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Metallic bone implant devices are commonly used to tackle a wide array of bone failures in human patients. The success of such implants relies on the biomechanical and functional bonding between the living bone tissue and implant, a process defined as osseointegration. However, the mechanism of osseointegration is still under debate in the scientific community. One efficient method to help understand this complex process is to characterize the interface between human bones and implant devices after the osseointegration has been established, while another approach is to visualize mineralization in real-time under simulated body conditions. Both of these approaches to understand mineralization have been explored in this thesis. Firstly, due to the inhomogeneous nature of bone and complex topography of implant surfaces, a suitable sample geometry for three-dimensional (3D) characterization was required to fully understand osseointegration. Electron tomography has been proven as an efficient technique to visualize the nanoscale topography of bone-implant interface in 3D. However, resulting from the thickness and shadowing effects of conventional transmission electron microscope (TEM) lamellae at high tilt angles and the limited tilt-range of TEM holders, “missing wedge” artifacts limit the resolution of final reconstructions. In Chapter 3, the exploration of a novel sample geometry to explore osseointegration is reported. Here, on-axis electron tomography based on a needle-shaped sample was applied to solve the problem of the “missing wedge”. This resulted in a near artifact-free 3D visualization of the structure of human bone and laser-modified titanium implant, showing bone growth into the nanotopographies of the implant surface and contributing to the evolution of the definition of osseointegration towards nano-osseointegration. One of the key issues regarding the mechanism of osseointegration that remains is that of the chemical structure at the implant interface, namely distribution of calcium-based and carbon-based components at the interface and their origins. Thus, the second objective of this thesis aimed to push characterization techniques further to four dimensions (4D), by incorporating chemical information as the fourth dimension after the spatial X,Y,Z coordinates. In Chapter 4, correlative 4D characterization techniques including electron energy-loss spectroscopy (EELS) tomography and atom probe tomography (APT) and other spectroscopy techniques were used to probe the nanoscale chemical structure of the bone-implant interface. This work uncovered a transitional biointerphase at the bone-implant interface, consisting of morphological and chemical differences compared to bone away from the interface. Also, a TiN layer between the surface oxide and bulk metal was identified in the laser-modified commercial dental implant. Both findings have implications for the immediate and long-term osseointegration. Since bone formation at the implant interface is a dynamic process, which includes calcium phosphates (CaP) biomineralization as a basis of these reactions, the third objective of this work focused on exploring real-time mineralization processes. Liquid-phase transmission electron microscopy (LP-TEM) is a promising technique to enable real-time imaging with nanoscale spatial resolution and sufficient temporal resolution. In Chapter 5, by using this technique, we present the first real-time imaging of CaP nucleation and growth, which is a direct evidence to demonstrate that CaP mineralization occurs by particle attachment. Overall, this thesis has applied state-of-the-art advanced microscopy techniques to enhance the knowledge and understanding of osseointegration mechanisms by investigating established biointerfaces and real-time mineralization. The developed correlative 4D tomography workflow is transferable to study other interfacial applications in materials science and biological systems, while the LP-TEM work forms a basis for further mineralization research. / Thesis / Doctor of Philosophy (PhD)

