Human umbilical cord perivascular cells (HUCPVCs) derived from regions surrounding the umbilical cord vessels represent an attractive cell source for cellular therapies, given their proliferative potential and the accessibility of donor material compared with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). However, these cells remain poorly characterised. Using flow cytometry, HUCPVCs were shown to express conventional MSC markers CD29, CD44, CD73, CD90, CD105, CD146, CD166 and integrins alpha1 to -5, alphaV, alphaVβ3, alphaVβ5, β1 and β3, but not CD14, CD34, CD45, STRO-1 or integrin alphaVβ6. HUCPVC marker profiles were consistent between three donors and at different passage numbers. Immunostaining for smooth muscle cell (SMC) markers; alpha-SMA, SM22alpha and smoothelin revealed that HUCPVCs shared expression of these markers with SMCs. However, in comparison with SMCs, HUCPVCs deposited more extensive fibronectin-rich matrices. When compared with hBM-MSCs, HUCPVCs differentiated along adipogenic and osteogenic lineages more slowly and did not progress to terminal phenotypes. mRNA expression of recently identified mesenchymal progenitor cell markers, ROR2, EPHA2, PLXNA2, CDH13 and CD9, was confirmed in HUCPVCs from two donors. In addition, all these markers (except EPHA2) were detected in the umbilical cord vessel wall cells of three donors, confirming their expression in both cultured HUCPVCs and cells of the primary tissue. To determine the roles of these markers in HUCPVCs, they were depleted individually using siRNA. Knockdown (KD) efficiencies of 90-97% were achieved. CD9 KD cells appeared elongated compared to cells treated with control siRNA, and these cells along with ROR2, EPHA2 and PLXNA2 KD cells exhibited larger cell areas than controls. All KD cells also showed decreased proliferative potential by day 6 compared with control siRNA or lipofectamine treated cells. A decrease in total β1 integrins was detected in the CD9 KD cells. Up-regulation of ROR2 and PLXNA2 mRNA expression was detected in HUCPVCs from two donors, when they underwent osteogenic differentiation. ROR2 and PLXNA2 knockdown resulted in increases in PLXNA2 and ROR2 mRNAs respectively, when cells were cultured in osteogenic medium compared with basal conditions. In addition, each individual knockdown revealed that the KD cells showed trends in increasing RUNX2 mRNA expression after 13-16 days in osteogenic medium. These data suggest that ROR2 and PLXNA2 may co-operate in promoting an osteogenic phenotype. Culturing HUCPVCs on non-mineralised BVSMC-derived matrices had very little impact on their differentiation status. In contrast, when HUCPVCs were cultured on mineralised BVSMC-derived matrices in osteogenic medium, their ability to further deposit mineralised matrix was enhanced by 7 days; no accompanying changes in RUNX2, ROR2 or PLXNA2 mRNA expression were detected. Taken together, early up-regulation of RUNX2, ROR2 and PLXNA2 appears to be important in driving osteogenic differentiation in HUPCVCs, whilst subsequent down-regulation of these markers may be required for mineralisation to occur. HUCPVCs express ROR2, PLXNA2, CDH13 and CD9 in vitro and in situ; these markers have distinct roles in regulating cell proliferation, shape and differentiation which may be regulated via changes in β1 integrins. It is not known why HUCPVCs might differentiate along adipogenic and osteogenic lineages more incompletely than hBM-MSCs. Further comparative characterisation of HUCPVCs and hBM-MSCs is a prerequisite for exploiting their vast clinical potential.

