Acute lung injury (ALI) is a major cause of mortality after lung transplantation. Recent studies indicate protein kinase C delta (PKCδ) could be an effective target to treat ALI. We have developed a gold nanoparticle (GNP)-peptide hybrid that can inhibit PKCδ signaling. PKCδ inhibitor peptide (PKCi) and 95P2P4 stabilizing peptides were conjugated onto GNP. Physicochemical properties of the nanoformulations were examined. A lung transplant-simulated cell culture model was used to evaluate therapeutic efficacy in vitro. A pulmonary ischemia-reperfusion (IR) model was used to test therapeutic efficacy in vivo. GNP-Peptide hybrids showed good stability with high cellular uptake. GNP-PKCi formulations demonstrated anti-inflammatory and anti-apoptotic effects in vitro. When administered to rats under IR stress, GNP-PKCi formulation improved blood oxygenation, reduced pulmonary edema and histological lung injury. In conclusion, we have successfully formulated a clinically-applicable nanoparticle with therapeutic potential to ameliorate lung injury and inflammation. Our formulation strategy could be used to deliver other peptide-based drugs.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2008" Includes bibliographical references.

Thesis (Ph. D.)--Lunds universitet, 2005. / Title from title screen. Description based on contents viewed May 20, 2005. Includes bibliographical references (p. [36]-[48]).

Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 123-141). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Made available in DSpace on 2016-06-02T20:21:35Z (GMT). No. of bitstreams: 1 6077.pdf: 10672804 bytes, checksum: 8a7ba5e1820e96664e3cbb7f872f6f3e (MD5) Previous issue date: 2013-10-28 / Financiadora de Estudos e Projetos / Over the recent years, the incidence of human fungal infections has shown a significant increase. Aspergillus fumigatus is a filamentous fungus opportunistic pathogen responsible for many human respiratory diseases, including invasive pulmonary aspergillosis, which is the most serious form of infection . Studies show that A. fumigatus virulence has a multifactorial process associated with its structure, capacity for growth, adaptation to stress conditions, evasion mechanisms of the immune system and ability to cause harm to the host. CWI (via cell wall integrity ) is a signaling cascade activated in yeast cells under conditions of cell wall stress and plays a role in the adaptation of various fungal pathogens in the human host . In many fungi , CWI is triggered by activation of protein kinase C ( PKC ) and that this pathway is associated with the transcription of genes related to maintaining the integrity of the cell wall and its redevelopment. In this work, a mutant Gly579Arg (G579R) was constructed by transformation mediated by inserting a gene replacement cassette comprising a G2044C transversion located in the cysteine-rich domain controller C1B pkcA of A. fumigatus. From the phenotypic analysis of the mutant strain was observed in the involvement of pkcAG579R CWI since the mutant showed high sensitivity to agents such as CR (congo red) and CFW (calcofluor white) . Furthermore, pkcA is also involved in tolerance to oxidative stress caused by paraquat and menadione. Additionally it was found to increase the sensitivity of the mutant pkcAG579R temperature variations as well as the inhibitor of Hsp90 radicicol. Como CWI is related to the transcriptional activation of biosynthetic genes and rugged cell wall (such as glucan synthase, glucanosil chitin synthases and transferases) the abundance of major genes coding for these enzymes was analyzed by RT-PCR in real time. Based on the tests can be α -1 ,3 glucan synthase ( agsA-C ) dependent signaling mediated PkcA for correct expression. Furthermore, genes such as β-1,3 glucan synthase (fksA) glucanosyltransferase (gelA-C) and some chitin synthases (chsB-E-C) appear not to be dependent function and CWI PkcA . These data demonstrated the role of pkcA signaling cascade in the maintenance of cell wall and thermotolerance in A. fumigatus. This work was the first in which a systematic analysis of gene pkcA was conducted in the human opportunistic fungal pathogen A. fumigatus. / Ao longo dos últimos anos, a ocorrência de infecções fúngicas humanas vem apresentando um aumento expressivo. Aspergillus fumigatus é um fungo filamentoso patógeno oportunista responsável por diversas doenças respiratórias humanas, incluindo aspergilose pulmonar invasiva, que é a forma de infecção mais grave. Estudos demonstram que o A. fumigatus possui um processo de virulência multifatorial associado a sua estrutura, capacidade de crescimento, adaptação em condições de estresse, mecânismos de evasão do sistema imune e capacidade de causar danos ao hospedeiro. A CWI (via de integridade da parede celular) é uma cascata de sinalização ativada nas células fúngicas sob condições de estresse de parede celular e desempenha um papel na adaptação de vários fungos patogênicos no hospedeiro humano. Em muitos fungos, CWI é desencadeada através da ativação da proteína quinase C (PKC) sendo que esta via está associada à transcrição de genes relacionados com a manutenção da integridade da parede celular e sua remodelação. Neste trabalho o mutante Gly579Arg (G579R) foi construído através da transformação mediada pela inserção de um cassete de substituição gênica que compreende uma transversão G2044C localizado no domínio regulador rico em cisteína C1B da pkcA de A. fumigatus. A partir da análise fenotípica desse mutante foi possível observar o envolvimento de pkcAG579R na CWI uma vez que a linhagem mutante mostrou alta sensibilidade a agentes como o CR (congo red) e CFW (calcofluor white). Além disso, pkcA está envolvido também na tolerância ao estresse oxidativo causado por menadiona e paraquat. Adicionalmente verificou-se o aumento da sensibilidade da linhagem mutante pkcAG579R à variações de temperatura bem como ao inibidor de Hsp90, radicicol. Como a CWI está relacionada à ativação transcricional de genes de biossíntese e reforço de parede celular (como por exemplo glucanas sintases, quitinas sintases e glucanosil transferases), a abundância dos principais genes que codificam essas enzimas foi analisada através de RTPCR em tempo real. Baseado nos testes pode-se verificar que as α-1,3 glucana sintase (agsA-C) dependem da sinalização mediada por PkcA para sua expressão. Por outro lado, genes como a β-1,3 glucana sintase (fksA), glucanosiltransferases (gelA-C) e algumas quitinas sintases (chsB-C-E) parecem não ser dependente da CWI e da função de PkcA. Esses dados demostraram parte do papel de pkcA na cascata de sinalização da manutenção da parede celular e termotolerância em A. fumigatus. Este trabalho foi o primeiro no qual uma análise sistemática do gene pkcA foi conduzida no fungo patógeno oportunista humano A. fumigatus.