This dissertation work focuses on the processability improvement of B4C, especially the compaction and sintering improvement of B4C by applying a Ni-B nanolayer coating on individual B4C particles. A modified electroless coating procedure was proposed and employed to coat nanometer Ni-B layer onto micron-sized B4C particles. The thickness was able to be tuned and controlled below 100 nm. Key parameters, including the amount of nickel source, the amount of the surface activation agent (PdCl2), the amount of the complexing agent (C2H8N2), and the addition rate of the reducing agent (NaBH4) were studied. When the targeted thickness was 5 nm, a continuous and uniform nanolayer coating was obtained with the optimal condition of individual parameter combined. Reduction of the as-coated B4C powder in a H2-Ar atmosphere was studied between 400-900C to reduce the surface oxides' Ni2O3 and B2O3. Reduction at 800C in hydrogen atmosphere was found to be the most effective condition to remove oxygen in the coating layer, with Ni2B as the reduction product. Compaction of the as-received, separated and uncoated, and separated with Ni-B coating B4C powders using uniaxial die compaction and combustion driven compaction (CDC) techniques was studied. CDC technique showed the advantage over the traditional uniaxial die compaction by yielding much higher green density and green strength (73% vs. 53.8% green density for the Ni-B coated B4C). Among compacts obtained from the same technique, Ni-B coated B4C compact yielded the densest packing with crack-free compact surface and the highest strength, demonstrating more bonding between B4C particles provided by Ni-B surface coating. Sintering of the Ni-B coated B4C in an Ar atmosphere between 1150 - 1600C with soaking time of 2 hrs and 10 hrs was studied. Liquid phase was found to form during the sintering process. Density measurement showed that the liquid phase Ni-B formed greatly facilitated B4C densification. Considerable density increase and inter-granular connection was achieved when sintered at 1600C for 10 hrs. The density enhancement by Ni-B coating was supported by transmission electron microscopy-energy dispersive spectroscopy (TEM-EDS) examination which showed that there was B4C species diffusion into liquid Ni-B phase. This liquid phase enhanced the diffusion of B4C species and formed strong bonding between B4C grains by dissolving small B4C particles and sharp edge and corners of B4C particles. Strength test demonstrated that the Ni-B coating dramatically improved the strength of B4C compacts by yielding a much higher strength of the Ni-B coated samples than the uncoated samples (13.97 vs. 1.79 MPa for the uinaxial die compacted samples, 27.03 vs. 2.21 MPa for the CDC samples). Electrical conductivity Ni-B coated B4C samples was also shown to be improved with the electrical resistivity being reduced from infinite for pure B4C samples to 1.8Ã 10-3 Ω·m for the Ni-B coated samples. This research work has shown that with the Ni-B coating, B4C densification can start at a temperature as low as 1600C via a liquid phase sintering process. / Ph. D.

The effects of polymer concentration and solvents on cocrystal morphology of low solubility drugs were investigated, both of which had an impact. The melting temperatures also decreased with increasing polymer concentration. Placing the binding agent, benzene, at different interfaces induced morphological changes, such as formation of porous cocrystals. Previously liquid-liquid phase separation (LLPS) has been reported as a hindrance in the crystallisation process impeding further development. A phase diagram was constructed, and different phases were categorised into 4 types. After separation of the highly concentrated amorphous Oil Phase II, it was prone to gradual crystallisation. Crystallisation took place over 30-60 minutes; this allowed the in-situ monitoring. A novel cocrystallisation technique was developed; from (LLPS). Cocrystals of indomethacin with saccharin and nicotinamide were obtained by mixing Oil Phase II with the coformers. In-situ monitoring by spectroscopic had gradual changes in spectra; characteristic peaks increased in height and area with the formation of crystals until the reaction was complete. With crystal formation, the XRD spectra gradually had a sharper baseline due to a decrease in the amorphous indomethacin. The photoluminescence (PL) spectra displayed several peaks coupling into one large hump together with increasing intensity as the sample crystallised. There was a shift in the peak absorbance of the pure drug crystals obtained from LLPS and the indomethacin:saccharin cocrystal obtained from LLPS. Amorphous stabilisation was achieved by mixing polymer (PVP) with Oil Phase II. There were no changes to the XRD diffractogram as the sample did not undergo crystallisation.

