De nouveaux nano-objets anisotropes fluorescents sont obtenus par l’assemblage d’acènes spécifiquement conçus. Dans des cristaux, nano-rubans et nanoparticules anisotropes de 2,3-dialkyldiphenylanthracènes, les efficacités et la polarisation de l’émission bleue sont remarquables. La couleur de l’émission est accordée par le dopage avec des émetteurs verts et oranges (di- et tétra-phényltétracènes). La microscopie confocale de fluorescence permet d’étudier les cinétiques des états excités et des transferts d’énergie photo-induits, ainsi que la dispersion et les orientations des émetteurs. Pour la première fois, l’influence de la largeur de nano-rubans sur la cinétique d’annihilations triplet-triplet de tétracènes est mise en évidence. La microscopie révèle également le polymorphisme inhabituel d’un dérivé diéthynylphényl-anthracène. Ce travail ouvre des perspectives pour le développement et l’étude de processus fondamentaux de nano-matériaux luminescents. / New fluorescent anisotropic nano-objects are obtained by the assembly of specifically designed acenes. In crystals, nano-ribbons and anisotropic nanoparticles of 2,3-dialkyldiphenylanthracenes, the efficiencies and the polarization of the blue emission is remarkable. The color of the emission is tuned by doping with green and orange emitters (di-and tetra-phenyltetracenes). Confocal fluorescence microscopy is used to study the kinetics of excited states and photo-induced energy transfers, as well as the dispersion and orientation of the emitters. For the first time, the influence of the width of the nano-ribbons on the kinetics of tetracene triplet-triplet annihilations is highlighted. Microscopy also reveals the unusual polymorphism of a diethynylphenyl anthracene derivative. This work opens perspectives for the development and study of fundamental processes of luminescent nano-materials.

A terapia fotodinâmica (TFD) utilizando ácido 5-aminolevulinico (ALA) e derivados em aplicação tópica e, como precursor da protoporfirina IX (PPIX) apresenta alguns limitantes relativos a baixa permeação das substâncias na pele. Comportamento este que afeta a produção e homogeneidade da distribuição da PPIX na superfície e camadas mais profundas da pele. Para resolver essa limitação muitos autores propõem alternativas modificando a molécula do ALA e derivados, bem como modificando as propriedades químicas da fase externa da emulsão (mais hidrofílica ou hidrofóbica) ou então o sistema de entrega para a emulsão. O objetivo desse estudo é avaliar qual a proporção de ALA e metil-5-aminolevulinato (MAL) que quando misturados levam ao aumento da quantidade e uniformidade da formação da PPIX na superfície e em profundidade na pele. Para esse estudo foi realizada análises de fluorescência e histologia. O estudo foi conduzido in vivo e ex vivo usando biópsias de pele de porco cultivadas in vitro. A produção de PPIX foi monitorada utilizando espectroscopia de fluorescência, imagem de fluorescência de campo amplo e microscopia confocal de fluorescência. E para a aplicação da TFD os parâmetros usados foram de 125 mW/cm2 de intensidade e 150 J/cm2 de dose. A análise do dano causado pela irradiação foi realizada por meio de histologia da pele após 24 e 48 horas da aplicação da TFD. O ALA e MAL na concentração de 20% foram misturados nas seguintes proporções: ALA ou M, M2 (80% ALA - 20% MAL), M3 (60% ALA 40% MAL), M4 (50% ALA MAL), M5 (40% ALA 60% MAL), M6 (20% ALA 80% MAL) e MAL como M7. As diferentes proporções foram incorporadas em emulsões óleo em água (O/A) e água em óleo (A/O). De acordo com os resultados, as misturas M3, M4 e M5 mostraram maior produção de PPIX na superfície da pele segundo as medidas de fluorescência em 3h de incubação e, no estudo da cinética mostraram produzir PPIX em menor tempo. No estudo de permeação do creme in vitro em pele ex vivo, por microscopia confocal de fluorescência, observou-se que as misturas M3, M4 e M5 produziram mais PPIX nas camadas da pele do que ALA e MAL. As análises histológicas das misturas apresentaram maior dano fotodinâmico na superfície e