Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on April 24, 2010). Includes bibliographical references.

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The vertebrate embryonic limb is a key model in elucidating the genetic basis underlying the three dimensional morphogenesis of structures. Despite the wealth of insights that have been generated from this model, many long-standing questions remain. For example, it has been known for over 70 years that the apical ectodermal ridge (AER) of the embryonic limb is essential for distal outgrowth and patterning of the adjacent limb mesenchyme. The mechanisms whereby the AER does accomplish outgrowth and patterning are still poorly understood. We propose that secreted FGFs from the AER activate Wnt5a expression in gradient fashion, which in turn provides an instructional cue to direct outgrowth in the direction of increasing Wnt5a expression (i.e. toward the distal tip of the limb). In vivo and in vitro models were used to test this hypothesis. We placed Wnt5a expressing L-cell implants into stage 23 chick limb buds and demonstrate that labeled mesenchyme cells grow toward the source of Wnt5a. Purified Wnt5a soaked heparin bead implants have only a marginal effect on directed growth of the adjacent mesenchyme, whereas a greater effect was seen with beads soaked in Wnt5a conditioned media. Using an in vitro model where cultured limb mesenchyme cells were subjected to a gradient of conditioned Wnt5a media or purified Wnt5a, we show no specific migratory direction. However, clusters of cells tended to move toward the source of Wnt5a indicating that it might be necessary for the cells to be in complete contact to respond to the Wnt5a signal. Taken together, our results suggest that Wnt5a is sufficient to direct limb mesenchyme. This finding has given support to a new model of limb development proposed by our lab and referred to as the Mesenchyme Recruitment Model.

Cai, Ganhui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 160-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Mesenchymal stromal cells (MSC) are adherent, clonogenic, fibroblast-like cells endowing with unique multipotent differentiation potential and immunosuppressive properties. They are considered as promising candidates for regenerative medicine and immunotherapy. <p>MSC can be isolated from different tissue sources including bone marrow (BM), adipose tissue (AT) and Wharton’s Jelly (WJ). Although fulfilling the ISCT criteria required to be recognized as MSC, MSC from these different sources could disclose some differences taking into account their different anatomical origin and ontogeny as well.<p>In the present work, we investigated the influence of MSC source on their immunosuppressive as well as differentiation properties. We further extended our study to the role of the microenvironment (infection and inflammation) on these features. <p>We show that BM-MSC express Toll-like receptors (TLR) from TLR1 to TLR6. In an inflammatory environment, TLR2, 3 and 4 are significantly upregulated. By upregulating TLR3 and TLR4 transcription, inflammation increases BM-MSC responsiveness to LPS (TLR4 ligand) and poly(I:C) (TLR3 ligand) leading to a pro-inflammatory shift of their cytokine profile. The effect of TLR ligation on BM-MSC osteogenic potential is donor dependent. Inflammation as well as stimulation with LPS and poly(I:C) result in a decrease of BM-MSC immunosuppressive capabilities. <p>We further observed that BM-, AT- and WJ-MSC do not have the same pattern of TLR expression and consequently do not respond the same way to bacterial or viral infection. WJ-MSC do not express TLR4 and although TLR3 is present at the protein level it is not functional as its ligation do not trigger cytokine expression. Inflammation modulates this TLR pattern expression by upregulating TLR3 in all three MSC types and TLR4 only in BM-MSC. TLR ligation increases the production of inflammatory cytokines in BM- and AT- but not in WJ-MSC and augments anti-inflammatory cytokines in AT-MSC. Although inflammation increases in all MSC types the secretion of inflammatory cytokines, additional TLR triggering does not further affect WJ-MSC. The immunosuppressive potential of WJ-MSC on mixed leucocytes reaction (MLR) is not affected either by inflammation or by TLR triggering.