The work presented has the ultimate aim to increase dynamic mechanical properties by improvements in density and optimisation of microstructure of ultra high carbon PM steels by careful selection of processes, i.e. mixing, binding, alloying, heating profile and intelligent heat treatment. ThermoCalc modelling was employed to predict liquid phase amounts for two different powder grades, Astaloy 85Mo or Astaloy CrL with additive elements such as (0.4-0.6wt%)Si, (1.2-1.4wt%)C and (1-1.5wt%)Mn, in the sintering temperature range 1285-1300ºC and such powder mixes were pressed and liquid phase sintered. In high-C steels carbide networks form at the prior particle boundaries, leading to brittleness, unless the steel is heat-treated. To assist the breaking up of these continuous carbide networks, 0.4-0.6% silicon, in the form of silicon carbide, was added. The water gas shift reaction (C + H2O = CO + H2, start from ~500ºC) and Boudouard reaction (from ~500ºC complete ~930ºC) form CO gas in the early part of sintering and can lead to large porosity, which lowers mechanical properties. With the use of careful powder drying, low dew point atmospheres and optimisation of heating profiles, densities in excess of 7.70g/cm3 were attained. The brittle microstructure, containing carbide networks and free of cracks, is transformed by intelligent heat treatment to a tougher one of ferrite plus sub-micron spheroidised carbides. This gives the potential for production of components, which are both tough and suitable for sizing to improve dimensional tolerance. Yield strengths up to 410 MPa, fracture strengths up to 950 MPa and strains of up to 16 % were attained. Forging experiments were subsequently carried out for spheroidised specimens of Fe-0.85Mo+06Si+1.4C, for different strain rates of 10-3, 10-2, 10-1 and 1sec-1 and heated in argon to 700ºC, density ~7.8g/cm3 and 769 MPa yield strength were obtained.

A new solid phase microextraction system composed of nanotubes coating fixed on a stainless steel support is suggested. Thermal stability and selectivity of the system was examined. It was determined that the system can be used for direct and headspace extraction of volatile aromatic hydrocarbons. Possibilities of four liquid phase microextraction techniques – hollow fibre liquid phase microextraction, liquid phase microextraction based on the solidification of a floating drop, dispersive liquid-liquid microextraction and dispersion-solidification liquid-liquid microextraction – for the extraction of volatile aromatic hydrocarbons were investigated. Extraction conditions of the investigated methods were optimized and the main analytical characteristics were determined. For all the methods detection limits and repeatability of the results are close. An exception is dispersive liquid-liquid microextraction with slightly higher detection limits. An extraction is especially fast using dispersive liquid-liquid microextraction and dispersion-solidification liquid-liquid microextraction. The most time-consuming method is liquid phase microextraction based on the solidification of a floating drop. All the methods are suitable for clean sample extraction. For the extraction from complex matrices the most suitable methods are headspace solid phase microextraction and hollow fibre liquid phase microextraction. The prepared microextraction techniques were applied for water and snow... [to full text] / Pasiūlyta nauja kietafazės mikroekstrakcijos sistema, kurioje nerūdijančio plieno strypelis dengtas anglies nanovamzdeliais, ištirtas jos terminis stabilumas ir atrankumas, nustatyta, kad sistema tinka lakių aromatinių angliavandenilių ekstrakcijai iš tirpalo ir iš viršerdvės. Ištirtos keturių skysčių-skysčių mikroekstrakcijos metodų - skysčių-skysčių mikroekstrakcijos kapiliare, mikroekstrakcijos užšaldomu tirpiklio lašu, dispersinės skysčių-skysčių mikroekstrakcijos ir dispersinės skysčių-skysčių mikroekstrakcijos užšaldant ekstraktą - galimybės ekstrahuoti lakius aromatinius angliavandenilius. Optimizuotos tirtų metodų ekstrakcijos sąlygos, nustatytos pagrindinės analizinės charakteristikos. Visų metodų rezultatų pasikartojamumas ir aptikimo ribos artimi. Išimtis – dispersinė skysčių-skysčių mikroekstrakcija, kuria gautos kiek didesnės aptikimo ribos. Greičiausi ekstrakcijos metodai - dispersinė skysčių-skysčių mikroekstrakcija ir dispersinė skysčių-skysčių mikroekstrakcija užšaldant ekstraktą, ilgiausiai trunka mikroekstrakcija užšaldomu tirpiklio lašu. Švarių mėginių ekstrakcijai tinka visi tirti metodai, užterštiems mėginiams geriau tinka kietafazė mikroekstrakcija iš viršerdvės arba skysčių-skysčių mikroekstrakcija kapiliare. Paruoštos lakių aromatinių angliavandenilių mikroekstrakcijos metodikos pritaikytos vandens ir sniego mėginių analizei.