profundidade das camadas da pele após a TFD, independente da emulsão. A análise em até 48h observou-se predominantemente a fase do processo de reparo referente à fase inflamatória, mas existem indícios ao longo das análises tanto macroscópicas e histológicas que o processo de reparo referente as fases subsequentes de proliferação e remodelamento estão iniciando-se em paralelo. A mistura M4 em ambas as emulsões apresentou elevada quantidade de formação de PPIX em menor tempo de incubação. M4 em emulsão O/A apresentou menor dano fotodinâmico já que a evolução do processo reparo foi mais rápida sugerindo-se potencial de aplicação em TFD voltado para área cosmética-estética. Já M4 em emulsão A/O levou a um maior dano fotodinâmico já que a evolução do processo de reparo foi mais lenta sugerindo-se potencial de aplicação em TFD voltado para área oncológica e de doenças de pele. De modo geral o estudo proposto apresentou impacto positivo para a otimização da terapia fotodinâmica em aplicação tópica. / Photodynamic therapy (PDT) using 5-aminolevulinic acid and derivatives on topical application and as a precursor of protoporphyrin (PPIX) has some limitations for low permeation of substances into the skin. This behavior affects PPIX production and homogeneous distribution on the surface and deeper layers of the skin. To resolve this limitation, many authors propose alternatives such as modifying the molecule of ALA and its derivatives, as well as changing the chemical properties of the external phase of the emulsion (more hydrophilic or hydrophobic) or the delivery system to the emulsion. The aim of this study is to assess the proportion of ALA and methyl-5-aminolevulinate (MAL) that when mixed leads to an increase in the amount and uniformity of the PPIX formation on surface and deep skin. For this study we performed fluorescence analysis and histology. The studies were conducted in vivo and also using pig skin biopsies (ex vivo) cultured in vitro. The PPIX production was monitored using fluorescence spectroscopy, widefield fluorescence imaging, and fluorescence confocal microscopy. For the application of PDT an intensity of 125 mW/cm2 and a dose 150 J/cm2 were used. Analysis of the damage caused by irradiation was performed through skin histology after 24 and 48 hours after PDT application. ALA and MAL in concentration of 20% were mixed in the following proportions: ALA or M, M2 (80% ALA - 20% MAL), M3 (60% ALA - 40% MAL), M4 (50% ALA - MAL) M5 (40% ALA - 60% MAL), M6 (20% ALA - 80% MAL) MAL and as M7. Different proportions were incorporated in oil-in-water emulsions (O/W) and water-in-oil (W/O). The fluorescence measurements for 3h of incubation showed better PPIX production in the skin surface for mixtures M3, M4 and M5. Moreover, the kinetics study showed PPIX production in less time for these mixtures. In the study of cream permeation of ex vivo skin in vitro by confocal fluorescence microscopy, we observed that the mixtures M3, M4 and M5 produced more PPIX in the skin layers than ALA and MAL. The histological analyses of the mixtures showed higher photodynamic damage on the surface and deeper layers of the skin after PDT, independent of the emulsion. The analysis in 48 hours predominantly observed the phase of the healing process regarding the inflammatory phase but there are signs along both macroscopic and histological analysis that the healing process concerning the subsequent stages of proliferation and remodeling are initiating in parallel. The mixture M4 in both emulsions had high amounts of PPIX formation in shorter incubation time. M4 emulsion O/A showed a lower photodynamic damage since the evolution of the healing process was faster suggesting to potential application in PDT facing cosmetic-aesthetic area. M4 already in W/O emulsion led to a greater photodynamic damage since the evolution of the healing process was slower suggesting to potential application in PDT facing oncology and skin diseases. Overall the proposed study had a positive impact on the optimization of photodynamic therapy for topical application.

