This project introduces a novel ultrasonic shearing-based fabrication method for synthesizing nanobubbles, which can then be utilized as a platform for any theranostic applications in clinical medicine, such as drug/gene delivery systems. Our standard in situ sonochemical synthesis of nanobubbles incorporates a perfluorocarbon gas core (300 μl) and an albumin outer shell, which are then incorporated into phosphate-buffered saline (4 ml) and later sonicated with a US probe. The initial optimization phase consisted of experimenting with various amounts of human serum albumin (HSA), which stabilizes the nanobubble gas core. Of the parameters (20 mg, 40 mg, and 80 mg HSA), 40 mg HSA significantly outperformed (pin vivo imaging nanobubbles in the liver. A continuation of this project will continue to optimize and expand on the theranostic applications of the US sheared nanobubbles in vivo and ex vivo in osteoporosis and the bone.

Hypertension is the chronic elevation in blood pressure that is regulated in part through the retention and regulation of sodium retention and excretion in the kidneys. Hence the kidney has been considered the organ that regulates blood pressure. There are a cohort of patients that suffer with high blood pressure due to lack of 11β-hydroxysteroid dehydrogenase-type 2 (11β-HSD2) expression (which inactivates glucocorticoids (GCs), allowing selective activation of mineralocorticoid receptors (MR) by aldosterone) that results in hypertensive and increased salt appetite phenotypes - a condition known as syndrome of apparent mineralocorticoid excess (SAME). This disorder can be recapitulated in the mouse through the global deletion of 11β-HSD2, which results in over activation of the MR driving an elevation in blood pressure. However, the distinction between blood pressure elevation because of kidney dysfunction with loss of 11β-HSD2 or increased salt appetite due to loss of brain 11β-HSD2 expression is not clear from the global 11β-HSD2 knockout model. Salt appetite is regulated by regions of the brain out-with the blood-brain barrier, known as circumventricular organs. In the mouse, salt appetite is controlled by aldosterone-sensitive cells in the nucleus of the solitary tract (NTS) in the brain stem, where 11β-HSD2 is expressed to provide mineralocorticoid selectivity. However, in the fetal brain, 11β-HSD2 is widely expressed, protecting against adverse GC action that alters brain development and increases susceptibility to psychiatric disorders as adults. 11β-HSD2 deletion solely in the brain from embryonic day 12 resulting in GC fetal programming (HSD2BKO) causes effects on both behaviour and salt appetite. To determine the role of developmental versus adult expression of brain 11β- HSD2, mice with deletion of brain 11β-HSD2 from mid gestation (HSD2BKO) and mice with adult deletion of 11β-HSD2 in the NTS using lentivirus (HSD2.v- BKD) were compared. The phenotypes (salt appetite, blood pressure (BP), baroreceptor response (BRR) and cognition), can be categorised as either due to GC fetal programming (as indicated by HSD2BKO groups), or increased activation of MR in adult 11β-HSD2 expressing neurons (recapitulated in the HSD2.v-Cre groups). Salt appetite increased in both HSD2BKO and HSD2.v-BKD cohorts (mean percentage increase 65% n=8 and 46% n=6, compared to their respective controls), leading to an increased BP in both groups (+12% and +8%, respectively) as well as an impaired BRR, indicating all phenotypes are mediated by adult NTS neurons. However, spatial recognition memory (Object-in-Place task) is abolished in HSD2BKO mice, whereas, HSD2.v-BKD mice still retain short-term memory. Our data suggest that neural 11β-HSD2 protects against inappropriate activation of MR by corticosterone to regulate salt appetite and salt-induced rises in blood pressure. However, spatial recognition memory is not influenced by deletion of 11β-HSD2 in the adult brain, confirmation that this phenotype is underpinned by developmental programming by GCs, which is observed in the 11β-HSD2 brain KO. Salt appetite has been shown to be centrally regulated through the adult deletion of 11β-HSD2. From this, our data suggest that an increased salt appetite is due to adult loss of function of 11β-HSD2 rather than GC programming during development. Highlighting the NTS as a region for drug delivery to try and control salt appetite in salt sensitive individuals who struggle with administering a recommended change in diet. To develop this further, minimally invasive modes of delivery of viruses and drugs into the brain were investigated. In so doing, a non-invasive and reversible method to temporarily disrupt the blood brain barrier (BBB) was optimised. The technique required acoustic insonation of ultrasonic contrast agents (CAs) (gas microbubbles) adjacent to the BBB. These microbubbles (SonoVueTM, Bracco) were delivered via tail vein injection into the vasculature. To target the BBB, an ultrasonic transducer was suspended and focused through coupling gel onto the area of interest in the brain with skull the intact. The optimisation of this technique required determination of the focal position of the 3.5MHz transducer that was utilised, in addition to optimisation of the pulse length, pulse repetition frequency and power output of the ultrasound beam to enable the BBB to be disrupted. In addition, measurement of the attenuation of the ultrasound beam through ex vivo mouse skulls were measured. These results showed a 50% reduction in pressure amplitude from the baseline of 335.2mV (Baseline