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.

<p dir="ltr">This thesis describes advancements in both chemical imaging and optical manipulation methodologies for their application in tandem monitoring and control of biochemical processes. We developed a fast acquisition multimodal nonlinear imaging platform based on pulse-picking to minimize photoperturbation to the sample during imaging. By frequency doubling the imaging source, through acousto-optic modulation and simple comparator circuitry, we developed a comprehensive platform that uses chemical specific signals generated during imaging to control the pixel location for laser activation for reaction control. This feedback loop allows for advanced decision logic on a pixel by pixel basis.</p>

1) Aims To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. 2) Methods Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. 3) Results 3View-SEM image stacks as large as 59µm x 59µm x 17µm (xyz) at a resolution of 16nm x 16nm x 50 nm and high resolution (5nm x 5nm x 10nm) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium sheet-like morphology. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. 4) Conclusions Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.

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.

Entender os processos em superfície/interface de filmes e seus efeitos sobre as propriedades ópticas e elétricas de materiais orgânicos é de grande importância tecnológica. Esta pesquisa descreve a fabricação e caracterização de filmes poliméricos extremamente finos (espessura <10 nm) e homogêneos compostos por camadas de polímero/polieletrólitos e estruturas com modulação de energia ou poços quânticos. O objetivo principal foi o estudo dos processos de transferência de carga e energia em tais estruturas. Os polímeros luminescentes utilizados foram poli(9,9 dioctilfluoreno) (PFO) poli(p-fenileno vinileno (PPV). O PPV foi obtido a partir do precursor poli(cloreto de tetraidrotiofeno de xililideno) (PTHT). A técnica de deposição denominada deposição camada por camada assistida por spin (SA-LbL) foi utilizada para obtenção dos filmes. Medidas de absorbância confirmaram o crescimento linear das camadas para as interfaces polieletrólito/polieletrólito e polímero/polieletrólito. Com o objetivo de entender a transferência do elétron &pi; do polímero conjugado para o polieletrólito, as configurações das estruturas poliméricas foram alteradas através da deposição de diferentes monocamadas de polieletrólito sobre o filme polimérico. Observamos que os elétrons &pi; foram efetivamente transferidos para os polieletrólitos que possuem alta afinidades eletrônica. Este efeito interfere fortemente na absorção bem como nas características de condução do filme polimérico ultrafino. A absorção é restabelecida após a conversão de PTHT em PPV. Medidas de fotoluminescência (PL) em filmes PFO/PPV resultam em curvas de emissão com picos característicos de ambos os polímeros, o que confirma que a técnica SA-LbL permite a deposição de estruturas poliméricas multicamadas. As várias configurações de filmes obtidas elucidaram os processos de transferência que ocorrem em diferentes interfaces, tais como: mudança da sequencia de deposição do polieletrólito, número de camadas duplas PTHT/DBS e a introdução de camadas separadoras. Além disso, um único poço quântico, ou seja, estruturas formadas por uma camada PPV cercada por barreiras de PFO com 10 nm de espessura foram obtidas. Medidas de absorbâcia, PL e excitação mostraram uma eficiente migração estado excitado da barreira de PFO para o PPV (poço). A homogeneidade da imagem confocal, demonstrou um rigoroso controle da camada de cobertura ao nível de um única monocamada e sem contaminação pelos materiais depositados sequencialmente. A microscopia confocal de fluorescência (CFM) e espectroscopia de