Establishing a Robust In Vitro Embryonic Stem Cell Differentiation Assay to Monitor the Hematopoietic Potential of DELES Clones

Afin d’effectuer des études fonctionnelles sur le génome de la souris, notre laboratoire a généré une bibliothèque de clones de cellules souches embryonnaires (ESC) présentant des suppressions chromosomiques chevauchantes aléatoires – la bibliothèque DELES. Cette bibliothèque contient des délétions couvrant environ 25% du génome murin. Dans le laboratoire, nous comptons identifier de nouveaux déterminants du destin des cellules hématopoïétiques en utilisant cet outil. Un crible primaire utilisant la benzidine pour démontrer la présence d'hémoglobine dans des corps embryoïdes (EBS) a permis d’identifier plusieurs clones délétés présentant un phénotype hématopoïétique anormal. Comme cet essai ne vérifie que la présence d'hémoglobine, le but de mon projet est d'établir un essai in vitro de différenciation des ESC permettant de mesurer le potentiel hématopoïétique de clones DELES. Mon hypothèse est que l’essai de différenciation hématopoïétique publié par le Dr Keller peut être importé dans notre laboratoire et utilisé pour étudier l'engagement hématopoïétique des clones DELES. À l’aide d’essais de RT-QPCR et de FACS, j’ai pu contrôler la cinétique de différenciation hématopoïétique en suivant l’expression des gènes hématopoïétiques et des marqueurs de surface comme CD41, c-kit, RUNX1, GATA2, CD45, β-globine 1 et TER-119. Cet essai sera utilisé pour valider le potentiel hématopoïétique des clones DELES candidats identifiés dans le crible principal. Mon projet secondaire vise à utiliser la même stratégie rétro-virale a base de Cre-loxP utilisée pour générer la bibliothèque DELES pour générer une bibliothèque de cellules KBM-7 contenant des suppressions chromosomiques chevauchantes. Mon but ici est de tester si la lignée cellulaire leuémique humaine presque haploïde KBM-7 peut être exploitée en utilisant l'approche DELES pour créer cette bibliothèque. La bibliothèque de clones KBM-7 servira à définir les activités moléculaires de drogues anti-leucémiques potentielless que nous avons identifiées dans le laboratoire parce qu’elles inhibent la croissance cellulaire dans plusieurs échantillons de leucémie myéloïde aiguë dérivés de patients. Elle me permettra également d'identifier les voies de signalisation moléculaires qui, lorsque génétiquement perturbées, peuvent conférer une résistance à ces drogues.

Mast cells in human periodontal disease

Recently, mast cells have been shown to produce cytokines which can direct the development of T-cell subsets. The aim of the present study was to determine the relationship between mast cells and the Th1/Th2 response in human periodontal disease. Tryptase+ mast cell numbers were decreased in chronic periodontitis tissues compared with healthy/gingivitis lesions. Lower numbers of c-kit+ cells, which remained constant regardless of clinical status, indicate that there may be no increased migration of mast cells into periodontal disease lesions. While there were no differences in IgG2+ or IgG4+ cell numbers in healthy/gingivitis samples, there was an increase in IgG4+ cells compared with IgG2+ cells in periodontitis lesions, numbers increasing with disease severity. This suggests a predominance of Th2 cells in periodontitis, although mast cells may not be the source of Th2-inducing cytokines.

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E-17) in the rat such that embryonic day 19 (E-19) wounds do not re-epithelialize. Moreover, wounds created in E-17 fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E-17 and E-19 skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E-17 and E-19 skin. c-fos and c-jun induction was transient in E-17 skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E-19 skin, AP-11 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E-17 skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.