Transient liquid phase (TLP) bonding has proven to be the preferred method for joining extremely difficult-to-weld advanced materials, including similar and dissimilar superalloys. In this work, an approach that combines experiments and theoretical simulations are used to investigate the effect of temperature gradient (TG) in a vacuum furnace on the temperature distribution in TLP bonded samples. When joining similar materials by this technique, the simulated results with experimental verifications show that, irrespective of where the samples are placed inside the vacuum furnace, a TG in the furnace can translate into a symmetric temperature distribution in bonded samples provided the diffusion direction is parallel to the source of heat emission. In addition, the effects of TLP bonding parameters on the joint microstructure were investigated during the joining of nickel-based IN738 and CMSX-4 single crystal (SX) superalloys. An increase in holding time and reduction in gap size reduces the width of eutectic product that forms within the joint region. It was also found that Liquid-state diffusion (LSD) can occur and have significant effects on the microstructure of dissimilar TLP bonded joints even though its influence is often ignored during TLP bonding. The occurrence of LSD produced single crystal joint when a SX and polycrystal substrate were bonded. This formation of a SX joint which cannot be exclusively produced by solid-state diffusion has not been previously reported in the literature. / February 2017

Os benzodiazepínicos (BDZ) são amplamente utilizados no tratamento da ansiedade. Aumentam a neurotransmissão GABAérgica por ação em sítios receptores específicos para BDZ presentes no complexo ionóforo no canal do receptor GABAA, onde modulam alostericamente suas atividades. Porém, além de atuar nesses receptores, têm sido relatado, também, a existência de receptores periféricos para benzodiazepínicos (PBR). Evidências sugestivas de uma ação imunomodulatória direta para os BZD emergem de estudos que demonstraram a presença de PBR em células imunes/inflamatórias. Este trabalho avaliou os efeitos da administração de diazepam sobre parâmetros imunológicos de ratos e desenvolveu um método para dosagem plasmática desse benzodiazepínico utilizando a técnica de microextração em fase líquida. Foram realizados tratamentos agudo e prolongado (21 dias) com as doses de 1 e 10 mg/kg. Os resultados mostraram que a dose de 1 mg/kg não alterou qualquer um dos parâmetros imunes analisados; porém, diminuiu a quantidade de hemoglobina e de hemácias, assim como o valor de hematócrito, quando administrado prolongadamente. Entretanto, a dose de 10 mg/kg, após ambos os tratamentos, foi capaz de diminuir as células apoptóticas e, conseqüentemente, de aumentar a porcentagem de células viáveis, aumentar a porcentagem de células Thelper e Tcitotóxicas, diminuir a porcentagem de células B e aumentar os níveis séricos de corticosterona. Além destes, observou-se, também, um aumento do número de reticulócitos e um aumento do número das células segmentadas no sangue periférico, após administração prolongada do diazepam, assim como um aumento do número de células segmentadas no baço, após administração aguda. A menor dose de diazepam (1 mg/kg) também não foi capaz de induzir tolerância, diferente da maior dose (10 mg/kg), que levou ao desenvolvimento de tolerância funcional. Esses resultados sugerem que a administração de diazepam em baixas doses (< 1mg/kg) não interfira com os parâmetros imunes analisados. Entretanto, o uso de diazepam em doses mais elevadas (> 10 mg/kg) é capaz de alterar tais parâmetros. Muito provavelmente esses efeitos tenham a ver com o número de receptores PBR presentes nas células imunes avaliadas e/ou com a dose de diazepam administrada. Com relação ao método analítico, encontrou-se linearidade na faixa de interesse (intervalo dinâmico de 25 a 2000 ng/mL) para o diazepam e o desmetildiazepam. Os limites de detecção e quantificação foram de 20 e 25 ng/mL, respectivamente, e os parâmetros analisados mostraram-se satisfatórios. Desta forma, o método desenvolvido utilizando