mean = 100% +/-SEM 0 n=3 (No skull), five regions across the skull averaged 47.79% +/-SEM 1.913 n=25 (using 5 different animals). In in vivo mice, after co-injection of the microbubbles with Evans Blue and insonation of the brain, disruption of the BBB was confirmed by the presence of Evans Blue dye in the brain, with no measurable damage occurring in the brain. This was confirmed by cell and nuclear morphology with no red blood cell extravasation into the surrounding tissue. The parameters used to open the BBB used a peak negative pressure of 2.1MPa (single pulse), transducer frequency 3.5MHz, 35,000 cycles over a 10ms burst at a pulse repetition frequency of 10Hz. The technique when applied in vivo in recovery animals is speculated to work by the focused ultrasound causing the microbubbles to oscillate within the vasculature adjacent to the BBB, resulting in high-shear stresses being generated on the tight junctions within the BBB. The resultant gaps in the BBB allow free circulating compounds (e.g. large dye molecules (Evans Blue - 960.8g/mol molecular weight) and adeno-associated-viruses (25nm with a packing capacity of 4.5kb) within the blood to pass into the brain, but there is no penetration of red blood cells (7μm). Longitudinal mouse experiments demonstrated that within 12-hours these gaps close with no long-term damage observed. Currently, utilising this technique, successful passage of an adeno-associated virus expressing GFP (as a marker) has been shown to pass into the brain (n=6 for each cohort including control) - indicating that the virus requires the ultrasound and microbubbles to facilitate its movement into the brain. Further technique optimisation is being explored looking at the role of CAs used in the opening and disruption of the BBB, comparing composition and size of the CAs. Microbubbles (2-3μm) and nanobubbles (200nm) were compared as well as lipid and non-ionic surfactant surface compositions, using volume of drug delivery and degree of disruption as outputs. Using this technique, the hydrophilic drug mimic calcein was delivered into the brain (n=5 non-ionic surfactant nanobubble, n=5 lipid nanobubble). Results have indicated that the delivery of calcein is most efficient when using non-ionic surfactant nanobubbles as opposed to lipid nanobubbles - with a greater volume of the drug being delivered into the cerebral tissue. Furthermore, the concentration and surface composition of the nanobubble have an effect as to the size and potential damage to the brain when opening the BBB. In conclusion, it has been shown that it is possible to non-invasively open the BBB and deliver viruses and dye into the brain. In addition, this thesis has investigated the use of nanobubbles as both facilitators to opening the BBB and delivery vectors for potentially therapeutic drugs. Finally, a non-invasive opening of the BBB has been achieved using focused ultrasound. Ultimately this non-invasive opening of the BBB can be used to achieve delivery of larger molecules (such as antibodies and viruses) into the brain to target treatments. Focused ultrasound brain targeting can be applied to the potential treatment of salt appetite regulation in the NTS. For the individuals who suffer from salt sensitive hypertension, the NTS can be targeted to reduce the drive to ingest high salt diets. Furthermore, the continuation of research into the central control of BP, salt appetite and baroreceptor reflex control can become better understood, using less invasive delivery techniques to the brain.

O presente trabalho teve como objetivo avaliar as interações entre bolhas com distintos tamanhos (diâmetros) e partículas minerais e o efeito de combinações destas bolhas na flotação de minérios, em escala de bancada. As bolhas empregadas nesses estudos foram: i) Nanobolhas – NBs, diâmetro médio de 150 – 200 nm; ii) Microbolhas – MBs, diâmetro médio de 70 μm; e iii) Macrobolhas – MaBs, diâmetro médio de 1 mm. As dispersões de NBs e MBs foram geradas a partir da despressurização de água saturada com ar e cavitação hidrodinâmica em válvula agulha. As pressões de saturação foram, respectivamente, 2,5 e 4,0 bar. Os estudos de interação bolhas-partícula mineral foram desenvolvidos utilizando um sistema fotográfico que permitiu avaliar a adesão de bolhas às partículas de quartzo (grão de alta pureza) e apatita em função do tipo de bolhas injetadas. Os resultados mostraram que a maior adesão de bolhas no grão de apatita ocorreu com a combinação de NBs + MBs + MaBs e, no caso do quartzo, uma expressiva concentração de MaBs aderidas foi obtida após “condicionamento” do mineral com NBs. As NBs ficaram confinadas às superfícies dos grãos minerais devido à dissipação da energia livre superficial dos sólidos em função de sua rugosidade e, desta forma, aumentaram o mecanismo de adesão de outras bolhas (MBs e/ou MaBs). O estudo de avaliação das associações de bolhas na flotação foi realizado com os sistemas minerais quartzo e minério fosfático. No caso do minério fosfático, constituído por 35% de partículas finas (< 37 μm), os estudos avaliaram a combinação de i) NBs + MaBs; ii) MBs + MaBs; e iii) NBs + MBs + MaBs, nos parâmetros cinéticos e de separação na flotação, em relação ao ensaio standard, que emprega o uso de apenas MaBs. Os efeitos foram avaliados em quatro situações que compreenderam variação de porcentagem de sólidos (28 e 35% em peso) e concentração de reagentes (1000 g.t-1 de coletor + 600 g.t-1 de depressor; e 500 g.t-1 de coletor + 300 g.t-1 de depressor; sendo que o coletor de apatita