Made available in DSpace on 2014-06-11T19:31:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-12-09Bitstream added on 2014-06-13T19:01:25Z : No. of bitstreams: 1 oliveira_pgg_dr_botfmvz.pdf: 525074 bytes, checksum: 52d948b8418c2241104df84f8dd60b54 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Lesões tendíneas, especialmente do tendão flexor digital superficial dos membros torácicos, são importantes enfermidades que acometem a espécie equina, e apresentam alto índice de recidivas e afastamento da atividade atlética. Os avanços médicos e pesquisas relacionadas têm mostrado crescente interesse na utilização da terapia celular em tratamentos dessas lesões que apresentam uma cicatrização lenta ou ineficaz. Existem dúvidas quanto à persistência e comportamento das células tronco mesenquimais (CTM) implantadas no local da lesão e de sua migração a outros locais inflamados. Sendo assim, este estudo teve como objetivo identificar CTM marcadas com nanocristais antes e após o implante em lesões experimentais do tendão flexor digital superficial (TFDS) de eqüinos, observando a possibilidade de migração das CTM para outro foco de lesão realizado no membro contralateral do animal. Para isso, foram utilizados cinco equinos, submetidos à indução de lesões no TFDS em ambos os membros torácicos e, após sete dias, foram implantadas as CTM autólogas marcadas com nanocristais Qtracker 655 em um dos membros do animal. Sete dias após o implante, foram realizadas biópsias dos tendões, congelados em nitrogênio, sendo posteriormente realizados cortes histológicos para visualização em microscópio de fluorescência. Paralelamente, células marcadas com nanocristal foram mantidas em cultivo por 24 horas e, após este período, avaliou-se a sua viabilidade. Nas lesões que receberam células marcadas, foram visualizados sinais fluorescentes nas amostras, enquanto estes eram ausentes nas lesões dos membros contralaterais. As CTM marcadas e injetadas no tecido tendíneo mantiveram sua fluorescência sete dias após o implante in vivo, não sendo observado migração para o membro contralateral. As células mantiveram sua viabilidade após 24 horas de incubação com o marcador Qtracker / Tendon lesions, especially of the superficial digital flexor tendon (SDFT) on forelimb, are important injuries in equine, because have high reinjury incidence and can cause retirement of athletic activities. Medical researches have shown growing interest on using stem cell therapy in the treatment of lesion with slow or ineffective repair. There are doubts regarding the persistency of the mesenchymal stem cells (MSC) implanted on the lesion site and of its migration to other injury sites. This study aims to identify marked MSC with nanoparticles quantum dots (QD) before and after the implant in equine induced tendinitis and to verify if there is a migration to another injury site on the contralateral. Five equines have a SDFT tendinitis induced on both forelimb and, after seven days, have seeded autologous MSC, marked with quantum dots Qtracker 655 in one of the limbs. Seven days after the implant, biopsies of the tendons were conducted, in which histological cuts were made for visualization in fluorescence microscope. The viability of the cells marked with nanoparticles and kept in cultivation was evaluated after 24 hours of incubation. Fluorescent signs were visualized on samples of the lesions that received marked cells, while they were absent on the lesions of the contralaterals. The marked MSCs kept their fluorescent emission, making it possible to identify them after seven days of in vivo implant, not occurring migration to the opposite limb. The cells maintained their viability in cultivation after 24 hours of incubation with the Qtracker marker

Orientador: Fernanda da Cruz Landim e Alvarenga / Banca: Sony Dimas Bicudo / Banca: Nereu Carlos Prestes / Banca: José Antonio Visintin / Banca: Claudia Barbosa Fernandes / Resumo: A célula-tronco (CT) é definida como uma célula com capacidade de gerar diferentes tipos celulares e reconstituir diversos tecidos. Além disso, a CT apresenta propriedades de auto-renovação, gerando cópias idênticas a si mesma. De acordo com sua origem, as células-tronco podem ser chamadas de "adultas" e "embrionárias". As células-tronco adultas (CTA) mais utilizadas nas clínicas de terapia celular são as células-tronco hematopoiéticas e as células tronco mesenquimais, encontradas principalmente na medula óssea, tecido adiposo e no sangue do cordão umbilical. As células-tronco embrionárias (CTE) são derivadas da massa celular interna de embriões no estágio de blastocisto. Desta maneira este trabalho teve como objetivo desenvolver uma metodologia adequada para o isolamento, cultivo e caracterização de células tronco embrionárias e mesenquimais de eqüinos, além de verificar a capacidade que as células possuem em se diferenciar in vitro em outros tipos célulares. Foi coletado sangue da medula óssea de eqüinos entre 8 e 15 anos de idade. As células tronco mesenquimais foram isoladas após a primeira e segunda passagem. As células foram caracterizadas com marcadores de superfície CD34 (mononucleares) e CD44 (mesenquimais). Após isolamento e caracterização, as células tronco mesenquimais foram diferenciadas para as linhagens