<p>On the differentiation side, WJ-MSC has the lower potential to differentiate into osteoblast as compared to BM- and AT-MSC, as revealed by alkaline-phosphatase (ALP) activity and by measuring extracellular Ca2+ deposits. However, inflammation is able to strongly increase the osteogenic differentiation of WJ-MSC as calcification and ALP activity appears as early as at day 7. However this latter enzymatic activity remains much lower than that disclosed by BM-MSC. TLR3 or TLR4 triggering does not affect the osteogenesis of WJ-MSC while it increases it in AT- and also, although to lesser extent, in BM-MSC.<p><p>Our work establishes that the source from which MSC is derived is of major importance for the design of MSC based immunointervention. WJ-MSC appear to be the most attractive cell type when an immunosuppressive action is required in an inflammatory or infectious context. Although WJ-MSC are poorly osteogenic, a complete osteogenic differentiation can be obtained under inflammatory conditions. Taking into account their easy accessibility as well as their huge proliferative potential, these data open an avenue for using these cells in regenerative medicine particularly in clinical settings where chronic inflammation or infection have to be considered. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

[ES] Las poliaminas son metabolitos esenciales para el crecimiento de las células eucariotas y su metabolismo está frecuentemente desregulado en cáncer. Una de las dianas moleculares de las poliaminas es el factor de elongación de la traducción eIF5A, una proteína esencial y conservada evolutivamente. eIF5A es la única proteína conocida que contiene el aminoácido hipusina, que deriva de la poliamina espermidina. En humanos existen dos isoformas, eIF5A1 y eIF5A2. EIF5A2 se encuentra en el cromosoma 3q26, una región frecuentemente amplificada en muchos tumores y que está altamente expresada en varios tipos de cáncer, incluyendo el cáncer de pulmón no microcítico (CPNM). eIF5A2 es esencial para el mantenimiento de la proliferación celular y su inhibición la suprime en algunos tumores. Recientemente se ha correlacionado la sobreexpresión de eIF5A2 con la invasión y como biomarcador de mal pronóstico en algunos cánceres, y se ha observado que eIF5A2 induce la transición epitelio-mesenquimal (EMT) en CPNM. La EMT es un proceso complejo y reversible que induce la diferenciación de las células epiteliales a células mesenquimales migrantes con capacidad de invasión. Numerosos estudios han demostrado que la EMT está relacionada con la progresión del cáncer, metástasis y mal pronóstico en tumores. Por tanto, la determinación de un método eficaz para inhibir la EMT en CPNM podría mejorar significativamente los tratamientos actuales. La naturaleza altamente selectiva de la hipusinación de eIF5A2 y su susceptibilidad a la inhibición farmacológica sugieren que eIF5A2 es una diana terapéutica muy atractiva. Actualmente, se dispone de un análogo de poliamina, GC7, que se utiliza para inhibir la hipusinación y se ha demostrado que frena el crecimiento de células cancerosas. El presente trabajo de tesis doctoral tiene como objetivo caracterizar el papel patológico de eIF5A2 en el desarrollo del CPNM. Para ello, hemos estudiado, mediante modificaciones genéticas por silenciamiento y sobreexpresión, el papel de eIF5A2 en la proliferación, motilidad e invasión celular utilizando líneas celulares