[Truncated abstract] Articular cartilage (AC) covers the surface of synovial joints providing a nearly frictionless bearing surface and distributing mechanical load. Joint trauma can damage the articular surface causing pain, loss of mobility and deformation. Currently there is no uniform treatment protocol for managing focal cartilage defects, with most treatment options targeted towards symptomatic relief but not limiting the progression into osteoarthritis (OA). Autologous chondrocyte implantation (ACI) and more recently matrix-induced autologous chondrocyte implantation (MACI), have emerged as promising methods for producing hyaline or hyaline-like repair tissue, however there remains some controversy regarding the exact histological nature of the tissue formed. Histological characterisation of AC repairs requires destructive tissue biopsy potentially inducing further joint pathology thereby negating the treatment effect. OA is recognised as a major cause of pain, loss of function and disability in Western populations, however the exact aetiology is yet to be elucidated. The assessment of both OA and cartilage repair has been limited to macroscopic observation, radiography, magnetic resonance imaging (MRI) or destructive biopsy. The development of non-destructive AC assessment modalities will facilitate further development of AC repair techniques and enable early monitoring of OA changes in both experimental animal models and clinical subjects. Confocal laser scanning microscopy (CLSM) is a type of fluorescence microscopy that generates high-resolution three-dimensional images from relatively thick sections of tissue. ... Biomechanical analysis suggested that the mechanical properties of MACI tissue remain inferior for at least three months. This study showed the potential of a multi-site sheep model of articular cartilage defect repair and validated its assessment via LSCA. Finally, the LSCA was used to arthroscopically image the cartilage of an intact fresh frozen cadaveric knee from a patient with clinically diagnosed OA. Images were correlated to ICRS (Outerbridge) Grades I-IV and histology. The LSCA gave excellent visualization of cell morphology and cell density to a depth of up to 200'm. Classical OA changes including clustering chondrocytes, surface fibrillation and fissure formation were imaged. Fair to moderate agreement was demonstrated with statistically significant correlations between all modalities. This study confirmed the viability of the LSCA for non-destructive imaging of the microstructure of the OA cartilage. In conclusion, the LSCA identified histological features of orthopaedic tissues, accurately quantified chondrocyte morphology and demonstrated classical OA changes. While the development and investigation of an ovine model of cartilage repair showed the treatment benefit of MACI, some biomechanical issues remain. Ultimately, the LSCA has been demonstrated as a reliable nondestructive imaging modality capable of providing optical histology without the need for mechanical biopsy. Medical Subject Headings (MESH): articular cartilage; autologous chondrocyte implantation; matrix-induced autologous chondrocyte implantation; biomechanics; cartilage; confocal microscopy; diagnosis; histology; image analysis; immunohistochemistry; magnetic resonance imaging; microscopy; osteoarthritis

No description available.

A terapia fotodinâmica (TFD) utilizando ácido 5-aminolevulinico (ALA) e derivados em aplicação tópica e, como precursor da protoporfirina IX (PPIX) apresenta alguns limitantes relativos a baixa permeação das substâncias na pele. Comportamento este que afeta a produção e homogeneidade da distribuição da PPIX na superfície e camadas mais profundas da pele. Para resolver essa limitação muitos autores propõem alternativas modificando a molécula do ALA e derivados, bem como modificando as propriedades químicas da fase externa da emulsão (mais hidrofílica ou hidrofóbica) ou então o sistema de entrega para a emulsão. O objetivo desse estudo é avaliar qual a proporção de ALA e metil-5-aminolevulinato (MAL) que quando misturados levam ao aumento da quantidade e uniformidade da formação da PPIX na superfície e em profundidade na pele. Para esse estudo foi realizada análises de fluorescência e histologia. O estudo foi conduzido in vivo e ex vivo usando biópsias de pele de porco cultivadas in vitro. A produção de PPIX foi monitorada utilizando espectroscopia de fluorescência, imagem de fluorescência de campo amplo e microscopia confocal de fluorescência. E para a aplicação da TFD os parâmetros usados foram de 125 mW/cm2 de intensidade e 150 J/cm2 de dose. A análise do dano causado pela irradiação foi realizada por meio de histologia da pele após 24 e 48 horas da aplicação da TFD. O ALA e MAL na concentração de 20% foram misturados nas seguintes