a técnica de microextração em fase líquida mostrou-se prático, de baixo custo e com grande potencial para extração prévia à injeção no CG. / Benzodiazepines (BZD) are widely used for the treatment of anxiety. They enhance GABA-ergic neurotransmission through binding on specific BDZ recognition sites, within the GABAA receptor-ion channel complex, where they allosterically modulate its activity. However, recents studies showed that BZD also act on peripheral benzodiazepine receptor sites (PBR). Evidence for a direct immunomodulatory action for BZD emerged from studies that demonstrated the presence of PBR on immune/inflammatory cells. The present experiment was designed to analyze the effects of diazepam on rat immune parameters and to develop a method to detect diazepam on rat plasma through a liquid-phase microextraction technique (LPME). The effects of both acute and long-term (21 days) diazepam (1 and 10 mg/kg/day) administrations were evaluated. Results showed that diazepam (1 mg/kg) treatment did not change the immune parameters analyzed; however, long-term diazepam treatment decreased erytrocytes, hemoglobin and hematocrit values. Both diazepam (10mg/kg) acute and long-term treatments decreased the number of apoptotic cells and, consequently, enhanced the percentage of viable cells; they also increased the percentage of Thelper and Tcitotoxic cells; decreased the percentage of B cells and increased the corticosterone serum levels. Long-term diazepam (10 mg/kg) treatment enhanced reticulocytes and segmented cells in the peripheric blood; however, only acute diazepam (10mg/kg) treatment increased the number of segmented cells on spleen. The low dose of diazepam used (1 mg/kg) was unable to induce tolerance, differently of that found after the high dose of diazepam (10 mg/kg). This latter treatment indicated the development of functional tolerance, at least for diazepam effects on corticosterone serum levels. These results suggested that low doses of diazepam (< 1 mg/kg) were not able to change the immune parameters analyzed; however, high doses of diazepam (> 10 mg/kg) changed many of these immune parameters. It is possible that the observed effects were related to the number of PBR receptor sites present on immune cells and/or to the levels of serum diazepam after the treatments used. The analytical method provided to be linear in the interest range (dynamic range from 25 to 2000 ng/mL) for diazepam and its main metabolite, desmethyldiazepam. The limits of detection and quantification were 20 and 25 ng/mL, respectively, and the parameters analyzed provided to be satisfactory. Finally, the method developed utilizing the liquid-phase microextraction technique showed to be practical, with low costs and thus presenting potential to be used previously to the extraction procedures, i.e., prior to the injection into the gas-chromatography apparatus.

O uso de plantas aromáticas ricas em óleos essenciais (OE) como agentes saneantes ou conservantes remonta às civilizações antigas. Nos dias atuais, o aumento nos surtos de infecções em hospitais e creches, bem como da resistência bacteriana, aumentou também a demanda por desinfetantes. Os OE\'s são reconhecidos por sua atividade antimicrobiana, mas a maioria dos estudos foi realizada na fase líquida. Buscando aproveitar a característica de alta volatilidade dos OE´s, desenvolvemos uma metodologia para a avaliação da atividade antimicrobiana na fase vapor. Nesse sentido, utilizamos o OE extraído de Hesperozygis myrtoides (St.Hil. ex Benth.) Epling (Lamiaceae), uma espécie nativa com ocorrência nos estados do sudeste do Brasil. A planta foi coletada em Campos do Jordão e o óleo teve um rendimento médio de 1,99% (m/m), sendo os componentes majoritários pulegona (48,8%) e isomentona (16,2%). A atividade antimicrobiana foi comparada na fase líquida, pelo método da microdiluição em placa, com a fase vapor, pelo método da placa invertida modificado. Os resultados indicaram uma atividade mais potente para a fase vapor do que na fase líquida. Staphylococcus aureus apresentou CIM de 0,392 mg. L-1 na fase vapor e 19 mg. L-1 na líquida, já