de CPNM. Así mismo, se ha estudiado el efecto del inhibidor GC7 en líneas celulares de CPNM y modelos murinos para determinar si previene o revierte la EMT, y reduce la migración y la invasión de células de CPNM. Por último, se ha analizado la correlación entre la expresión de eIF5A2, las variables clínico-patológicas y la supervivencia de los pacientes en una colección de muestras de pacientes con CPNM. Los resultados obtenidos sugieren la existencia de una regulación entre las isoformas eIF5A1 y eIF5A2 para compensar la expresión de ambos homólogos. Además, nuestros datos apuntan a una coordinación temporal y posicional entre las vías de TGFß1 y eIF5A2 para impulsar la traducción de proteínas requerida para el reordenamiento del citoesqueleto y la motilidad de las células cancerosas invasivas. Hemos demostrado con modelos de ratón in vivo, que los tumores generados mediante xenotrasplante de células que sobreexpresan eIF5A2 tienen mayor capacidad invasiva. Finalmente, mostramos la existencia de una correlación positiva entre la expresión de eIF5A2 y el marcador de proliferación Ki67 en tejido de tumores de CPNM, y que la tasa de supervivencia es menor en aquellos pacientes que expresan niveles elevados de eIF5A2. Los resultados obtenidos en este trabajo confirman que eIF5A2 podría ser empleado como un biomarcador de mal pronóstico en CPNM y su inhibición farmacológica podría emplearse como una posible herramienta terapéutica, sola o en combinación con otros fármacos, en aquellos casos en los que eIF5A2 se encuentre sobreexpresado. / [CA] Les poliamines són metabòlits essencials per al creixement de les cèl·lules eucariotes i el seu metabolisme està frequentment desregulat en càncer. Una de les dianes moleculars de les poliamines és el factor d'elongació de la traducció eIF5A, una proteïna essencial i conservada evolutivament. eIF5A és l'única proteïna cel·lular coneguda que conté l'aminoàcid hipusina, que deriva de la poliamina espermidina. En humans hi ha dues isoformes, eIF5A1 i eIF5A2. EIF5A2 es troba al cromosoma 3q26, una regió freqüentment amplificada en molts tumors, i que està altament expressada en diferent tipus de càncer, incloent el càncer de pulmó no microcític (CPNM). eIF5A2 és essencial per al manteniment de la proliferació cel·lular i la seva inhibició la suprimeix en alguns tumors. Recentment s'ha correlacionat la sobreexpressió d'eIF5A2 amb la invasió i com a biomarcador de mal pronòstic a alguns càncers i s'ha observat que eIF5A2 indueix la transicició epiteli-mesenquima (EMT) en CPNM. L'EMT és un procés complex i reversible que indueix la diferenciació de les cèl·lules epitelials a cèl·lules mesenquimals migrants amb capacitat d'invasió. Nombrosos estudis han demostrat que l'EMT està relacionada amb la progressió del càncer, la metàstasi i el mal pronòstic en molts tumors. Per tant, la determinació d'un mètode eficaç per inhibir l'EMT a CPNM podria millorar significativament els règims dels tractaments actuals. La naturalesa altament selectiva de la hipusinació d'eIF5A2 i la susceptibilitat a la inhibició farmacològica fan d'aquesta proteina una diana terapèutica molt atractiva. Actualment, es disposa d'un anàleg de poliamina, anomenat GC7, que s'utilitza per inactivar la reacció d'hipusinació i s'ha demostrat que inhibeix el creixement de cèl·lules canceroses. Aquest treball de tesi doctoral té com a objectiu caracteritzar el paper patològic d'eIF5A2 en el desenvolupament del CPNM. Per això, hem estudiat, mitjançant modificacions genètiques per silenciament i sobreexpressió, el paper d'eIF5A2 en la proliferació, la motilitat i la invasió cel·lular utilitzant línies cel·lulars de càncer de pulmó. Així mateix, s'ha estudiat l'efecte de l'inhibidor GC7 en línies cel·lulars de CPNM i models murins per determinar si augmenta la quimiosensibilitat de les cèl·lules, prevé o reverteix l'EMT i redueix la