Structural and cellular characterization of Idiopathic Pulmonary Fibrosis

La Fibrosi Polmonare Idiopatica (IPF) è una malattia polmonare cronica, irreversibile la cui eziologia risulta essere ignota, caratterizzata da un processo fibrotico progressivo che inizia nel tratto respiratorio inferiore. Le persone affette da IPF presentano età media compresa tra 55 e 77 anni. L’incidenza annuale di IPF è stata recentemente stimata tra 14 e 42,7 casi per 100.000 persone e tale dato risulta essere in aumento. IPF fa parte delle malattie Polmonari Idiopatiche Interstiziali (IIP) che comprendono patologie con quadri istologici e clinici differenti. Le affezioni su cui si concentrerà questo studio sono: UIP (Usual Interstitial Pneumonia) caratterizzata da fibrosi interstiziale e dalla presenza di foci fibrotici connessi alla pleura e corrispondente al quadro anatomopatologico della maggior parte dei casi di IPF; NSIP (Non Specific Interstitial Pneumonia) simile alla UIP ma con maggiore uniformità temporale e spaziale delle manifestazioni; Sarcoidosi, malattia granulomatosa ad eziologia ignota. Attualmente la gravità della IPF, che implica una mortalità del 50% dei pazienti a 5 anni dall’esordio, e la scarsa efficacia farmacologica nel rallentarne la progressione vedono il trapianto polmonare come unica possibilità di sopravvivenza nelle forme più severe. Al momento non è chiaro il meccanismo patogenetico di insorgenza e progressione della IPF anche se sono stati individuati alcuni fattori scatenanti quali fumo di sigaretta, infezioni respiratorie e inquinanti atmosferici; tuttavia nessuno di tali elementi può da solo determinare un così esteso e progressivo rimodellamento del parenchima polmonare. Numerose sono le evidenze di come il substrato genetico, le alterazioni del rapporto morte/proliferazione cellulare e le citochine svolgano un ruolo nella genesi e nella progressione della malattia, ma non sono ancora chiari i fenomeni biologico-cellulari che la sostengono e, quindi, quali siano i punti di attacco per poter incidere terapeuticamente nel modificare l’evoluzione della IPF. Poiché il nostro laboratorio ha partecipato alla scoperta dell’esistenza di cellule staminali nel polmone umano normale, uno degli obiettivi finali di questo progetto si basa sull’ipotesi che un’alterazione del compartimento staminale svolga un ruolo cruciale nella eziopatogenesi di IPF. Per questo in precedenti esperienze abbiamo cercato di identificare nella IPF cellule che esprimessero antigeni associati a staminalità quali c-kit, CD34 e CD133. Questo lavoro di tesi si è proposto di condurre un’indagine morfometrica ed immunoistochimica su biopsie polmonari provenienti da 9 pazienti affetti da UIP, 3 da NSIP e 5 da Sarcoidosi al fine di valutare le alterazioni strutturali principali imputabili alle patologie. Preparati istologici di 8 polmoni di controllo sono stati usati come confronto. Come atteso, è stato osservato nelle tre patologie esaminate (UIP, NSIP e Sarcoidosi) un significativo incremento nella sostituzione del parenchima polmonare con tessuto fibrotico ed un ispessimento dei setti alveolari rispetto ai campioni di controllo. L’analisi dei diversi pattern di fibrosi presenti fa emergere come vi sia una netta differenza tra le patologie con una maggiore presenza di fibrosi di tipo riparativo e quindi altamente cellulata nei casi di UIP, e NSIP mentre nelle Sarcoidosi il pattern maggiormente rappresentato è risultato essere quello della fibrosi replacement o sostitutiva. La quantificazione delle strutture vascolari è stata effettuata tenendo separate le aree di polmone alveolare rispetto a quelle occupate da focolai sostitutivi di danno (componente fibrotica). Nei campioni patologici analizzati era presente un significativo riarrangiamento di capillari, arteriole e venule rispetto al polmone di controllo, fenomeno principalmente riscontrato nel parenchima fibrotico. Tali modifiche erano maggiormente presenti nei casi di NSIP da noi analizzati. Inoltre le arteriole subivano una diminuzione di calibro ed un aumento dello spessore in special modo nei polmoni ottenuti da pazienti affetti da Sarcoidosi. Rispetto ai controlli, nella UIP e nella Sarcoidosi i vasi linfatici risultavano inalterati nell’area alveolare mentre aumentavano nelle aree di estesa fibrosi; quadro differente si osservava nella NSIP dove le strutture linfatiche aumentavano in entrambe le componenti strutturali. Mediante indagini immunoistochimiche è stata documentata la presenza e distribuzione dei miofibroblasti, positivi per actina muscolare liscia e vimentina, che rappresentano un importante componente del danno tissutale nella IPF. La quantificazione di questo particolare fenotipo è attualmente in corso. Abbiamo inoltre analizzato tramite immunoistochimica la componente immunitaria presente nei campioni polmonari attraverso la documentazione dei linfociti T totali che esprimono CD3, andando poi a identificare la sottopopolazione di T citotossici esprimenti la glicoproteina CD8. La popolazione linfocitaria CD3pos risultava notevolmente aumentata nelle tre patologie analizzate soprattutto nei casi di UIP e Sarcoidosi sebbene l`analisi della loro distribuzione tra i vari distretti tissutali risultasse differente. Risultati simili si sono ottenuti per l`analisi dei linfociti CD8pos. La componente monocito-macrofagica è stata invece identificata tramite la glicoproteina CD68 che ha messo in evidenza una maggiore presenza di cellule positive nella Sarcoidosi e nella UIP rispetto ai casi di NSIP. I dati preliminari di questo studio indicano che il rimodellamento strutturale emo-linfatico e cellulare infiammatorio nella UIP si differenziano rispetto alle altre malattie interstiziali del polmone, avanzando l’ipotesi che il microambiente vascolare ed immunitario giochino un ruolo importante nella patogenesi della malattia