fluorescência resolvida no tempo (FLIM) foram utilizadas para caracterizar a dinâmica do exciton e o seu confinamento nos poços quânticos. As medidas de CFM demonstraram que excitons que são gerados na barreira de PFO são eficientemente transferidos para o PPV. Além disso, o tempo de decaimento da emissão PFO residual é fortemente reduzido devido a processos de migração concorrentes no poço. O tempo de decaimento de PPV diminui substancialmente para poços com espessuras abaixo de 5 nm como resultado da auto-aniquilação do exciton. Dessa forma, as estruturas de MQW obtidos pela técnica de SA-LbL podem ser usadas para estudar a transferência de energia, efeitos túneis e para a construção de novos dispositivos optoelectrónicos com maior eficiência. / The understanding of surface/interface processes and their effects on optical/electrical properties of organic materials is of strong technological importance. This research describes the fabrication and characterization of extremely thin (thickness <10 nm) and homogeneous multilayered polymeric structures including polymer/poly-electrolyte layers and structures with energy modulation such as quantum well. Our main purpose was the study of charge and energy transfer processes in such energy modulated structures. The luminescent polymers used were Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly(p-phenylenevinylene) (PPV). PPV has been obtained from the poly(xylyliden tetrahydrothiophenium chloride) (PTHT) precursor. The so-called Spin Self-Assembly Layer-by-Layer deposition method (SA-LbL) was utilized to obtain the films. Absorption measurements confirmed the linear growth of layers using for polyelectrolyte/polyelectrolyte and polymer/polyelectrolyte interfaces. In order to understand the &pi;-electron transfer from the conjugated polymer to charged states of the polyelectrolyte, the configurations of the polymeric structures were modified by depositing different polyelectrolyte monolayer on the polymer film. We observed that &pi;-electrons were effectively transferred to polyelectrolytes that have high electron affinities. This effect strongly affects both absorption and conduction features of such very thin polymeric film. The absorption is restored after the conversion of PTHT in PPV. Photoluminescence measurements on PFO/PPV films result in emission curves with characteristic peaks of both polymers, confirming that SA-LbL technique allows deposition of multilayer polymeric structures. The various film configurations elucidates the transfer processes occurring at different interfaces like: change of polyelectrolyte deposition order, number of PTHT/DBS bilayers and introduction of spacers. In addition, Single Quantum Well (SQW), i.e., structures consisted of PPV layer surrounded of 10 nm thick PFO barriers were obtained. Optical absorption, PL and excitation spectroscopy showed an efficient excited state migration from the PFO barrier to the PPV well. The confocal image homogeneity demonstrated the layer coverage control at a monolayer level and without layer intermixing of the sequentially deposited polymeric materials. High resolution Confocal Fluorescence Microscopy (CFM) and Fluorescence Life spectroscopy and Imaging (FLIM) were used to characterize the exciton dynamics and confinement in quantum well. The CFM measurements demonstrated that excitons generated at the PFO barrier are efficiently transferred to the PPV well. Furthermore, the decay time of the residual PFO emission is strongly reduced due to the competing migration process in the well. The decay time of PPV decreases substantially for well thicknesses below 5 nm as a result of exciton self-annihilation. Thus, the MQW structures obtained by SA-LbL technique can be used to study energy transfer, tunneling effects and to build up new optoelectronic devices with greater efficiency.