proporções: ALA ou M, M2 (80% ALA - 20% MAL), M3 (60% ALA 40% MAL), M4 (50% ALA MAL), M5 (40% ALA 60% MAL), M6 (20% ALA 80% MAL) e MAL como M7. As diferentes proporções foram incorporadas em emulsões óleo em água (O/A) e água em óleo (A/O). De acordo com os resultados, as misturas M3, M4 e M5 mostraram maior produção de PPIX na superfície da pele segundo as medidas de fluorescência em 3h de incubação e, no estudo da cinética mostraram produzir PPIX em menor tempo. No estudo de permeação do creme in vitro em pele ex vivo, por microscopia confocal de fluorescência, observou-se que as misturas M3, M4 e M5 produziram mais PPIX nas camadas da pele do que ALA e MAL. As análises histológicas das misturas apresentaram maior dano fotodinâmico na superfície e profundidade das camadas da pele após a TFD, independente da emulsão. A análise em até 48h observou-se predominantemente a fase do processo de reparo referente à fase inflamatória, mas existem indícios ao longo das análises tanto macroscópicas e histológicas que o processo de reparo referente as fases subsequentes de proliferação e remodelamento estão iniciando-se em paralelo. A mistura M4 em ambas as emulsões apresentou elevada quantidade de formação de PPIX em menor tempo de incubação. M4 em emulsão O/A apresentou menor dano fotodinâmico já que a evolução do processo reparo foi mais rápida sugerindo-se potencial de aplicação em TFD voltado para área cosmética-estética. Já M4 em emulsão A/O levou a um maior dano fotodinâmico já que a evolução do processo de reparo foi mais lenta sugerindo-se potencial de aplicação em TFD voltado para área oncológica e de doenças de pele. De modo geral o estudo proposto apresentou impacto positivo para a otimização da terapia fotodinâmica em aplicação tópica. / Photodynamic therapy (PDT) using 5-aminolevulinic acid and derivatives on topical application and as a precursor of protoporphyrin (PPIX) has some limitations for low permeation of substances into the skin. This behavior affects PPIX production and homogeneous distribution on the surface and deeper layers of the skin. To resolve this limitation, many authors propose alternatives such as modifying the molecule of ALA and its derivatives, as well as changing the chemical properties of the external phase of the emulsion (more hydrophilic or hydrophobic) or the delivery system to the emulsion. The aim of this study is to assess the proportion of ALA and methyl-5-aminolevulinate (MAL) that when mixed leads to an increase in the amount and uniformity of the PPIX formation on surface and deep skin. For this study we performed fluorescence analysis and histology. The studies were conducted in vivo and also using pig skin biopsies (ex vivo) cultured in vitro. The PPIX production was monitored using fluorescence spectroscopy, widefield fluorescence imaging, and fluorescence confocal microscopy. For the application of PDT an intensity of 125 mW/cm2 and a dose 150 J/cm2 were used. Analysis of the damage caused by irradiation was performed through skin histology after 24 and 48 hours after PDT application. ALA and MAL in concentration of 20% were mixed in the following proportions: ALA or M, M2 (80% ALA - 20% MAL), M3 (60% ALA - 40% MAL), M4 (50% ALA - MAL) M5 (40% ALA - 60% MAL), M6 (20% ALA - 80% MAL) MAL and as M7. Different proportions were incorporated in oil-in-water emulsions (O/W) and water-in-oil (W/O). The fluorescence measurements for 3h of incubation showed better PPIX production in the skin surface for mixtures M3, M4 and M5. Moreover, the kinetics study showed PPIX production in less time for these mixtures. In the study of cream permeation of ex vivo skin in vitro by confocal fluorescence microscopy, we observed that the mixtures M3, M4 and M5 produced more PPIX in the skin layers than ALA and MAL. The histological analyses of the mixtures showed higher photodynamic damage on the surface and deeper layers of the skin after PDT, independent of the emulsion. The analysis in 48 hours predominantly observed the phase of the healing process regarding the inflammatory phase but there are signs along both macroscopic and histological analysis that the healing process concerning the subsequent stages of proliferation and remodeling are initiating in parallel. The mixture M4 in both emulsions had high amounts of PPIX formation in shorter incubation time. M4 emulsion O/A showed a lower photodynamic damage since the evolution of the healing process was faster suggesting to potential application in PDT facing cosmetic-aesthetic area. M4 already in W/O emulsion led to a greater photodynamic damage since the evolution of the healing process was slower suggesting to potential application in PDT facing oncology and skin diseases. Overall the proposed study had a positive impact on the optimization of photodynamic therapy for topical application.