empregado foi óleo de soja saponificado com hidróxido de sódio e o depressor das partículas de ganga - magnetita, carbonatos, micas, quartzo, diopsídio e perovskita/anatásio - foi amido de milho gelatinizado com hidróxido de sódio). O emprego combinado de bolhas apresentou incrementos na recuperação de P2O5, com pequena queda no teor de P2O5 e elevada cinética, em relação aos ensaios STD em praticamente todas as condições estudadas, com valores variando entre 0,2 e 8.9%. Os incrementos quando a porcentagem de sólidos foi de 35% p/p (até 2,7%) foram consideravelmente menores que os obtidos com 28% de sólidos (até 8,9%). Em algumas condições, o uso concomitante de MBs + MaBs e NBs + MBs + MaBs foi capaz de superar, já na primeira coleta (1,5 min de ensaio), a recuperação total de fosfato obtida no ensaio standard. No caso do quartzo, os ensaios de microflotação (realizados em tubo de Hallimond modificado) com e sem a presença de NBs mostraram que a associação de NBs às MaBs aumentou a recuperação de quartzo (até 39%) em praticamente todas as condições estudadas, mostrando sua efetividade tanto com partículas finas quanto com as mais grossas. Já os ensaios realizados em célula mecânica com e sem adição de NBs, mostraram que o emprego de NBs foi responsável pelo aumento de 23 pontos percentuais na recuperação de quartzo. Ainda, a cinética de flotação aumentou significativamente e já no primeiro min do ensaio com NBs obteve-se cerca do dobro da recuperação de quartzo do ensaio standard. Os resultados obtidos na dissertação mostram o elevado potencial das NBs combinadas a outras distribuições de tamanhos de bolhas na melhoria do processo de adesão bolha-partícula e na flotação de minérios fosfáticos finos e de frações granulométricas distintas de quartzo, principalmente em tempos curtos. / The objective of this work was to evaluate the interactions between bubbles with different sizes (diameters) and mineral particles and the effect of the combinations of these bubbles on the flotation of ores, on a bench scale. The bubbles used in these studies were: i) Nanobubbles - NBs, mean diameter of 150-200 nm; ii) Microbubbles - MBs, average diameter of 70 μm; and iii) Macrobubbles - MaBs, average diameter of 1 mm. Dispersions of NBs and MBs were generated after depressurizing-cavitation of the saturated water in air. The saturation pressures were, respectively, 2.5 and 4.0 bar. The bubble-particle mineral interaction studies were developed using a photographic system that allowed to evaluate the adhesion of bubbles to the particles of quartz (high purity grain) and apatite according to the type of bubbles injected. The results showed that the highest adhesion of bubbles in the apatite grain occurred with the combination of NBs + MBs + MaBs and, in the case of quartz, an expressive concentration of adhered MaBs was obtained after "conditioning" of the mineral with NBs. The NBs were confined to the surfaces of the mineral grains as a result of the dissipation of the free surface energy of the solids due to their roughness and, therefore, it increased the adhesion mechanism of other bubbles (MBs and / or MaBs). The study of the evaluation of the associations of bubbles in the flotation was carried out with the mineral systems quartz and phosphate ore. In the case of phosphate ore, consisting of 35% fine particles (<37 μm), the studies evaluated the combination of i) NBs + MaBs; ii) MBs + MaBs; and iii) NBs + MBs + MaBs, in the kinetics and flotation separation parameters, in relation to the standard test, which employs the use of only MaBs. The effects were evaluated in four situations involving percentage of solids (28 and 35% by weight) and concentration of reagents (1000 g·t-1 collector + 600 g·t-1 depressant and 500 g·t-1 collector + 300 g·t-1 of depressor; the apatite collector used was a saponified soybean oil with sodium hydroxide and the depressant of the ganga - magnetite, carbonates, micas, quartz, diopside and perovskite / anatase particles - was a gelatinized corn starch with sodium hydroxide). The combined use of bubbles presented increases in the recovery of P2O5, with a small drop in P2O5 content and high kinetics, in relation to the STD tests in practically all the studied conditions, with values varying between 0.2 and 8.9%. The increments when the percentage of solids was 35% w/w (up to 2.7%) were considerably lower than those obtained with 28% solids (up to 8.9%). In some conditions, the concomitant use of MBs + MaBs and NBs + MBs + MaBs was able to overcome, in the first min and a half of the test, the total phosphate recovery obtained in the standard tests. Regarding the quartz, the microflotation tests (performed in modified Hallimond tube) with and without the presence of NBs showed that the association of NBs with MaBs increased the recovery of quartz (up to 39%) in practically all the studied conditions, showing its effectiveness with both fine and coarse particles. However, the tests performed in mechanical cell with and without addition of NBs showed that the use of NBs was responsible for the increase of 23 percentage points in quartz recovery. Also, flotation kinetics increased significantly and ealier in the first min of the NBs test, about twice the quartz recovery of the standard test was achieved. The results obtained in the dissertation show the high potential of NBs combined with other bubble size distributions in the improvement of the bubble-particle adhesion process and the flotation of fine phosphatic or quartz granulometric fractions, especially in short times.