Entender os processos em superfície/interface de filmes e seus efeitos sobre as propriedades ópticas e elétricas de materiais orgânicos é de grande importância tecnológica. Esta pesquisa descreve a fabricação e caracterização de filmes poliméricos extremamente finos (espessura <10 nm) e homogêneos compostos por camadas de polímero/polieletrólitos e estruturas com modulação de energia ou poços quânticos. O objetivo principal foi o estudo dos processos de transferência de carga e energia em tais estruturas. Os polímeros luminescentes utilizados foram poli(9,9 dioctilfluoreno) (PFO) poli(p-fenileno vinileno (PPV). O PPV foi obtido a partir do precursor poli(cloreto de tetraidrotiofeno de xililideno) (PTHT). A técnica de deposição denominada deposição camada por camada assistida por spin (SA-LbL) foi utilizada para obtenção dos filmes. Medidas de absorbância confirmaram o crescimento linear das camadas para as interfaces polieletrólito/polieletrólito e polímero/polieletrólito. Com o objetivo de entender a transferência do elétron &pi; do polímero conjugado para o polieletrólito, as configurações das estruturas poliméricas foram alteradas através da deposição de diferentes monocamadas de polieletrólito sobre o filme polimérico. Observamos que os elétrons &pi; foram efetivamente transferidos para os polieletrólitos que possuem alta afinidades eletrônica. Este efeito interfere fortemente na absorção bem como nas características de condução do filme polimérico ultrafino. A absorção é restabelecida após a conversão de PTHT em PPV. Medidas de fotoluminescência (PL) em filmes PFO/PPV resultam em curvas de emissão com picos característicos de ambos os polímeros, o que confirma que a técnica SA-LbL permite a deposição de estruturas poliméricas multicamadas. As várias configurações de filmes obtidas elucidaram os processos de transferência que ocorrem em diferentes interfaces, tais como: mudança da sequencia de deposição do polieletrólito, número de camadas duplas PTHT/DBS e a introdução de camadas separadoras. Além disso, um único poço quântico, ou seja, estruturas formadas por uma camada PPV cercada por barreiras de PFO com 10 nm de espessura foram obtidas. Medidas de absorbâcia, PL e excitação mostraram uma eficiente migração estado excitado da barreira de PFO para o PPV (poço). A homogeneidade da imagem confocal, demonstrou um rigoroso controle da camada de cobertura ao nível de um única monocamada e sem contaminação pelos materiais depositados sequencialmente. A microscopia confocal de fluorescência (CFM) e espectroscopia de fluorescência resolvida no tempo (FLIM) foram utilizadas para caracterizar a dinâmica do exciton e o seu confinamento nos poços quânticos. As medidas de CFM demonstraram que excitons que são gerados na barreira de PFO são eficientemente transferidos para o PPV. Além disso, o tempo de decaimento da emissão PFO residual é fortemente reduzido devido a processos de migração concorrentes no poço. O tempo de decaimento de PPV diminui substancialmente para poços com espessuras abaixo de 5 nm como resultado da auto-aniquilação do exciton. Dessa forma, as estruturas de MQW obtidos pela técnica de SA-LbL podem ser usadas para estudar a transferência de energia, efeitos túneis e para a construção de novos dispositivos optoelectrónicos com maior eficiência. / The understanding of surface/interface processes and their effects on optical/electrical properties of organic materials is of strong technological importance. This research describes the fabrication and characterization of extremely thin (thickness <10 nm) and homogeneous multilayered polymeric structures including polymer/poly-electrolyte layers and structures with energy modulation such as quantum well. Our main purpose was the study of charge and energy transfer processes in such energy modulated structures. The luminescent polymers used were Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO) and poly(p-phenylenevinylene) (PPV). PPV has been obtained from the poly(xylyliden tetrahydrothiophenium chloride) (PTHT) precursor. The so-called Spin Self-Assembly Layer-by-Layer deposition method (SA-LbL) was utilized to obtain the films. Absorption measurements confirmed the linear growth of layers using for polyelectrolyte/polyelectrolyte and polymer/polyelectrolyte interfaces. In order to understand the &pi;-electron transfer from the conjugated polymer to charged states of the polyelectrolyte, the configurations of the polymeric structures were modified by depositing different polyelectrolyte monolayer on the polymer film. We observed that &pi;-electrons were effectively transferred to polyelectrolytes that have high electron affinities. This effect strongly affects both absorption and conduction features of such very thin polymeric film. The absorption is restored after the conversion of PTHT in PPV. Photoluminescence measurements on PFO/PPV films result in emission curves with characteristic peaks of both polymers, confirming that SA-LbL technique allows deposition of multilayer polymeric structures. The various film configurations elucidates the transfer processes occurring at different interfaces like: change of polyelectrolyte deposition order, number of PTHT/DBS bilayers and introduction of spacers. In addition, Single Quantum Well (SQW), i.e., structures consisted of PPV layer surrounded of 10 nm thick PFO barriers were obtained. Optical absorption, PL and excitation spectroscopy showed an efficient excited state migration from the PFO barrier to the PPV well. The confocal image homogeneity demonstrated the layer coverage control at a monolayer level and without layer intermixing of the sequentially deposited polymeric materials. High resolution Confocal Fluorescence Microscopy (CFM) and Fluorescence Life spectroscopy and Imaging (FLIM) were used to characterize the exciton dynamics and confinement in quantum well. The CFM measurements demonstrated that excitons generated at the PFO barrier are efficiently transferred to the PPV well. Furthermore, the decay time of the residual PFO emission is strongly reduced due to the competing migration process in the well. The decay time of PPV decreases substantially for well thicknesses below 5 nm as a result of exciton self-annihilation. Thus, the MQW structures obtained by SA-LbL technique can be used to study energy transfer, tunneling effects and to build up new optoelectronic devices with greater efficiency.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance – PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol – using 2H solidstate nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

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.

No description available.