migració i la invasió de cèl·lules de CPNM. Finalment, s'ha analitzat la correlació entre l'expressió d'eIF5A2, les variables clinicopatològiques i la supervivència dels pacients en una col·lecció de mostres de pacients amb CPNM. Els resultats obtinguts suggereixen que hi ha una regulació entre les isoformes eIF5A1 i eIF5A2 per compensar l'expressió dels dos homòlegs. A més, les nostres dades apunten a una coordinació temporal i posicional entre les vies de TGFß1 i eIF5A2 per impulsar la traducció requerida de proteïnes per als reordenaments del citosquelet i les característiques de motilitat de les cèl·lules canceroses invasives. Hem demostrat amb models de ratolí in vivo que els tumors generats mitjançant xenotrasplantament de cèl·lules que sobreexpressen eIF5A2 tenen més capacitat invasiva. Finalment, mostrem l'existència una correlació positiva entre l'expressió d'eIF5A2 i el marcador de proliferació Ki67 en teixit de tumors de CPNM, i que la taxa de supervivència és menor en aquells pacients que expressaven alts nivells d'eIF5A2. Els resultats obtinguts en aquest treball confirmen que eIF5A2 podria ser emprat com un biomarcador de mal pronòstic a CPNM i la seva inhibició farmacològica podria utilitzar-se com una possible eina terapèutica, sola o en combinació amb altres fàrmacs, en aquells casos en què eIF5A2 es trobe sobreexpressat. / [EN] Polyamines are essential metabolites for eukaryotic cells growth, and their metabolism is frequently deregulated in cancer. One of the molecular targets of polyamines is the translation elongation factor eIF5A, an essential and evolutionarily conserved protein. eIF5A is the only protein known that contains the amino acid hypusine, which is derived from the polyamine spermidine. In humans there are two isoforms, eIF5A1 and eIF5A2. EIF5A2 is located on chromosome 3q26, a region frequently amplified in different types of cancer, including non-small cell lung cancer (NSCLC). eIF5A2 is essential for the maintenance of cell proliferation and its inhibition suppresses it in many tumors. Recently, eIF5A2 overexpression has been correlated with invasion and as a biomarker of poor prognosis in some cancers, and it has been observed that eIF5A2 induces epithelial-mesenchymal transition (EMT) in NSCLC. EMT is a complex and reversible process that induces the differentiation of epithelial cells into migrant mesenchymal cells with invasive capacity. Numerous studies have shown that EMT is related to cancer progression, metastasis, and poor prognosis in many tumors. Therefore, the determination of an effective method to inhibit EMT in NSCLC could significantly improve current treatment regimens. The highly selective nature of eIF5A2 hypusination and its susceptibility to pharmacological inhibition make this process a very attractive therapeutic target. Currently, a polyamine analog, called GC7, is available and is used to inactivate the hypusination reaction and has been shown to inhibit cancer cell growth. The objective of this doctoral thesis is to characterize the pathological role of eIF5A2 in the development of NSCLC. For this, we have studied, through genetic alterations by silencing and overexpression, the role of eIF5A2 in cell proliferation, motility and invasion using lung cancer cell lines. Likewise, the effect of the GC7 inhibitor in NSCLC cell lines and murine models has been studied to determine if it increases the chemosensitivity of cells, prevents or reverses EMT, and reduces migration and invasion of NSCLC cells. Finally, the correlation between the expression of eIF5A2, clinicopathological variables and patient survival has been analyzed in a collection of samples from patients with NSCLC. The results obtained suggest the existence of a regulation between the eIF5A1 and eIF5A2 isoforms to compensate the expression of both homologues. Furthermore, our data point to a temporal