O presente trabalho teve como objetivo avaliar as interações entre bolhas com distintos tamanhos (diâmetros) e partículas minerais e o efeito de combinações destas bolhas na flotação de minérios, em escala de bancada. As bolhas empregadas nesses estudos foram: i) Nanobolhas – NBs, diâmetro médio de 150 – 200 nm; ii) Microbolhas – MBs, diâmetro médio de 70 μm; e iii) Macrobolhas – MaBs, diâmetro médio de 1 mm. As dispersões de NBs e MBs foram geradas a partir da despressurização de água saturada com ar e cavitação hidrodinâmica em válvula agulha. As pressões de saturação foram, respectivamente, 2,5 e 4,0 bar. Os estudos de interação bolhas-partícula mineral foram desenvolvidos utilizando um sistema fotográfico que permitiu avaliar a adesão de bolhas às partículas de quartzo (grão de alta pureza) e apatita em função do tipo de bolhas injetadas. Os resultados mostraram que a maior adesão de bolhas no grão de apatita ocorreu com a combinação de NBs + MBs + MaBs e, no caso do quartzo, uma expressiva concentração de MaBs aderidas foi obtida após “condicionamento” do mineral com NBs. As NBs ficaram confinadas às superfícies dos grãos minerais devido à dissipação da energia livre superficial dos sólidos em função de sua rugosidade e, desta forma, aumentaram o mecanismo de adesão de outras bolhas (MBs e/ou MaBs). O estudo de avaliação das associações de bolhas na flotação foi realizado com os sistemas minerais quartzo e minério fosfático. No caso do minério fosfático, constituído por 35% de partículas finas (< 37 μm), os estudos avaliaram a combinação de i) NBs + MaBs; ii) MBs + MaBs; e iii) NBs + MBs + MaBs, nos parâmetros cinéticos e de separação na flotação, em relação ao ensaio standard, que emprega o uso de apenas MaBs. Os efeitos foram avaliados em quatro situações que compreenderam variação de porcentagem de sólidos (28 e 35% em peso) e concentração de reagentes (1000 g.t-1 de coletor + 600 g.t-1 de depressor; e 500 g.t-1 de coletor + 300 g.t-1 de depressor; sendo que o coletor de apatita empregado foi óleo de soja saponificado com hidróxido de sódio e o depressor das partículas de ganga - magnetita, carbonatos, micas, quartzo, diopsídio e perovskita/anatásio - foi amido de milho gelatinizado com hidróxido de sódio). O emprego combinado de bolhas apresentou incrementos na recuperação de P2O5, com pequena queda no teor de P2O5 e elevada cinética, em relação aos ensaios STD em praticamente todas as condições estudadas, com valores variando entre 0,2 e 8.9%. Os incrementos quando a porcentagem de sólidos foi de 35% p/p (até 2,7%) foram consideravelmente menores que os obtidos com 28% de sólidos (até 8,9%). Em algumas condições, o uso concomitante de MBs + MaBs e NBs + MBs + MaBs foi capaz de superar, já na primeira coleta (1,5 min de ensaio), a recuperação total de fosfato obtida no ensaio standard. No caso do quartzo, os ensaios de microflotação (realizados em tubo de Hallimond modificado) com e sem a presença de NBs mostraram que a associação de NBs às MaBs aumentou a recuperação de quartzo (até 39%) em praticamente todas as condições estudadas, mostrando sua efetividade tanto com partículas finas quanto com as mais grossas. Já os ensaios realizados em célula mecânica com e sem adição de NBs, mostraram que o emprego de NBs foi responsável pelo aumento de 23 pontos percentuais na recuperação de quartzo. Ainda, a cinética de flotação aumentou significativamente e já no primeiro min do ensaio com NBs obteve-se cerca do dobro da recuperação de quartzo do ensaio standard. Os resultados obtidos na dissertação mostram o elevado potencial das NBs combinadas a outras distribuições de tamanhos de bolhas na melhoria do processo de adesão bolha-partícula e na flotação de minérios fosfáticos finos e de frações granulométricas distintas de quartzo, principalmente em tempos curtos. / The objective of this work was to evaluate the interactions between bubbles with different sizes (diameters) and mineral particles and the effect of the combinations of these bubbles on the flotation of ores, on a bench scale. The bubbles used in these studies were: i) Nanobubbles - NBs, mean diameter of 150-200 nm; ii) Microbubbles - MBs, average diameter of 70 μm; and iii) Macrobubbles - MaBs, average diameter of 1 mm. Dispersions of NBs and MBs were generated after depressurizing-cavitation of the saturated water in air. The saturation pressures were, respectively, 2.5 and 4.0 bar. The bubble-particle mineral interaction studies were developed using a photographic system that allowed to evaluate the adhesion of bubbles to the particles of quartz (high purity grain) and apatite according to the type of bubbles injected. The results showed that the highest adhesion of bubbles in the apatite grain occurred with the combination of NBs + MBs + MaBs and, in the case of quartz, an expressive concentration of adhered MaBs was obtained after "conditioning" of the mineral with NBs. The NBs were confined to the surfaces of the mineral grains as a result of the dissipation of the free surface energy of the solids due to their roughness and, therefore, it increased the adhesion mechanism of other bubbles (MBs and / or MaBs). The study of the evaluation of the associations of bubbles in the flotation was carried out with the mineral systems quartz and phosphate ore. In the case of phosphate ore, consisting of 35% fine particles (<37 μm), the studies evaluated the combination of i) NBs + MaBs; ii) MBs + MaBs; and iii) NBs + MBs + MaBs, in the kinetics and flotation separation parameters, in relation to the standard test, which employs the