osteogênica, adipogênica, condrogênica e neurogênica. A confirmação da diferenciação das células tronco foi realizada por marcadores teciduais específicos. Estas células também, foram capazes de expressarem marcadores neurais. Para o isolamento das células tronco embrionária eqüina, embriões com oito a nove dias foram coletado e a massa celular interna (MCI) isolada mecanicamente. Após o isolamento, a MCI foi transferida para a placa de cultivo previamente preparada com monocamada de fibroblastos para o desenvolvimento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The stem cell (SC) is defined as cells with the capacity of generate different cellular types and rebuild various tissues. Moreover, the SC has a selfregenerate ability, generating identical copies of itself. According to its origins, the SC can be named as "adult" or "embryonic". The adult stem cell (ASC) more often used in clinical trials and cellular therapy, are the hematopoietic stem cells and the mesenchymal stem cells, isolated mainly from the marrow bone, adipose tissue and umbilical cord blood. The embryonic stem cells (ESC) are obtained from the inner cell mass of embryos at the blastocyst stage. In this way the present study had as objective to develop an adequate methodology of isolation, culture and characterization of embryonic and mesechymal stem cells from horses, verifying the capacity of those cells to differentiate in vitro into different cells types. Bone marrow blood was collected from horses, aging from 8 to 15 years and filtered with a donation blood kit filter, to avoid clots. The mesenchymal stem cells were isolated after the first and the second passage. The SC were characterized using surface markers CD34 (monuclear) and CD44 (mesenchymal). After the isolation and characterization, the mesenchymal stem cells were differenced into osteogenic, adipogenic, condrogenic and neurogenic lineage. The cells differentiations were confirmed using specific tissue markers. To isolate the embryonic stem cells equine embryos with 8 to 9 days were used. The inner cell mass (ICM) were extract mechanically and transferred to a culture dish previously prepared with fibroblasts monolayer to colony formation and development. The colonies were characterized with pluripotency markers and then submitted to a differentiation process into neurogenic lineage, confirmed by specific neural tissue markers / Doutor

Hypoxia (low tissue oxygenation) is an important signalling cue for many cell types. The study of its effects has direct relevance to surgery since hypoxic gradients are generated with every cut. On a cellular level, changes in molecular oxygen are sensed by the Hypoxia-Inducible Factors (HIFs). The HIFs are a family of transcription factors that are master regulators of over 100 genes and can effect changes in multiple cellular processes including migration, survival and differentiation. The broad nature of the response to hypoxia means that study of the HIF system is also important in cancer; where many tumour cells have found ways of subverting the HIF response to ensure their continued growth and survival. This thesis explores the role of hypoxia and the HIF system in the regulation of migration, survival and differentiation in both cancer and stem cells. The first experimental chapter examines the role of hypoxia and the HIF system in the regulation of migration and three-dimensional organisation in several cancer cell lines. Using biochemical and functional assays, the HIF system is shown to exert a pleiotropic effect across a panel of cancer cell lines. In particular, HIF 1α is shown to activate proliferation in a prostate cancer cell line in findings that may be useful to inform future clinical strategies for the management of this disease. In the second experimental chapter, the first epigenetic mechanism involving histone modification for the specific regulation of HIF 2α expression is characterised. Here the family with sequence similarity 60, member A (Fam60a) protein is shown to repress expression of the HIF 2α gene through its association with the class 1 Sin3-HDAC co-repressor complex, achieving specificity by co-operation with the SP1 transcription factor. This novel mechanism is demonstrated to be important in the regulation of the basal expression of HIF 2α. Modification of HIF 2α expression through this mechanism is shown to alter cell migration, three dimensional organisation and angiogenesis in vitro. The clinical importance of these findings is demonstrated in a series of 45 patients suffering from colorectal cancer of known stage. In this cohort, the reciprocal relationship between Fam60a and HIF 2α is maintained, and both are identified as potential novel biomarkers for the development of this disease. In the final experimental chapter, the role of hypoxia in the regulation of differentiation is explored. These effects are documented in mesenchymal progenitors primarily derived from human fat. Here, hypoxia is shown to regulate differentiation in a context-dependent manner, promoting osteogenic and retarding adipose and neural differentiation in-vitro. The roles of Fam60a and HIF 2α are explored in this system. These data may be useful in optimising future surgical engraftment of these cells for regenerative purposes.