and positional coordination between the TGFß1 and eIF5A2 pathways to drive the required translation of proteins for cytoskeletal rearrangements and motility characteristics of invasive cancer cells. We have demonstrated with in vivo mouse models that tumors generated by xenotransplantation of cells that overexpress eIF5A2 have a greater invasive capacity. Finally, we show the existence of a positive correlation between the expression of eIF5A2 and the proliferation marker Ki67 in NSCLC tumor tissue, and that the survival rate is lower in those patients who expressed high levels of eIF5A2. The results obtained in this work confirm that eIF5A2 could be used as a biomarker of poor prognosis in NSCLC and its pharmacological inhibition could be used as a possible therapeutic tool, alone or in combination with other drugs, in those cases in which eIF5A2 is found overexpressed. / Martínez Férriz, A. (2023). Funciones del factor de inicio de la traducción eucariota 5A2 en el cáncer de pulmón [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/193293

Abstract Wnt4, a member of the Wnt family of secreted factors, is essential for kidney organogenesis since the kidney fails to develop in its absence. Besides the kidney, Wnt4 signaling is involved in the control of development of several other organs such as the gonads, adrenal glands and pituitary gland. In the context of the embryonic kidney, Wnt4 signaling induces mesenchymal to epithelial transition of the progenitor cells in the metanephric mesenchyme, an early step in nephrogenesis. Wnt4 signaling may also be relevant in the development of a childhood kidney tumor, the Wilms’ tumor, that involves the function of Wilms’ tumor suppressor protein 1 (WT1). Wilms’ tumor is thought to arise from the early metanephric mesenchymal cells of the embryonic kidney, but the detailed mechanisms are not known. The main aim of this project was to study the mechanisms that regulate expression of the Wnt4 gene by using immortalized embryonic kidney mesenchyme-derived mK4 cells as a model. The Wnt4 gene expression was also analyzed in vivo in the frog embryonic pronephros. Through the use of reporter assays and a two-hybrid screen, Sox11, a member of the SoxC family of transcription factors, was identified as a synergistic protein that interacts with WT1. Immunoprecipitation studies provided further evidence that Sox11 and WT1 may physically interact with each other in the developing embryonic kidney. Indeed, Sox11 and WT1 may regulate the Wnt4 gene expression in vivo since the morpholino-based knock-down of either WT1 or Sox11 led to notable downregulation of the Wnt4 gene expression in the frog embryonic pronephros. The other general aim of this thesis was to develop novel tissue targeting and therapy tools to the cell lineages regulated by the Wnt4 signals, including the podocytes. For this purpose, we utilized mice carrying a floxed expression cassette for the avidin-LDL receptor fusion protein, Lodavin, in the constitutively active Rosa-26 locus. Three Cre driver mice, including the Wnt4-Cre knock-in line, were used to activate Lodavin expression in the respective cells of the embryonic kidney. Moreover, we generated a podocyte injury model by expressing the human receptor for diphtheria toxin specifically in the podocytes. This was achieved by crossing mice containing a floxed expression cassette for this receptor in the Rosa-26 locus with those expressing the Cre recombinase under the nephrin promoter. Administration of diphtheria toxin led initially to podocyte damage only, followed by a progression to glomerular sclerosis. As a summary, Sox11 and WT1 serve as synergistic transcription factors that may regulate expression of the Wnt4 gene in vivo. The transgenic mouse models generated and used provide the basis to generate acute and chronic kidney disease models and the potential to purify