use of only MaBs. The effects were evaluated in four situations involving percentage of solids (28 and 35% by weight) and concentration of reagents (1000 g·t-1 collector + 600 g·t-1 depressant and 500 g·t-1 collector + 300 g·t-1 of depressor; the apatite collector used was a saponified soybean oil with sodium hydroxide and the depressant of the ganga - magnetite, carbonates, micas, quartz, diopside and perovskite / anatase particles - was a gelatinized corn starch with sodium hydroxide). The combined use of bubbles presented increases in the recovery of P2O5, with a small drop in P2O5 content and high kinetics, in relation to the STD tests in practically all the studied conditions, with values varying between 0.2 and 8.9%. The increments when the percentage of solids was 35% w/w (up to 2.7%) were considerably lower than those obtained with 28% solids (up to 8.9%). In some conditions, the concomitant use of MBs + MaBs and NBs + MBs + MaBs was able to overcome, in the first min and a half of the test, the total phosphate recovery obtained in the standard tests. Regarding the quartz, the microflotation tests (performed in modified Hallimond tube) with and without the presence of NBs showed that the association of NBs with MaBs increased the recovery of quartz (up to 39%) in practically all the studied conditions, showing its effectiveness with both fine and coarse particles. However, the tests performed in mechanical cell with and without addition of NBs showed that the use of NBs was responsible for the increase of 23 percentage points in quartz recovery. Also, flotation kinetics increased significantly and ealier in the first min of the NBs test, about twice the quartz recovery of the standard test was achieved. The results obtained in the dissertation show the high potential of NBs combined with other bubble size distributions in the improvement of the bubble-particle adhesion process and the flotation of fine phosphatic or quartz granulometric fractions, especially in short times.

Master of Science / Food Science Institute - Animal Sciences and Industry / Randall K. Phebus / A variety of antimicrobial processes are used to reduce pathogen risks on commercially processed raw beef. Little research has evaluated chlorinated water on beef tissues, especially in a processing water dip scenario. Interest in nanobubble technology has increased due to its proposed surfactant properties, but it is undetermined whether this improves antimicrobial effectiveness of chlorine-based solutions in food applications. Benchtop studies were conducted to evaluate chlorinated nanobubble waters (0 to 11.94 ppm) against Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O121, O145, and O157:H7 (STEC-7), Salmonella spp., and USDA-approved non-pathogenic STEC surrogates 1) in pure culture with the goal of characterizing the lethality contributions of pH (5 or 7), temperature, free available chlorine level (FAC), inclusion of nanobubbles, or a combination thereof; 2) in select chlorinated nanobubble “red water” (water containing 0.1% beef purge) solutions; and 3) on the surface of lean and fat beef tissue. In pure culture solutions, surrogates demonstrated greater resistance (P ≤ 0.05) to chlorinated solutions (3.4-5.5 log CFU/mL reductions) with increased reductions at the higher (11.94 ppm) FAC levels. STEC-7 and Salmonella population reductions were also notably reduced (3.3-7.1 log CFU/mL) by the higher FAC concentrations. No definitive impacts of temperature, nanobubble inclusion, or acidic pH were observed. At an average 5.23 ppm FAC in red water, all microbial populations were reduced by > 6 log CFU/mL after 60 minutes. Reductions of target organisms on inoculated lean and fat tissues were ≤ 1 log CFU/g in red water; likely due to the inability to maintain FAC levels above 0.7 ppm in the presence of organic loading. An in-plant antimicrobial validation study of a proprietary raw beef manufacturing process was conducted to determine the effectiveness of a recirculating acidic nanobubble water system, chlorinated to 5 ppm FAC using EO water generated concentrate, against the USDA-approved STEC surrogates. Preliminarily, inoculated beef trim was introduced into the system targeting 5 ppm FAC; chlorine concentrate reinfusion rates were determined to establish applicable operational parameters and sampling strategies for the system. An optimized in-plant study was conducted. Meat inoculated at ~ 7 log CFU/g was introduced into the recirculating chlorinated nanobubble system every other day over 6 days, achieving an average 1.6 log CFU/g surrogate reduction on inoculated meat throughout the manufacturing process. Approximately 2.7 log CFU/g of residual surrogates were recovered on non-inoculated meat ~35 minutes after inoculated meat entered the system, indicating that harborage of microbial contamination on processing equipment can lead to subsequent contamination carry-over that must be controlled during processing. Surrogate organisms were recovered by enrichment only from non-inoculated meat 24 h after inoculated meat processing on alternate days, likely stemming from inadequately sanitized processing equipment after inoculated batch processing. Control of the residual surrogate population in the system following inoculation was accomplished through daily equipment sanitation and boosting recirculated processing water to 50 ppm during a 4-h sanitation period (no beef entering system). The optimized study will be used as an antimicrobial process validation against STEC and Salmonella spp. in beef manufacturing.