Introduction. Safe closure of gastric luminal defects remains a big challenge for development of gastric endoscopic surgery. The aims of this thesis are to assess the effect and efficiency of Eagle Claw VIII (endoscopic suturing device)and adipose tissue-derived mesenchymal stem cells (ADMSCs) for closure and enhancing healing of gastric luminal defects. / Methods and Results. 1. Endoscopic suturing is superior to endoclips for closure of gastrotomy after NOTES. A 2cm linear incision on the body of porcine stomach was closed by hand suturing, Eagle Claw VIII or endoclips, respectively (n=17 for each group). The results indicated that all gastrotomies were successfully closed. Closure time was significantly longer in Eagle Claw VIII group. Bursting pressure of gastrotomies for Eagle Claw VIII was significantly higher than endoclips, but lower than hand suturing. Besides, both Eagle Claw VIII and endoclip closure encountered significantly technical challenges. This study suggested that Eagle Claw VIII had potential for endoscopic closure of gastrotomies, but need further refinement. / 2. ADMSCs for Acceleration of Healing of Sutured Gastric Perforation(SGP). ADMSCs were isolated and expanded in vitro, and characterized by stromal differentiations and cell surface markers. A 2cm SGP was produced on gastric body of rats. 5×10⁶ ADMSCs were transplanted into SGP by local injection (LI-ADMSCs) or topical spraying (TS-ADMSCs). Healing of SGP was assessed. LI-ADMCs significantly decreased peritoneal adhesion and wound dehiscence, and increased bursting pressure of SGP, when comparing to other experimental groups. Histologic analysis indicated that SGPs in LI-ADMSCs group had more re-epithelialization and collagen regeneration, and less inflammation. Expression of TGF-β1 was up-regulated, while IL-6 was down-regulated in LI-ADMSCs group, when comparing to fibrin and control groups. This study suggested that local injection of ADMSCs is an effective approach for accelerating the healing of SGP. / 3. Promoting Effect of ADMSCs on Healing of Gastric Ulcer is abrogated by NSAIDs. Gastric ulcer model in rats was successfully produced by using 70% acetic acid. A total of 1×10⁷ ADMSCs was locally injected into ulcer lesion. Ulcer area was measured at different time points. Therapeutic potentialof ADMSCs was assessed when NSAIDs was simultaneously administrated. The results demonstrated that ADMSCs significantly decreased ulcer area. Histologic assessment indicated that ADMSCs increased re-epithelialization, angiogenesis and collagen deposition, and suppressed inflammation. Transplanted ADMSCs homed into gastric ulcer lesion and differentiated into endothelial and smooth muscle cells. In addition, ADMSCs treatment increased the gene expressions for wound healing, and activated COX-2-PGE₂ and Erk1/2-MAPK signaling pathways. Repeated administration of Indomethacin reduced cell proliferation and angiogenesis, and eliminated ADMSCs-induced ulcer healing on day 10. The results suggested that ADMSCs promoted the healing of peptic ulcer, which is eliminated by NSAIDs. / Conclusions. Endoscopic suturing by Eagle Claw VIII is feasible for closure of gastrotomy, when comparing to endoclips. ADMSC promotes the healing of gastric luminal defects including SGP and ulcer. The promoting effect of ADMSC is PGE₂-dependent, and attenuated by NSAIDs. These evidences implied that combined use of endoscopic suturing and ADMSCs is a helpful approach for safe closure of gastrotomy and gastric perforation. / 引言：胃傷口癒合是胃消化內鏡手術發展的障礙之壹。本課題之目的是評價和探索Eagle Claw VIII和脂肪幹細胞(ADMSCs)縫合和促進胃內傷口癒合的效果和作用。 / 方法和結果：1. 內鏡縫合器Eagle Claw VIII閉合經胃自然腔道手術後傷口的效果評價體外豬胃體上造2cm的胃傷口模型，使用手工縫合、內鏡下Eagle Claw VIII縫合或內鏡夾閉合胃傷口；每組17個樣本。本研究提示所有胃傷口均成功閉合。Eagle Claw VIII縫合胃傷口時間顯著長於其他兩組的閉合時間。Eagle Claw VIII縫合的胃傷口破裂壓顯著高於內鏡夾閉組，但是明顯低於手工縫合組。此外，內鏡縫合和夾閉都面臨較大的技術難度。