the respective cells for developing cell-based therapy avenues for the kidney. Moreover, the Lodavin-based approaches may enable targeted delivery of biotinylated small compounds, proteins, viruses or even cells and novel means for in vivo imaging and functional studies. / Tiivistelmä Wnt-4 kuuluu signaloivien proteiinien Wnt-perheeseen ja sen toiminta on välttämätöntä munuaisen kehityksessä. Ilman Wnt-4 proteiinia munuainen ei kehity. Munuaisen lisäksi Wnt4-signalointi on mukana useiden muiden elinten, kuten sukurauhasten, lisämunuaisen ja aivolisäkkeen säätelyssä. Alkion munuaisessa Wnt4-signalointi saa aikaan mesenkymaalisen kantasolukon epitelisoitumisen, edustaen näin ollen nefronin kehityksen varhaisia vaiheita. Wnt4-signaloinnilla on myös merkittävä asema lapsuusiän munuaiskasvaimen, niin kutsutun Wilmsin kasvaimen kehittymisessä. Tämän tyyppisessä kasvaimessa keskeisenä on Wilmsin tuumoriproteiinin WT1:n toiminta, mutta myös Wnt4:n toiminnalla voi olla merkitystä. Wilmsin kasvaimen arvellaan saavan alkunsa varhaisista sikiöaikaisista jälkimunuaisen soluista, mutta yksityiskohtaisia mekanismeja ei vielä tunneta. Tämän projektin tarkoituksena oli tutkia Wnt4-geenin ilmentymistä sääteleviä mekanismeja käyttäen mallina mK4-soluja eli alkion munuaisesta saatuja, immortalisoituja soluja. Wnt4-geenin ilmentymistä analysoitiin myös in vivo sammakon alkion alkumunuaisessa. Tuplahybridi-analysoinnin avulla tunnistettiin transkriptiotekijäperhe SoxC:n jäsen Sox11 samantoimiseksi proteiiniksi transkriptiotekijä WT1:n kanssa Wnt4-geenin ilmentymisen säätelyssä. Immunopresipitaatiotutkimukset tukivat ajatusta, että Sox11 ja WT1 voisivat olla fyysisessä vuorovaikutuksessa säädellessään nefroninmuodostuksen alullepanossa ratkaisevan Wnt4-geenin ilmentymistä. Sox11 ja WT1 voivat mahdollisesti säädellä Wnt4-geenin ilmentymistä myös in vivo, sillä morfoliineihin perustuvissa kokeissa sekä WT1:n että Sox11:n hiljennys laski Wnt4-geenin ilmentymistasoa sammakon alkumunuaisessa. Tämän väitöstutkimuksen toinen yleinen tavoite oli kehittää uusia kudoskohdennus- ja terapiakeinoja Wnt4-signaloinnin säätelemille solulinjoille, kuten podosyyteille. Tätä tarkoitusta varten kloonattiin siirtogeeninen hiiri, jossa floksattu avidiini-LDL -reseptorifuusioproteiini, Lodavin, kohdennettiin jatkuvasti aktiiviseen Rosa-26 -lokukseen. Kolmea eri Cre-hiirilinjaa käytettiin aktivoimaan Lodavinin ilmentyminen kussakin tietyssä alkion munuaisen solupopulaatiossa. Yksi näistä Cre-linjoista oli Wnt4-Cre. Jotta kyettäisiin vahingoittamaan samoja soluja, jotka ilmentävät Lodavinia, hyödynnettiin difteriamyrkyn ihmisen reseptoria (iDTR). IDTR:n ilmentäminen tietyissä hiiren soluissa tekee ne alttiiksi tappavalle difteriamyrkylle. IDTR-perusteisen munuaisvauriomallin kehittämiseksi käytettiin floksattua iDTR-hiirimallia, ja geenin ilmentyminen aktivoitiin Wnt4-indusoiduissa munuaissolulinjoissa, erityisesti podosyyteissä Nephrin Cre -välitteisesti. NephrinCre;R26RiDTR hiiriä altistettiin difteriamyrkylle ja niiden munuaiskerästen muutoksia seurattiin. Tutkimukset antavat viitteitä siitä, että R26R-floksatut iDTR-hiiret toimivat hyvänä mallina kehitettäessä sekä akuutteja että kroonisia munuaistautimalleja. Yhteenvetona voidaan todeta, että Sox-11 ja WT-1 ovat samantoimisia transkriptiotekijöitä, jotka voivat säädellä Wnt4-geenin ilmentymistä in vivo. Tutkimuksessa kehitetyt ja käytetyt siirtogeeniset hiirimallit tarjoavat perustan kehittää sekä akuutteja että kroonisia munuaistautimalleja. Samalla ne mahdollistavat kulloistenkin solujen eristämisen uusien soluperusteisten hoitomenetelmien kehittelemiseksi. Lisäksi Lodavin-perusteiset lähestymistavat voivat mahdollistaa biotinyloitujen pienten yhdisteiden, proteiinien, virusten tai jopa solujen kuljetuksen kohdennetusti sekä avata uusia mahdollisuuksia in vivo -kuvantamiselle ja toiminnallisille tutkimuksille.