O presente trabalho teve como objetivo avaliar as interações entre bolhas com distintos tamanhos (diâmetros) e partículas minerais e o efeito de combinações destas bolhas na flotação de minérios, em escala de bancada. As bolhas empregadas nesses estudos foram: i) Nanobolhas – NBs, diâmetro médio de 150 – 200 nm; ii) Microbolhas – MBs, diâmetro médio de 70 μm; e iii) Macrobolhas – MaBs, diâmetro médio de 1 mm. As dispersões de NBs e MBs foram geradas a partir da despressurização de água saturada com ar e cavitação hidrodinâmica em válvula agulha. As pressões de saturação foram, respectivamente, 2,5 e 4,0 bar. Os estudos de interação bolhas-partícula mineral foram desenvolvidos utilizando um sistema fotográfico que permitiu avaliar a adesão de bolhas às partículas de quartzo (grão de alta pureza) e apatita em função do tipo de bolhas injetadas. Os resultados mostraram que a maior adesão de bolhas no grão de apatita ocorreu com a combinação de NBs + MBs + MaBs e, no caso do quartzo, uma expressiva concentração de MaBs aderidas foi obtida após “condicionamento” do mineral com NBs. As NBs ficaram confinadas às superfícies dos grãos minerais devido à dissipação da energia livre superficial dos sólidos em função de sua rugosidade e, desta forma, aumentaram o mecanismo de adesão de outras bolhas (MBs e/ou MaBs). O estudo de avaliação das associações de bolhas na flotação foi realizado com os sistemas minerais quartzo e minério fosfático. No caso do minério fosfático, constituído por 35% de partículas finas (< 37 μm), os estudos avaliaram a combinação de i) NBs + MaBs; ii) MBs + MaBs; e iii) NBs + MBs + MaBs, nos parâmetros cinéticos e de separação na flotação, em relação ao ensaio standard, que emprega o uso de apenas MaBs. Os efeitos foram avaliados em quatro situações que compreenderam variação de porcentagem de sólidos (28 e 35% em peso) e concentração de reagentes (1000 g.t-1 de coletor + 600 g.t-1 de depressor; e 500 g.t-1 de coletor + 300 g.t-1 de depressor; sendo que o coletor de apatita empregado foi óleo de soja saponificado com hidróxido de sódio e o depressor das partículas de ganga - magnetita, carbonatos, micas, quartzo, diopsídio e perovskita/anatásio - foi amido de milho gelatinizado com hidróxido de sódio). O emprego combinado de bolhas apresentou incrementos na recuperação de P2O5, com pequena queda no teor de P2O5 e elevada cinética, em relação aos ensaios STD em praticamente todas as condições estudadas, com valores variando entre 0,2 e 8.9%. Os incrementos quando a porcentagem de sólidos foi de 35% p/p (até 2,7%) foram consideravelmente menores que os obtidos com 28% de sólidos (até 8,9%). Em algumas condições, o uso concomitante de MBs + MaBs e NBs + MBs + MaBs foi capaz de superar, já na primeira coleta (1,5 min de ensaio), a recuperação total de fosfato obtida no ensaio standard. No caso do quartzo, os ensaios de microflotação (realizados em tubo de Hallimond modificado) com e sem a presença de NBs mostraram que a associação de NBs às MaBs aumentou a recuperação de quartzo (até 39%) em praticamente todas as condições estudadas, mostrando sua efetividade tanto com partículas finas quanto com as mais grossas. Já os ensaios realizados em célula mecânica com e sem adição de NBs, mostraram que o emprego de NBs foi responsável pelo aumento de 23 pontos percentuais na recuperação de quartzo. Ainda, a cinética de flotação aumentou significativamente e já no primeiro min do ensaio com NBs obteve-se cerca do dobro da recuperação de quartzo do ensaio standard. Os resultados obtidos na dissertação mostram o elevado potencial das NBs combinadas a outras distribuições de tamanhos de bolhas na melhoria do processo de adesão bolha-partícula e na flotação de minérios fosfáticos finos e de frações granulométricas distintas de quartzo, principalmente em tempos curtos. / The objective of this work was to evaluate the interactions between bubbles with different sizes (diameters) and mineral particles and the effect of the combinations of these bubbles on the flotation of ores, on a bench scale. The bubbles used in these studies were: i) Nanobubbles - NBs, mean diameter of 150-200 nm; ii) Microbubbles - MBs, average diameter of 70 μm; and iii) Macrobubbles - MaBs, average diameter of 1 mm. Dispersions of NBs and MBs were generated after depressurizing-cavitation of the saturated water in air. The saturation pressures were, respectively, 2.5 and 4.0 bar. The bubble-particle mineral interaction studies were developed using a photographic system that allowed to evaluate the adhesion of bubbles to the particles of quartz (high purity grain) and apatite according to the type of bubbles injected. The results showed that the highest adhesion of bubbles in the apatite grain occurred with the combination of NBs + MBs + MaBs and, in the case of quartz, an expressive concentration of adhered MaBs was obtained after "conditioning" of the mineral with NBs. The NBs were confined to the surfaces of the mineral grains as a result of the dissipation of the free surface energy of the solids due to their roughness and, therefore, it increased the adhesion mechanism of other bubbles (MBs and / or MaBs). The study of the evaluation of the associations of bubbles in the flotation was carried out with the mineral systems quartz and phosphate ore. In the case of phosphate ore, consisting of 35% fine particles (<37 μm), the studies evaluated the combination of i) NBs + MaBs; ii) MBs + MaBs; and iii) NBs + MBs + MaBs, in the kinetics and flotation separation parameters, in relation to the standard test, which employs the use of only MaBs. The effects were evaluated in four situations involving percentage of solids (28 and 35% by weight) and concentration of reagents (1000 g·t-1 collector + 600 g·t-1 depressant and 500 g·t-1 collector + 300 g·t-1 of depressor; the apatite collector used was a saponified soybean oil with sodium hydroxide and the depressant of the ganga - magnetite, carbonates, micas, quartz, diopside and perovskite / anatase particles - was a gelatinized corn starch with sodium hydroxide). The combined use of bubbles presented increases in the recovery of P2O5, with a small drop in P2O5 content and high kinetics, in relation to the STD tests in practically all the studied conditions, with values varying between 0.2 and 8.9%. The increments when the percentage of solids was 35% w/w (up to 2.7%) were considerably lower than those obtained with 28% solids (up to 8.9%). In some conditions, the concomitant use of MBs + MaBs and NBs + MBs + MaBs was able to overcome, in the first min and a half of the test, the total phosphate recovery obtained in the standard tests. Regarding the quartz, the microflotation tests (performed in modified Hallimond tube) with and without the presence of NBs showed that the association of NBs with MaBs increased the recovery of quartz (up to 39%) in practically all the studied conditions, showing its effectiveness with both fine and coarse particles. However, the tests performed in mechanical cell with and without addition of NBs showed that the use of NBs was responsible for the increase of 23 percentage points in quartz recovery. Also, flotation kinetics increased significantly and ealier in the first min of the NBs test, about twice the quartz recovery of the standard test was achieved. The results obtained in the dissertation show the high potential of NBs combined with other bubble size distributions in the improvement of the bubble-particle adhesion process and the flotation of fine phosphatic or quartz granulometric fractions, especially in short times.