本研究提示Eagle Claw VIII有臨床運用的潛在價值，但需要進壹步改進。 / 2. 局部移植脂肪幹細胞促進胃穿孔癒合的實驗性研究：建立大鼠2cm胃體穿孔模型，局部註射或傷口表面塗抹法移植ADMSCs，觀察胃傷口癒合情況。局部註射移植ADMSCs顯著減輕胃傷口粘連和裂開發生率，增加胃傷口破裂壓。組織學分析提示ADMSCs治療促進傷口上皮和肉芽組織再生，抑制炎癥反應。此外，局部註射ADMSCs增加TGF-β1抑制IL-6表達。本研究提示局部註射移植ADMSCs是促進胃穿孔傷口癒合的有效方法。 / 3. 局部移植脂肪幹細胞促進胃饋瘍癒合的實驗性研究：使用70%醋酸建立大鼠胃體饋瘍模型；饋瘍病竈內局部註射移植1×107 ADMSCs。研究提示第10和15天ADMSCs顯著減小饋瘍面積。組織學研究提示ADMSCs增加饋瘍傷口上皮和血管再生，促進膠原蛋白分泌和抑制炎癥反應。移植的ADMSCs能夠在饋瘍病竈內成活，並分化成血管內皮細胞和平滑肌細胞。ADMSCs顯著提高促傷口癒合相關基因表達水準。此外，ADMSCs啟動COX-2-PGE2和Erk1/2-MAPK信號通路。第10天，和對照組相比，引哚美辛/ADMSCs組潰瘍病竈內細胞增殖和血管再生顯著降低、饋瘍癒合延遲。本研究提示脂ADMSCs促進胃饋瘍癒合；非甾體抗炎藥顯著減弱ADMSCs的促胃饋瘍癒合作用。 / 結論：與內鏡夾閉相比，Eagle Claw VIII內鏡縫合胃創口有可行性。ADMSCs促進胃穿孔和饋瘍癒合，且依賴於前列腺素E2；引哚美辛抑制前列腺素E2合成從而抑制ADMSCs促胃組織癒合之效能。本研究提示聯合使用內鏡縫合器和ADMSCs是促進胃傷口癒合的潛在有效方法。 / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 152-162). / Abstracts also in Chinese. / Title from PDF title page (viewed on 11, October, 2016). / Liu, Liu. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

As mitocôndrias são organelas semiautonômicas, portadoras do próprio DNA, o mtDNA e responsáveis pela produção de energia celular na forma de ATP, através do processo de fosforilação oxidativa. Atualmente, diferentes tipos de doenças, como distrofias musculares e diversos tipos de câncer, estão associadas à alteração nas moléculas de mtDNA. Na década de 70 um modelo a partir do cultivo celular com brometo de etídio (EtBr) foi desenvolvido com o objetivo de se criar uma linhagem celular depletada de cópias de mtDNA. Desde então os mais variados estudos foram realizados e diversos tipos celulares foram submetidos à depleção do mtDNA. Este projeto teve como objetivos criar um modelo de cultivo celular somático na espécie bovina com depleção de cópias de mtDNA para investigar a resposta da célula a esta condição; avaliar como as células depletadas se comportam na ausência de EtBr, além da utilização destas células no processo de reprogramação celular por indução gênica na tentativa de avaliar o efeito do numero de cópias de mtDNA na indução na espécie bovina. Para tanto foram desenvolvidos três experimentos; Experimento 1- Depleção de mtDNA a partir da utilização do brometo de etídeo; Experimento 2 Repleção do mtDNA; e Experimento 3 Utilização de células bovinas depletadas no sistema de reprogramação nuclear. Todos os experimentos foram avaliados quanto a quantidade de cópias de mtDNA e expressão gênica para os genes Bax, Bcl2 e Tfam. Ademais, os experimentos 1 e 2 foram avaliados quanto a viabilidade celular e apenas o experimento 1 foi avaliado quanto ao crescimento e morfologia celular. O experimento 1 foi avaliado durante o cultivo celular nos períodos D0, D4, D7, D10 e D13, com os grupos experimentais controle (EtBr-C) e tratado com 100 ng/mL de brometo de etídio (EtBr-T), quanto a núero de cópias do mtDNA, o grupo EtBr-T diferiu do grupo EtBr-C (P=0,0459), apresentando menor número de cópias de mtDNA; menor taxa crescimento celular (P<0,05), porém sem alteração na morfologia celular, e na expressão dos genes descritos acima. No experimento da repleção, não houve diferença no número de cópias de mtDNA, entre os grupos EtBr-T e EtBr-R, indicativo de que as células atingiram o estado rho 0 ou que necessitam de mais tempo para ativar a replicação do mtDNA; quanto a viabilidade celular, houve diferença entres os grupos, quanto a expressão gênica, com aumento do Bax e do Bcl-2 para o grupo EtBr-T; O grupo EtBr-R apresentou queda do Bcl-2; para o Tfam houve aumento para o grupo EtBr-T e uma queda para o grupo EtBr-R. Quanto ao experimento 3, não foi possível observar sinais de pluripotência, porém foi detectada uma queda na quantidade de mtDNA dos dois grupos tratados por EtBr (EtBr com e sem Stemcca) e o grupo controle com Stemcca. Para analise de expressão gênica, não houve diferenças entre os grupos em relação ao Tfam. Quanto ao Bax, os grupos controle com