para Candida albicans foi 0,833 mg. L-1 , na fase vapor e 94,4 mg. L-1 na fase líquida. Entretanto, para Escherichia coli, Pseudomonas aeruginosa, Bacilus subtillis e Aspergillus brasiliensis os valores da CIM, em ambas as fases, foram acima de 100 mg. L-1, sendo então considerado inativo. A atividade antimicrobiana na fase vapor é mais intensa devido aos OE´s se ligarem diretamente ao microrganismo sem a interferência do solvente. A toxicidade do OE foi avaliada frente a células tumorais humanas de mama (MCF-7) e próstata (CP-3) e em células de fibroblastos murinos normais (BALB/3T3) e indicaram uma DL50 superior a 2.014 mg.Kg-1, podendo ser considerado seguro. A atividade antimicrobiana associada à baixa toxicidade é um forte indicativo que este OE pode ser utilizado para descontaminação ambiental na fase vapor, inclusive em ambientes habitados. / Aromatic plants rich in essential oils (EO) have been used as sanitizing agents or preservatives since early civilizations. Nowadays, the increase in infectious outbreaks in hospitals and nurseries, as well as the bacterial resistance has also increased the demand for disinfectants. EO\'s are known for their antimicrobial activity, but most reports is in the liquid phase. Due to the EO high volatility, we have developed a methodology for evaluating the antimicrobial activity in the vapor phase. In this sense, we use the Hesperozygis myrtoides (St.Hil. Ex Benth.) Epling (Lamiaceae) EO, a native species occurring in southeastern Brazil. The plant was collected in Campos do Jordão (SP) and the oil had an average yield of 1.99% (m/m) and as major components pulegone (48.8%) and isomenthone (16.2%). Antimicrobial activity was compared in the liquid phase, microdilution method, with the vapor phase, modified inverted plate method. The results indicated that the vapor phase was a more potent than the liquid. Staphylococcus aureus afforded MIC 0.392 mg. L-1 for the vapor phase and 19 mg. L-1 for the liquid phase, while for Candida albicans it was 0.833 mg. L-1 for the vapor and 94.4 mg. L-1 for the liquid. However, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Aspergillus brasiliensis had in both phases MIC\'s higher than 100 mg. L-1, considered then inactive. The vapor phase antimicrobial activity is more intense because the EO are free to directly bind with the microorganism without the solvent interference. Toxicity was evaluated against human breast tumor cells (MCF-7) and prostate cancer (PC-3) and with normal murine fibroblast cells (BALB/3T3) indicating a DL50 higher 2,014 mg.Kg-1, and it was considered safe. The antimicrobial activity associated with the low toxicity is a strong indication that this EO vapors can be used for environmental decontamination, even in inhabited places.

Os benzodiazepínicos (BDZ) são amplamente utilizados no tratamento da ansiedade. Aumentam a neurotransmissão GABAérgica por ação em sítios receptores específicos para BDZ presentes no complexo ionóforo no canal do receptor GABAA, onde modulam alostericamente suas atividades. Porém, além de atuar nesses receptores, têm sido relatado, também, a existência de receptores periféricos para benzodiazepínicos (PBR). Evidências sugestivas de uma ação imunomodulatória direta para os BZD emergem de estudos que demonstraram a presença de PBR em células imunes/inflamatórias. Este trabalho avaliou os efeitos da administração de diazepam sobre parâmetros imunológicos de ratos e desenvolveu um método para dosagem plasmática desse benzodiazepínico utilizando a técnica de microextração em fase líquida. Foram realizados tratamentos agudo e prolongado (21 dias) com as doses de 1 e 10 mg/kg. Os resultados mostraram que a dose de 1 mg/kg não alterou qualquer um dos parâmetros imunes analisados; porém, diminuiu a quantidade de hemoglobina e de hemácias, assim como o valor de hematócrito, quando administrado prolongadamente. Entretanto, a dose de 10 mg/kg, após ambos os tratamentos, foi capaz de diminuir as células apoptóticas e, conseqüentemente, de aumentar a porcentagem de células viáveis, aumentar a porcentagem de células Thelper e Tcitotóxicas, diminuir a