L'étude des nanobulles de vapeur générées autour de nanoparticules métalliques chauffées par un laser dans de l'eau a connu un intérêt croissant au cours de la dernière décennie, motivé notamment par leur utilisation potentielle pour des applications biomédicales. Ces travaux sont majoritairement expérimentaux et il n'existe pas de description complète des phénomènes physiques régissant la génération et la dynamique des nanobulles. L'objet de cette thèse est de répondre à ces questions fondamentales par la résolution numérique d'un modèle fondé sur les équations de conservation locales dans le fluide (masse, quantité de mouvement et énergie) et prenant en compte la thermodynamique du fluide, les effets capillaires et la résistance thermique à l'interface or-fluide. Par la résolution de ce modèle, nous avons accès à la thermodynamique du fluide avant sa vaporisation et pendant la durée de l'existence des nanobulles, ce qui permet la description de leur dynamique. Dans un second temps, nous définissons le critère de vaporisation dans le fluide par le franchissement de la température spinodale du fluide. Enfin, nous effectuons le bilan énergétique de la production et de la croissance des nanobulles, pour optimiser le transfert énergétique entre le laser et la nanobulle. Nous nous intéressons enfin à la conductance thermique d'interface due au couplage électron-phonon entre un métal et un diélectrique. Après avoir souligné l'influence de ce type de couplage sur le transfert énergétique interfacial, nous présentons des résultats préliminaires concernant le chauffage d'un fluide par des nanoparticules de type cœur-coquille or-silicium / Nanobubbles produced around metal nanoparticles heated by a laser pulse have received an increasing interest over the last decade. This interest is motivated by the possible use of those nanobubbles as an agent for cancer therapy. Existing studies are mainly experimental and a complete description of the mechanisms controlling the nanobubbles generation and evolution is still lacking. The aim of this thesis is to answer those fundamental issues by numerically solving a model based on the conservation equations inside the fluid (mass, momentum and energy). This model accounts for the thermodynamics of the fluid, capillary effects and a thermal interface resistance across the particle-fluid interface. Solving this model gives information about the thermodynamics of the fluid before and after its vaporization, which allows for the description of the bubbles dynamics. Then, we can define a criterion for bubbles generation, which corresponds to the crossing of the spinodal temperature of the fluid. Finally, we investigate the role played by the interface thermal resistance arising from electron-phonon couplings between a metal and a dielectric. We present some preliminary results concerning the heating of a fluid with core-shell nanoparticles

Ce mémoire est consacré au développement et à l'application d'une méthode permettant de caractériser finement les propriétés physiques (densité d'hélium, pression, diamètre, morphologie) de bulles d'hélium de taille nanométrique pour in fine améliorer la compréhension du comportement de ces nano-systèmes. L'approche que nous avons choisie est basée sur la spectroscopie de pertes d'énergie des électrons et l'acquisition de spectres images en microscopie électronique en transmission filtrée en énergie. Les différentes étapes d'acquisition, de correction des aberrations, et de traitement des spectres sont détaillées. L'erreur sur la mesure est estimée, et des améliorations potentielles de la méthode sont discutées. Nous montrons de plus que cette approche permet non seulement de dépasser les limites imposées par la microscopie électronique en transmission à balayage habituellement utilisée, mais aussi d'aller au-delà, en terme de statistique notamment. Nous appliquons ensuite notre méthode pour déterminer les propriétés physiques de bulles d'hélium dans le silicium, lors de recuits thermiques in situ dans le microscope. L'évolution des caractéristiques morphologiques des bulles est mise en rapport avec la variation de la densité d'hélium qu'elles contiennent suite à ces recuits. Les valeurs de densité et de pression obtenues sont comparées aux valeurs disponibles dans la littérature par des méthodes expérimentales ou numériques. Enfin, le transfert de notre méthode pour l'étude de bulles dans d'autres matrices (germanium, carbure de silicium, euxénite) est discuté. / This thesis is dedicated to the development and application of a method allowing for the fine characterization of the physical properties (density, diameter, pressure and morphology) of helium bubbles at the nanometric scale, to eventually improve the understanding of the behavior of these nano-systems. The chosen approach is based on electron energy loss spectroscopy and the acquisition of spectral images by energy-filtered transmission electron microscopy. The acquisition, aberration correction, and data analysis steps are detailed. The measurement error is estimated, and potential improvements are discussed. We additionally show that this method is not only able to overcome the limits imposed by commonly used scanning transmission electron microscopy, but also to go further, notably in a statistical way. We then apply our method to determine the physical properties of helium bubbles in silicon, during in situ thermal annealing in the microscope. The evolution of the morphological characteristics of the bubbles is put in relation with the variation of the density of the helium contained following these annealings. The density and pressure values are compared to those available in the litterature through experimental and numerical methods. Finally, the translation of this method towards the study of bubbles in other matrices (germanium, silicon carbide, euxenite) is discussed.