Stemcca, controle sem Stemcca e EtBr sem Stemcca não diferiram, e o ultimo também não apresentou diferença quando comparado ao grupo EtBr com Stemcca. Para o Bcl-2, os grupos controle sem Stemcca e EtBr com Stemcca não apresentaram diferenças entre si; o grupo controle sem Stemcca não apresentou diferença quando comparado aos grupos controle com Stemcca e EtBr sem Stemcca. Concluindo, este trabalho no nosso conhecimento, descreve pela primeira vez a produção de células bovinas Rho 0 e discute sobre a relação da função mitocondrial e o processo de reprogramação celular. / Mitochondria are semi autonomic organelles which present their own DNA (mtDNA); are in charge of cell energy production as ATP through oxidative phosphorylation. Currently, different types of diseases like muscular distrofy; different types of cancer are associated to alterations of mtDNA molecules. In the 70\'s a model based on cell culture with ethidium bromide (EtBr) was developed in order to create a cell line depleted of mtDNA. Since then, a variety of studies were realized; diverse cell types were submited to mtDNA depletion. This project had as objective creating a model of somatic cell culture in bovine species with depletion of mtDNA copies, in order to investigate cell response to this condition; to analyze depleted cell behavior in the absence of EtBr, besides using this depleteded cell in a reprogramming cell process by genic induction in order evaluate the effect of the number of mtDNA copies during induction in bovine species. Therefore three experiments were developed: Experiment-1 Depletion of mtDNA using ethidium bromide. Experiment-2 repletion of mtDNA; Experiment-3 usage of depleted bovine cells in reprogramming nuclear system. Cell experiments were analyzed according to the quantity of mtDNA copies; genic expression for Bax, BCl2; Tfam genes. Also, experiments 1; 2 were analyzed on cell viability; only experiment 1 was analyzed regarding cell morphology; growth. Experiment-1 was analyzed during cell culture on periods D0, D4, D7, D10, D13, with control experimental groups (EtBr-C),; treated with 100 ng/mL ethidium bromide (EtBr-T); relating to mtDNA quantification the EtBr-T group differed from EtBr-C (P=0,0459) presenting a smaller number of mtDNA copies; smaller growth rate (P<0,05); although there was no differences on cell morphology as there was also no difference related to genic expression of the previous stated genes. Repletion experiment showed no differences about the number of mtDNA copies between EtBr-T; EtBr-R groups, indicating this cells reached Rho0 state or that they need more time to activate mtDNA replication; about cell viability, there were no differences among the groups; relating to genic expression there was an increase of Bax; BCl-2 for EtBt-T group; EtBr-R group showed decrease of BCl-2; for Tfam there was an increase for EtBr-T group; a decrease for EtBr-R. Relating to Experiment-3 it was impossible to notice signs of pluripotency, but we could see a decrease in the amount of mtDNA in both groups treated with EtBr (EtBr with; without STEMCCA) as in control group with STEMCCA. Genic expression analysis didn\'t show differences related to Tfam. Regarding to BAX, both control groups (with; without STEMCCA); EtBr without STEMCCA didn\'t differ from each other,; the last one also didn\'t show any difference when compared to EtBt with STEMCCA group. For BCl-2, control group without STEMCCA; EtBr with STEMCCA didn\'t show differences among each other; control group without SEMCCA didn\'t show differences when compared to control group with STEMCCA; EtBr without STEMCCA. Concluding, this work, regarding our knowledge, describes for the first time, production of bovine Rho0 cells; debates about the relationship among mitochondrial function; the process of cell reprogramation.