Made available in DSpace on 2016-06-02T19:04:00Z (GMT). No. of bitstreams: 1 6814.pdf: 27157124 bytes, checksum: ccc98f69d1fc4cdc487ac2e9917edfc0 (MD5) Previous issue date: 2015-03-09 / Microelectrode arrays (MEA) are devices that allow chemical and electrical stimulation and recording of the extracellular electrical activity from entire neuronal cultures over long periods of time, such as several weeks. Some MEA models have transparent substrate, which enables the imaging of culture using optical microscopy. The images are taken from two channels: fluorescence light and transmitted light channels. In the first one, it is possible to visualize the neurons, while in the other one, it is possible to observe the microelectrodes. The objective of this work is to develop methods that enable performing quantitative analysis of the dissociated culture of rat dorsal root ganglion (DRG) neurons plated on MEA by means of the processing of the images, obtained from confocal fluorescence microscopy. We proposed and developed the following methods in order to achieve this objective: (A) A method to automatically identify the microelectrodes in the transmitted light channel using circular Hough Transform and error correction based on the Delaunay triangulation; (B) the registration of a number of images taken at different parts of the MEA in order to generate a unique and high-resolution representation of the whole culture; (C) the segmentation of the neuron in 2D images taken from the fluorescence channel, composed by the steps: preprocessing, thresholding, morphological filtering, neurons occlusion correction, watershed transform and object classification; (D) 2D quantitative analysis based on the identified microelectrodes and on the segmented neurons; (E) a method for generating 3D polygonal models of the neurons from the volumetric images, to be used for visualizing the culture on the MEA by different points of view and zoom levels; and (F) 3D quantitative analysis performed by the processing of the polygonal surfaces in conjunction with the information about the microelectrodes positioning. The results show that the methods are capable to identify the neurons and microelectrodes on the 2D images efficiently. In the 3D images, the preprocessing step which uses information from the 2D segmentation method, showed to be capable to generate correct polygonal models efficiently. Most of the studies involving the analysis of neuron cultures on MEAs consider only qualitative analysis or simple quantitative measures. However, the methods proposed in this thesis enables to obtain important measures related to the neuron culture, such as: the density and morphology of the neurons, and the spatial and topological distribution of the neurons and microelectrodes. The information about neuron morphology is important because they are related to the behavior of this kind of neuron. The spatial and topological distribution of neurons and microelectrodes are used for providing models of the interface between these elements, for supporting the analysis of the electrophysiological signal recorded by the microelectrodes, as well as in the computational simulations of the neuron culture behavior. / Matrizes de Microeletrodos (MEAs) são dispositivos que permitem estimular quimicamente ou eletricamente e registrar a atividade elétrica extracelular de culturas de neurônios durante um longo período de tempo, da ordem de várias semanas. Modelos de MEAs com o substrato transparente permitem imagear a cultura por meio de microscopia óptica. As imagens são obtidas em dois canais: um de luz de fluorescência e outro de luz de transmissão. O primeiro permite visualizar os neurônios, enquanto o segundo os microeletrodos. O objetivo deste trabalho é desenvolver métodos que permitam realizar análises quantitativas de culturas dissociadas de neurônios de gânglio da raiz dorsal (Dorsal Root Ganglion DRG) de ratos em MEAs por meio do processamento de imagens obtidas por microscopia confocal de fluorescência. Os seguintes métodos foram propostos e desenvolvidos para atingir este objetivo: (A) Identificação automática dos microeletrodos nas imagens do canal de luz de transmissão utilizando a transformada de Hough circular e correção de erros baseado na triangulação de Delaunay; (B) Registro de várias imagens tomadas de diferentes regiões da MEA para gerar uma única imagem em alta resolução que contemple a cultura toda; (C) Segmentação dos neurônios em imagens 2D obtidas a partir do canal de fluorescência, composto por etapas de pré-processamento, segmentação, filtragem morfológica, correção da oclusão de neurônios, transformada watershed e classificação de objetos; (D) Análise quantitativa 2D baseada nos microeletrodos identificados e nos neurônios segmentados; (E) Método para geração de modelos poligonais 3D dos neurônios a partir de imagens volumétricas, modelos os quais são utilizados para visualização da cultura na MEA por diferentes pontos de vista e níveis de zoom; e (F) Análise quantitativa 3D realizada por meio do processamento das superfícies poligonais juntamente com as informações sobre a posição dos microeletrodos. Os resultados mostram que os métodos são capazes de identificar com eficiência os neurônios e microeletrodos presentes nas imagens 2D. Nas imagens 3D, a etapa de pré-processamento utilizando informações resultantes do método de segmentação 2D se mostrou eficiente na geração dos modelos poligonais corretos. Enquanto a maioria das análises de imagens de culturas de neurônios em MEA consideram apenas análises quantitativas simples, os métodos aqui propostos permitem obter importantes medidas quantitativas relacionadas às culturas, tais como: a densidade e morfologia dos neurônios, assim como a distribuição espacial e topológica dos neurônios em relação aos microeletrodos. As informações sobre a morfologia são importantes, pois estão relacionadas com o comportamento desse tipo de neurônio. A distribuição espacial e topológica dos neurônios e microeletrodos permitem modelar a interface entre neurônios e microeletrodos e auxiliar nos estudos dos sinais eletrofisiológicos capturados pelos microeletrodos, assim como em simulações computacionais do comportamento dessas culturas.