Plaquetas apresentam um papel importante no desenvolvimento de metastases tumorais. Os eventos que levam à ativação plaquetária, como agragação e secreção de proteínas, podem significar etapas importantes neste papel. O agonista plaquetário trombospondina está envolvido no processo de agragação plaquetária. Com o intuito de clonar o receptor de trombospondina GpIIIb, produziu-se um soro policlonal contra uma banda eluída de SDS PAGE de extrato proteico de plaquetas, que apresentava peso molecular igual ao de GpIIIb (denominada banda 80kD). Uma biblioteca de cDNA de endotélio de cordão umbilical humano construída em lambda gt11 foi varrida com este soro anti-80kD. Dois clones diferentes foram isolados, seus insertos foram subclonados no vetor pGEM-3Z e sequenciados. Através de consulta ao Genbank observou-se que um dos clones não apresentou homologia significativa com nenhuma proteína até então clonada. O outro clone, por sua vez, apresentou 100% de homologia com a proteína 80K-H, substrato de proteína quinase C. Levando em consideração o fato de que as vias detransdução de sinal que utilizam PKC apresentam extrema importância nos processos de ativação plaquetária decidiu-se prosseguir com a caracterização de 80K-H. Para isto foi produzido um soro policlonal contra a proteína de fusão 80K-H, que foi utilizado em ensaios bioquímicos e imunoquímicos que permitiram caracterizar a proteína 80K-H quanto a alguns aspectos como distribuição em diferentes tipos celulares, localização celular e fosforilação. Além de estar presente em plaquetas, a proteína 80K-H foi encontrada em todas as linhagens celulares testadas, parecendo portanto ser uma proteína ubíqua. Os dados obtidos indicaram que, apesar de apresentar uma sequência N-terminal que é clivada \"in vivo\" muito semelhante a um peptídeo sinal, 80K-H não é secretada nem é de membrana plasmática, mas sim citoplasmática. Em ensaios de fosforilação \"in vivo\" não se detectou fosforilação de 80K-H. Portanto, apesar de 80K-H ser um bom substrato para PKC \"in vitro\", ela não o é \"in vivo\", ao menos nas células analisadas, ou é fosforilada de uma forma extremamente rápida e transiente. / Abstract not available.

