterites de origem alimentar em humanos em diversos países, tendo como principal veículo de transmissão a carne de frango. Devido ao aumento da frequência com que é isolada a partir de humanos, animais, alimentos e água, esta bactéria tem sido foco crescente de atenção nos últimos 30 anos. No Brasil, ainda são limitadas as informações sobre esta bactéria na cadeia de produção de aves, não existindo legislação que contemple o controle de Campylobacter. A alta incidência na avicultura, a presença natural deste patógeno nos animais e os graves problemas de saúde pública gerados tornam essa bactéria alvo de esforços para prevenção e controle. O objetivo do presente estudo foi avaliar se a metodologia de contagem direta proposta pela normativa MLG 41.02 teria eficiência na monitoria em diferentes amostras avícolas e paralelamente comparar o desempenho de dois ágares (mCCDA e Campy-Cefex) na contagem de Campylobacter spp. Foram realizadas quatro tomadas de amostras em um frigorífico da região Sul do país, durante um mês, sendo a amostragem composta por suabes de cloaca, carcaças pré-chiller, carcaças pós-chiller, água pré-chiller, água do chiller, e amostras de água de abastecimento. O ágar Campy-Cefex obteve maior frequência de isolamento de Campylobacter spp. em diferentes amostras avícolas quando comparado com o ágar mCCDA. Houve redução significativa de contaminação ao longo da linha de abate, com níveis de 9,8 x 102 UFC/mL em carcaças préchiller e 1,5x102 UFC/mL em carcaças pós-chiller. Do total de amostras em que foram realizadas a PCR, 72% foram positivas para Campylobacter jejuni e 38% positivas para Campylobacter coli. A metodologia mostrou-se eficiente e possível de ser aplicada na indústria avícola, em diferentes materiais, para monitoria de Campylobacter. / In recent decades the Campylobacter spp. species are recognized as important agents of foodborne gastroenteritis in humans in several countries and the broiler meat as are pointed as the main vehicle of transmission. Due the increase in frequency isolation from humans, animals, food and water, this bacteria has received great attention in the last 30 years. The information about this bacteria are still limited in Brazil and in the poultry production chain, there are no laws to the Campylobacter’s control. The high incidence in poultry industry, the natural presence of this pathogen in animals and the serious concern in public health, lead this bacteria as target to prevention and control efforts. The aim of this study was to evaluate whether the direct counting methodology proposed by MLG 41.02 rules would be efficient on monitoring different poultry samples, as well as to compare two agars plate (mCCDA and Campy-Cefex) performance’s to Campylobacter cell count. We carried out four samples taken in a slaughterhouse located at southern region of Brazil, during one month. The samples were composed by cloacal swabs, pre-chiller carcasses, post-chiller carcasses, pre-chiller water, chiller water, and water supply samples. The Campy-Cefex agar showed higher Campylobacter spp isolation frequency of among different poultry samples than mCCDA agar. There was a significant reduction in contamination along the slaughter line with levels of 9.8 x 102 CFU/mL in pre- chiller carcasses and 1.5 x102 CFU/mL in post-chiller carcasses. The samples typified by PCR, showed 72% of the samples as Campylobacter jejuni and 38% as Campylobacter coli. The methodology was efficient and also possible to be used in the poultry’s industry for different samples to Campylobacter monitoring program.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

Invasive growth through a substrate requires a massive amount of penetrative force, and this is generated in the space of a few microns in a growing tip. This process is known to be critical in the root hair, pollen tube, rhizoids, and the topic of this thesis, hyphal growth. However defining the mechanisms underlying the tip growth remains a contentious issue. Shortcomings in control of direction and regulation of growth began to undermine early turgor-based theories, and the cytoskeletal protein actin, ubiquitous in nature and with crucial roles in structure and motility became a target for investigation. A major breakthrough came with the discovery that a characteristic actin depleted zone (ADZ) occurs at the growing tip of hyphae during invasive but not non-invasive hyphal growth. The ADZ is likely to have an important role in generating the greater protrusive force required for invasive growth. However, since its discovery, little has been determined about the characteristics of the ADZ. Uncertainty in the description of the physical environment the hyphae face adds a layer of complexity to interpretation of results. This thesis aims to address this issue, studying the impact of increasing agarose substrate concentration on the presence and dimensions of the ADZ in the oomycete A. bisexualis. Furthermore, agarose is examined by compression and imaging to compare the physical characteristics of the agar samples over the range of concentrations, and determine whether increasing agarose concentration influences agarose gel structure. Results suggest a difference in the number of ADZ observed in non-invasive compared with invasive samples, however no significant differences in the number or dimensions of ADZ were found amongst the 1-4% w/v agarose concentrations. The 0% sample showed 20.7 percent of hyphae exhibited depleted zones, while 1, 2, 3 and 4% samples showed 56.9%, 48.8%, 40.9% and 54.2% respectively. ADZ dimensions did not correlate with agarose concentration. The average ADZ area:hyphal diameter ratio was 0.634, 0.526, 0.430, 1.09, and 0.65 for 0-4% agarose concentrations respectively. Additionally, investigation of gel compression forces revealed gel strength increases with agarose concentration. The force required to compress the agarose increased from 1.85 Psi in 1% agarose to 4.85, 7.09 and 12.22 Psi in 2, 3 and 4% agarose concentrations respectively. SEM imaging, however, suggests heterogeneity of the fibrous interconnected network of agarose gels at a microscopic scale with variable porous structure at all agarose concentrations. This scale is relevant to hyphal tip growth. In combination, these results suggest F-actin depletion may be a response mechanism to provide greater force for invasive growth. Additionally, this response is not dependent on the concentration of the agarose media, possibly due to the variability encountered within the media. These results contribute another important step forward in unraveling the elusive mechanism of tip growth.

Dissertation (Ph.D.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 139 pages. Includes vita. Includes bibliographical references.