porcentagem de células B e aumentar os níveis séricos de corticosterona. Além destes, observou-se, também, um aumento do número de reticulócitos e um aumento do número das células segmentadas no sangue periférico, após administração prolongada do diazepam, assim como um aumento do número de células segmentadas no baço, após administração aguda. A menor dose de diazepam (1 mg/kg) também não foi capaz de induzir tolerância, diferente da maior dose (10 mg/kg), que levou ao desenvolvimento de tolerância funcional. Esses resultados sugerem que a administração de diazepam em baixas doses (< 1mg/kg) não interfira com os parâmetros imunes analisados. Entretanto, o uso de diazepam em doses mais elevadas (> 10 mg/kg) é capaz de alterar tais parâmetros. Muito provavelmente esses efeitos tenham a ver com o número de receptores PBR presentes nas células imunes avaliadas e/ou com a dose de diazepam administrada. Com relação ao método analítico, encontrou-se linearidade na faixa de interesse (intervalo dinâmico de 25 a 2000 ng/mL) para o diazepam e o desmetildiazepam. Os limites de detecção e quantificação foram de 20 e 25 ng/mL, respectivamente, e os parâmetros analisados mostraram-se satisfatórios. Desta forma, o método desenvolvido utilizando a técnica de microextração em fase líquida mostrou-se prático, de baixo custo e com grande potencial para extração prévia à injeção no CG. / Benzodiazepines (BZD) are widely used for the treatment of anxiety. They enhance GABA-ergic neurotransmission through binding on specific BDZ recognition sites, within the GABAA receptor-ion channel complex, where they allosterically modulate its activity. However, recents studies showed that BZD also act on peripheral benzodiazepine receptor sites (PBR). Evidence for a direct immunomodulatory action for BZD emerged from studies that demonstrated the presence of PBR on immune/inflammatory cells. The present experiment was designed to analyze the effects of diazepam on rat immune parameters and to develop a method to detect diazepam on rat plasma through a liquid-phase microextraction technique (LPME). The effects of both acute and long-term (21 days) diazepam (1 and 10 mg/kg/day) administrations were evaluated. Results showed that diazepam (1 mg/kg) treatment did not change the immune parameters analyzed; however, long-term diazepam treatment decreased erytrocytes, hemoglobin and hematocrit values. Both diazepam (10mg/kg) acute and long-term treatments decreased the number of apoptotic cells and, consequently, enhanced the percentage of viable cells; they also increased the percentage of Thelper and Tcitotoxic cells; decreased the percentage of B cells and increased the corticosterone serum levels. Long-term diazepam (10 mg/kg) treatment enhanced reticulocytes and segmented cells in the peripheric blood; however, only acute diazepam (10mg/kg) treatment increased the number of segmented cells on spleen. The low dose of diazepam used (1 mg/kg) was unable to induce tolerance, differently of that found after the high dose of diazepam (10 mg/kg). This latter treatment indicated the development of functional tolerance, at least for diazepam effects on corticosterone serum levels. These results suggested that low doses of diazepam (< 1 mg/kg) were not able to change the immune parameters analyzed; however, high doses of diazepam (> 10 mg/kg) changed many of these immune parameters. It is possible that the observed effects were related to the number of PBR receptor sites present on immune cells and/or to the levels of serum diazepam after the treatments used. The analytical method provided to be linear in the interest range (dynamic range from 25 to 2000 ng/mL) for diazepam and its main metabolite, desmethyldiazepam. The limits of detection and quantification were 20 and 25 ng/mL, respectively, and the parameters analyzed provided to be satisfactory. Finally, the method developed utilizing the liquid-phase microextraction technique showed to be practical, with low costs and thus presenting potential to be used previously to the extraction procedures, i.e., prior to the injection into the gas-chromatography apparatus.