Background: Exosomes have great potential in regenerative medicine through the transfer of their bioactive cargos, such as RNA. tRF RNA and tiRNA are tRNAderived non-coding RNA. Here, we sought to identify the tRF/tiRNA profile in human mesenchymal stem cell (hMSC) exosomes. Methods: Bone marrow hMSCs were cultured with/without osteogenic differentiation medium and exosomes were harvested. RNA was extracted from: 1) control cells (Cell-NT); 2) control exosomes (EXO-NT); 3) differentiated cells (Cell-OM); 4) exosomes produced by differentiated cells (EXO-OM). RNA was sequenced to profile the small RNA with a focus on tRF/tiRNA. Results: tRF/tiRNA was highly enriched in hMSC exosomes. Less diversity was seen in the tRF/tiRNA profile in exosomes than that in parent cells. Selective tRF/tiRNA were packed into MSC exosomes and their profile is dependent on the cell maturation status. Conclusions: Our results suggest that tRF/tiRNA may play a role in mediating the function of exosomes in tissue regeneration.

Prostate cancer presents a significant paradox: it is very common, yet rarely fatal. To wit, the prostate is the most common non-skin tissue for cancer diagnosis in men in the United States. Despite its high incidence, fatal malignancy occurs in only a small fraction of diagnosed men. The fatal cases are characteristically defined by distant spread in the body, also known as metastasis. In order to metastasize a cancer cell must complete several sequential steps. These include degradation of and invasion through the epithelial basement membrane, typically through the loss of static intracellular adhesions with fellow epithelial cells; entrance into the blood stream (intravasation); survival within circulation; exit from the blood stream upon arrival at a new tissue (extravasation); and survival and colonization at the secondary site. At the time of diagnosis, it is not currently possible to accurately predict future metastasis and thereby clinicians cannot delineate those men at high risk for fatal disease from the vast majority of men who are likely to experience an indolent disease course. Consequently, we examined the behavior of cancer cells in several steps of the metastatic cascade. In doing so, we uncovered both molecular and biophysical characteristics of cancer cells that may facilitate successful metastatic dissemination and tumor outgrowth. Epithelial-to-mesenchymal transition (EMT) is physiological process of transdifferentiation that is normally initiated during vertebrate development, but has recently been implicated in tumor development, progression, and metastases. The EMT program results in dramatic changes, including the exchange of epithelial for mesenchymal markers, altered cellular morphology, and gain of motility. EMT-like cellular alterations have been implicated most strongly in the metastasis steps of invasion and survival of cells at primary tumors sites. How EMT-like changes may facilitate survival and growth in the microenvironment of a micrometastatic niche has been less clearly elucidated. Consequently, we evaluated how EMT-like changes may affect the survival and subsequent outgrowth of prostate cancer cell lines following restrictive growth conditions. We observed that EMT-like cells as compared to their more epithelial counterparts displayed enhanced maintenance of their proliferative potential following extended culture in nutrient restriction. This phenotype depended on an EMT-associated increase in autophagy. Notably, the post-stress outgrowth phenotype could be conferred through a paracrine signaling mechanism that may involve autophagy-derived exosome-like extracellular vesicles. These studies demonstrated that EMT-like cells have a resistance to nutrient restriction through enhanced autophagy and may have uncovered a novel autophagy-dependent exosomal secretion pathway. Metastatic efficiency is thought to be strongly limited by the destruction of circulating tumor cells by the hemodynamic shear forces within the vasculature. However, such a persistent belief has little appropriate published experimental evidence. We developed an in vitro assay to expose cells to fluid shear stress (FSS). By monitoring the viability of the cells, we determined that transformed cells had a highly conserved ability to resist even very high FSS. The mechanism depended on the capacity to patch membrane defects, extracellular calcium, and a dynamic cytoskeleton. We also observed a stiffening of cancer cell membranes after exposure to FSS. Taken together, these studies expand the understanding of how cancer cells survive in circulation and indicate that metastatic efficiency is less limited by hemodynamic forces than previously thought. The steps of hematogenous metastasis between intravasation and extravasation necessitate the existence of circulating tumor cells (CTCs). Collection, enumeration, and study of CTCs have the potential to serve as a "liquid biopsy" of the metastatic cascade. In prostate cancer, the enumeration of CTCs by detection of the expression of epithelial markers has displayed limited clinical utility. We hypothesized that the prognostic value of CTC number may be enhanced by detection of cells which have undergone the pro-metastatic EMT-like program. We developed a flow cytometry-based experimental assay for enumeration of CTCs using epithelial (EpCAM) and mesenchymal-like (N-cadherin) surface proteins. We detected from prostatectomy patients before and after surgery events expressing EpCAM, N-cadherin, and both. However, the detection of background events from healthy control subjects was unacceptably high. These studies support the idea of mesenchymal-like tumor cells in circulation, but will require further assay development for reliable conclusions to be drawn. In sum, the work described above has provided descriptive and mechanistic insight to molecular and biophysical properties that may facilitate prostate cancer metastasis. It is our hope that these data will result in the development of relevant preventative, diagnostic, and therapeutic clinical strategies for prostate cancer.