Le principal objectif de cette thèse a consisté au développement d’une méthode de screenning pour la découverte de nouvelles réactions de ligations bioorthogonales ainsi que son application sur une bibliothèque développée pour cette étude. Par conséquent, un système de screening a été conçu en trois étapes consistant au départ en une analyse HPLC, puis une évaluation basée sur la fluorescence de haute résolution et finalement un test de microscopie confocal in cellulo. Puis, nous avons standardisé toutes les analyses avec les réactions CuAAC et SpAAC. En outre, nous avons synthétisés 18 réactifs d’intérêts et effectué un screening de 58 expériences de ligation avec une évaluation par méthode HPLC. Parmi les 9 réponses positives obtenues figure 6 réactions impliquant de nouveaux réactifs et les analyses LC‐MS ont pu tous les valider comme des réactions de cycloaddition directe à l’exception d’une réaction. Finalement, nous avons pu appliquer la méthode in cellulo développée, afin d’évaluer la pertinence des réactions de chélation CuAAC pour une application sur cellules. / The main goal of this thesis was the development of a screening method for the discovery of new bioorthogonal ligation reactions as well as its application on a self‐designed library. Therefore we designed a three step screening system consisting of a preliminary HPLC assay, a high resolution fluorescence based assay and a final in cellulo confocal microscopy assay.Subsequently we standardized all assays with the highly established CuAAC and SpAAC. Furthermore, we successfully synthesized 18 reagents of interest and screened 58 ligation experiments with the help of the HPLC setup. The 9 positive hits from this screening contained 6 reactions involving novel reagents and LCMS analysis was able to validate all but one as straight forward cycloaddition reaction. Finally we were able to apply the newly developed in cellulo assay to assess the suitability of chelating CuAAC for in cell application.

Indiana University-Purdue University Indianapolis (IUPUI) / The young field of gene therapy offers the promises of significant progress towards the treatment of many different types of human diseases. Gene therapy has been proposed as an innovative way to treat Acute Kidney Injury (AKI). Through proteomic analysis, the upregulation of two enzymes, IDH2 and SULT1C2, within the mitochondrial fraction has been identified following ischemic preconditioning, a treatment by which rat kidneys are protected from ischemia. Using the hydrodynamic fluid gene delivery technique, we were able to upregulate the expression of IDH2 and SULT1C2 in the kidney. We found that the delivery of IDH2 plasmid through hydrodynamic fluid delivery to the kidney resulted in increased mitochondrial oxygen respiration compared with injured kidneys without gene delivery. We also found that renal ischemic preconditioning altered the membrane fluidity of mitochondria. In conclusion, our study supports the idea that upregulated expression of IDH2 in mitochondria can protect the kidney against AKI, while the protective function of upregulated SULT1C2 needs to be further studied.

Indiana University-Purdue University Indianapolis (IUPUI) / Acute Kidney Injury (AKI) is a prevalent and significant problem whose primary treatment is supportive care. Ischemic preconditioning is a strategy used to protect organs from ischemic injury via a prior injury. Ischemic preconditioning in the kidneys has been shown to confer protection onto kidneys from subsequent ischemic insults with attenuated serum creatinine values in treated rats. In the preconditioned kidneys, the enzyme IDH2 was discovered to be upregulated in the mitochondria. Hydrodynamic fluid delivery to the kidney was found to be a viable technique for delivering this gene to the kidney, resulting in artificially upregulated expression of IDH2. Via a two-pronged effort to discern the functional significance of ischemic preconditioning and hydrodynamic IDH2 fluid injections, we performed mitochondrial oxygen respiration assays on both preconditioned and injected kidneys. We found that renal ischemic preconditioning resulted in no significant difference between sham and preconditioned, subsequently injured kidneys, which is similar to the results from the serum creatinine studies. Hydrodynamically IDH2-injected, and subsequently injured kidneys respire significantly better than vehicle injected, and subsequently injured kidneys, which shows that hydrodynamic injections of IDH2 protects kidneys against injury, and partially mimics the effects of preconditioning.

The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api795. The uptake was reduced for both peptides by 50 and 80%, respectively, after inhibiting endocytotic uptake with dynasore. In summary, Api794 and Api795 were highly active against P. aeruginosa in vitro. Both peptides passed across the bacterial membrane efficiently, most likely then disturbing the ribosome assembly, and resulting in further intracellular damage. Api795 with its IOIO-motif, which was particularly active and only slightly toxic in vitro, appears to represent a promising third generation lead compound for the development of novel antibiotics against P. aeruginosa.