Interactions between hydrophobic surfaces at short separation distances (at the nanometer scale) are very important in a number of industrial applications. For example, in the froth flotation mineral separation process it is the interaction between the hydrophobic particle and the bubble which is paramount in separating the valuable minerals from the gangue. A number of studies, most notably using the atomic force microscope (AFM) and the surface force apparatus (SFA) have found the existence of a long range hydrophobic attractive force between hydrophobic surfaces that cannot be explained by classical colloidal science theories. In many cases, this force is an artefact due to the accumulation of sub-microscopic bubbles, the so called nanobubbles, at the liquid-hydrophobic solid interface. Thus, what was thought to be a hydrophobic force was actually a capillary force resulting from the gaseous bridge formed from the coalescence of nanobubbles, that is, the nanobubble bridging capillary force (NBCF). It is the purpose of this thesis to provide further insight into the accumulation of soluble gases at the liquid-hydrophobic solid interface and the resulting NBCF. Specifically, this thesis studies these phenomena from a fundamental standpoint and additionally relates the findings to froth flotation mineral separation. A systematic method to measure the NBCF by controlling the size of the gaseous capillary bridge was devised in this thesis. Control of the capillary bridge was achieved by utilising the solvent-exchange method to accumulate nanobubbles at the surface, followed by surface scanning of the colloidal probe over the flat surface to harvest nanobubbles. Thus, the NBCF has been controlled to allow for greater success in modelling the interaction, understanding the geometric parameters of the bridge, observing changes in friction force due to nanobubbles and understanding the influence of ethanol on the force. An outcome of this thesis was the development of a capillary force model which describes the NBCF. The model considers a constant volume and constant contact angle assumption for a gaseous capillary bridge of toroidal geometry. The model was very successful in describing the NBCF at long separation distances (>20nm) for both the approach and retract interactions. The close fitting between the experimental data and the model allowed accurate determinations of the advancing and receding contact angles, bridge geometry and volume. The successful implementation of the capillary force model allowed a link between the bridge volume, and the resulting adhesion to the friction force between hydrophobic solid surfaces in water. Additionally, the model allowed the change from an attractive to a repulsive NBCF to be described by a change from a concave to convex bridge geometry. Thus, this thesis has added considerable knowledge to the fundamental aspects of nanobubbles and the NBCF. The final chapters of this thesis utilised the knowledge gained from the fundamental studies to understand the influence of nanobubbles on flotation. In the first study, the influence of NaCl concentration on the morphology of gaseous domains on a graphite surface is discussed in relation to the increased recovery of coal in saline water. In the second study, methanol treatment of a ZnS ore was found to increase the floatability due to slime removal and the artificial formation of nanobubbles.

In recent years, the field of nanomedicine has provided promising alternatives to traditional ways of gene and drug delivery, such as nanobubbles instead of viral vectors. Manufacturing these nanomaterials is cost-effective and tends to be safer than previous forms of treatment for clinical use. In the realm of osteoporosis, a disease based around a higher rate of bone resorption to bone formation, leads to weak and brittle bones, leaving sufferers at risk for fractures, which are sudden and unexpected. These fractures are estimated to cost USD 22 Billion annually in the US alone. Reports show a significant effect on the quality of life after these fractures. Osteoclast-inhibiting oral medicines have been used to slow down bone resorption, but they have had severe side effects. This project aimed to study the impact of nanobubbles synthesized to target bone and filled with CTSK-siRNA to silence the Cathepsin K gene in an ovariectomized mice model. Cathepsin K is pivotal in osteoporosis as it is responsible for collagen degradation in bone resorption. Low-intensity pulsed ultrasound (LIPUS) for nanobubble destruction to facilitate targeted CTSK-siRNA release. LIPUS can also promote bone formation. We studied the effects of the treatment through histological analysis. Microarchitecture in the mice femur was highlighted through different types of staining such as Masson’s Trichrome, Hematoxylin & Eosin, and tartrate-resistant acid phosphatase staining. ImageJ was used for all measurements taken to interpret bone resorption in experimental groups. The major organs were studied to ensure the biocompatibility of nanobubble treatment. Our results show an increase in the average cortical thickness and trabecular spacing, but there was no significant increase in trabecular thickness in the group treated with CTSK-siRNA nanobubbles.