The purpose of this study was to identify conditioning strategies for mesenchymal stromal cells (MSC) which optimize cellular immunosuppressive potency. By identifying new treatment strategies and previously unidentified small molecules capable of stimulating MSC we hope to pave the way tailoring licensed MSC phenotypes to be used in a specific disease state, rather than a one size fits all package. We sought to determine how MSC act in response to a changing immune response or environmental condition. MSC are exquisitely sensitive to changes in their environmental conditions and we show that cellular transcriptome and secretome changes are conditionally responsive to their inflammatory stimulus. One of the main subjects of analysis here is the observations of how these cellular profiles evolve over time in the presence of an inflammatory environment. Similarly, this study observes how MSC behavior changes after an inflammatory event has been resolved to address, in part, the plasticity of MSC licensing and the ability of MSC to rapidly recall a previous immunosuppressive state upon secondary challenge with an inflammatory stimulus. Data was obtained from in vitro experiments with human bone marrow derived MSC and donor human peripheral blood mononuclear cells (PBMC), while in vivo data was obtained using C57BL6/J mice. Overall this research demonstrated that MSC potency can be bolstered by small molecule and drug treatment conditioning, and that certain disease conditions may be more effectively paired with specific MSC conditioning strategies to improve their therapeutic effectiveness.

Tubulointerstitial fibrosis is the key histological predictor of the progression of declining renal function and the final common pathway of progressive kidney disease, regardless of aetiology. Despite its significance, there are currently no treatments available to abrogate this process and those that suffer with this burden eventually succumb to renal failure. Tubulointerstitial fibrosis is largely mediated by fibroblasts and myofibroblasts present in the interstitium. In response to injury, activated fibroblasts differentiate into myofibroblasts which serves as a histological hallmark of fibrosis. Myofibroblasts are characterised as the key contributors to interstitial volume and their presence ultimately leads to loss of renal function. The pathological entities leading to fibrosis inextricably depend on complex signalling pathways. Whilst many of the well-known growth factors that exert effects on renal fibroblasts (such as FGF, EGF and PDGF) involve the activation of receptor tyrosine kinases, the intracellular signalling events dictating the response of fibroblasts remain undefined. The kinase mTOR, responsible for integrating stress and amino acids and controlling cell growth, is increasingly recognised for its ability to integrate growth factor signals mediated through the upstream serine/threonine kinase PI3K. A number of recent studies have highlighted the role of PI3K and mTOR in the regulation of key events relevant to fibrosis, serving as a basis for Chapter 3: The role of PI3K and mTOR in the regulation of fibroblast proliferation and collagen synthesis, and the first part of Chapter 5: The role of PI3K and mTOR in the regulation of myofibroblast differentiation. These studies have identified a key role for PI3K and mTOR in the regulation of fibroblast proliferation, differentiation and collagen synthesis. The work described within has also attempted to examine the derivation of myofibroblasts via EMT. EMT is a process that is integral to embryogenesis and may act as an important source of myofibroblasts during fibrosis. This process is examined in Chapter 4: Development and validation of an ex vivo model of EMT. This model aims to better represent the in vivo environment and has also been used to identify novel regulators involved in EMT being utilised in the second part of Chapter 5: The role of PI3K and mTOR in EMT. Although cytokines and growth factors are thought to be chiefly responsible for tubulointerstitial fibrosis, we now know that serine proteases of the coagulation cascade may also play roles in renal disease. However, unlike their role in glomerular diseases, the role of coagulation in tubulointerstitial fibrosis is less well-known. The work described in Chapter 6: Constituents of the coagulation cascade are spatially and functionally related to experimental tubulointerstitial fibrosis has examined temporal and spatial in vivo relationships of coagulation factors and markers of fibrosis that aid our understanding of mechanisms of fibrosis. The aim of this thesis was to examine those facets of renal fibroblast function that are most devastating to renal function and culminate in an expansion of the renal interstitium during fibrosis. This work hopes to provide useful information to aid the understanding of the multifaceted mechanisms involved in renal tubulointerstitial fibrosis.