Abstract This study examined the role of tumor suppressor neurofibromin and Protein kinase C (PKC) in urinary bladder cancer, and the effect of PKC inhibitors on cancer cell behaviour. Tumor suppressor protein neurofibromin is a product of the NF1 gene, a mutation of which causes the most common hereditary tumor syndrome, type 1 neurofibromatosis. NF1 gene mutations and changes in expression have been demonstrated in malignancies, unrelated to type 1 neurofibromatosis. The best known function of neurofibromin is its Ras GTPase accelerating function. Thus, it functions as a Ras inactivator. This study demonstrated for the first time that the NF1 gene is expressed in normal and malignant urinary bladder epithelium and in cultured bladder carcinoma cells in mRNA and at the protein level. Furthermore, neurofibromin expression is decreased during bladder carcinogenesis. It can be speculated that this may lead to increased Ras activity in urinary bladder cancer. The PKC family is composed of several different isoenzymes which are responsible for a number of important intracellular events and cellular functions. Many of these are also important in cancer development and progression. The results demonstrate changes in expression of PKC α and βI isoenzymes in urinary bladder carcinoma. Furthermore, the results relate the increased expression of isoenzymes to increased PKC enzyme activity and the high proliferation rate of the cancer cells. In addition, this study utilizes small molecular inhibitors of PKC isoenzymes in order to study the effect of the inhibition of these isoenzymes on cancer cell behaviour in vitro and in vivo. The study mainly focuses on the function of PKC α and βI isoenzymes and on the effects of inhibition of these by using Go6976. The results show that Go6976 inhibits cancer cell growth, migration and invasion in vitro, and tumor growth in a mouse model. The use of Go6976 induces changes in desmosomes and adherens junctions, and in focal adhesions and hemidesmosomes. The results also show that Go6976 functions as a cell cycle checkpoint abrogator and increases the cytotoxicity of two classical chemotherapeutic agents, doxorubicin and paclitaxel. In the future, it may be possible that Go6976 or related drugs could be used in clinical cancer treatments.

The mortality of cardiovascular diseases remains high and it likely tends to increase in the future. Although many ways how to increase the resistance against myocardial ischemia- reperfusion damage have been described, few of them were transferred into clinical practice. Cardioprotective effect of chronic hypoxia has been described during 60s of the last century. Its detailed mechanism has not been elucidated, but a number of components has been identified. One of these components presents protein kinase C (PKC). The role of PKC was described in detail in the mechanism of ischemic preconditioning, but its involvement in the mechanism of cardioprotection induced by chronic hypoxia remains unclear. One reason is the amount of PKC isoforms, which have often contradictory effects, and the diversity of hypoxic models used. The most frequently mentioned isoforms in connection with cardioprotection are PKCδ and PKCε. The aim of my thesis was to analyze changes in these PKC isoforms at two different cardioprotective models of hypoxia - intermittent hypobaric (IHH) and continuous normobaric hypoxia (CNH). We also examined the target proteins of PKCδ and PKCε after the adaptation to IHH, which could be involved in the mechanism of cardioprotection. These included proteins associated with apoptosis and...

Hydrogen peroxide (H2O2) is a reactive oxygen species generated by several metabolic pathways in mammalian cells. Endothelial cells are extremely susceptible to oxidative stress. H2O2 has been reported to increase the permeability in these cells. Using rat heart endothelial cell culture as a model system, we examined the effect of H2O2 on the gene expression of vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and a vascular permeability factor. By Northern blot analysis we found that VEGF mRNA responded to H2O2 in a dose-and time- dependent manner. The induction was superinduced by cycloheximide and blocked by actinomycin D. N-Acetylcysteine, a synthetic antioxidant, was able to suppress the induction. H7, a protein kinase C inhibitor, could also block the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors, AP-1 and NF-κB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after H2O2 stimulation. Our results demonstrate that VEGF gene expression is upregulated by H2O2 in these endothelial cells.

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ∼60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 μM calyculin A, or 1 μM N6-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl- channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.