Developmental and molecular aberrations associated with deterioration of oocytes during FSH-receptor deficiency

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Developmental and molecular aberrations associated with deterioration of oocytes during FSH-receptor deficiency

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation.

The c-kit proto-oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK-cells, and to early T-cell precursors and it is not expressed by normal B-cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkin's disease and anaplastic large-cell lymphoma. In the present paper we describe a case of a B-cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B-cell population. Further studies are necessary to explore both the functional role of c-kit expression in the neoplastic B-cells from this patient and its potential utility for the diagnosis and follow-up of patients with B-cell non-Hodgkin's lymphoma.

The interferon-inducible murine p48 (ISGF3γ) gene is regulated by protooncogene c-myc

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-γ-regulated element. In addition to the IFN-regulated element, a myc–max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor.

Avocado Information Kit. Agrilink, your growing guide to better farming guide

Each Agrilink kit has been designed to be both comprehensive and practical. As the kits are arranged to answer questions of increasing complexity, they are useful references for both new and experienced producers of specific crops. Agrilink integrates the technology of horticultural production with the management of horticultural enterprises. REPRINT INFORMATION - PLEASE READ! For updated information please call 13 25 23 or visit the website www.deedi.qld.gov.au (Select: Queensland Industries - Agriculture link) This publication has been reprinted as a digital book without any changes to the content published in 2001. We advise readers to take particular note of the areas most likely to be out-of-date and so requiring further research: see detailed reprint information on first page of the kit. Even with these limitations we believe this information kit provides important and valuable information for intending and existing growers. This publication was last revised in 2001. The information is not current and the accuracy of the information cannot be guaranteed by the State of Queensland. This information has been made available to assist users to identify issues involved in the production of avocadoes. This information is not to be used or relied upon by users for any purpose which may expose the user or any other person to loss or damage. Users should conduct their own inquiries and rely on their own independent professional advice. While every care has been taken in preparing this publication, the State of Queensland accepts no responsibility for decisions or actions taken as a result of any data, information, statement or advice, expressed or implied, contained in this publication.

Tropical Banana Information Kit. Agrilink, your growing guide to better farming guide

Each Agrilink kit has been designed to be both comprehensive and practical. As the kits are arranged to answer questions of increasing complexity, they are useful references for both new and experienced producers of specific crops. Agrilink integrates the technology of horticultural production with the management of horticultural enterprises. REPRINT INFORMATION - PLEASE READ! For updated information please call 13 25 23 or visit the website www.deedi.qld.gov.au (Select: Queensland Industries – Agriculture link) This publication has been reprinted as a digital book without any changes to the content published in 1998.. We advise readers to take particular note of the areas most likely to be out-of-date and so requiring further research: see detailed information on first page of the kit. Even with these limitations we believe this information kit provides important and valuable information for intending and existing growers. This publication was last revised in 1998. The information is not current and the accuracy of the information cannot be guaranteed by the State of Queensland. This information has been made available to assist users to identify issues involved in the production of tropical bananas. This information is not to be used or relied upon by users for any purpose which may expose the user or any other person to loss or damage. Users should conduct their own inquiries and rely on their own independent professional advice. While every care has been taken in preparing this publication, the State of Queensland accepts no responsibility for decisions or actions taken as a result of any data, information, statement or advice, expressed or implied, contained in this publication.

Potato Information Kit. Agrilink, your growing guide to better farming guide

Each Agrilink kit has been designed to be both comprehensive and practical. As the kits are arranged to answer questions of increasing complexity, they are useful references for both new and experienced producers of specific crops. Agrilink integrates the technology of horticultural production with the management of horticultural enterprises. REPRINT INFORMATION - PLEASE READ! For updated information please call 13 25 23 or visit the website www.daf.qld.gov.au This publication has been reprinted as a digital book without any changes to the content published in 1997. We advise readers to take particular note of the areas most likely to be out-of-date and so requiring further research: see detailed information on first page of the kit. Even with these limitations we believe this information kit provides important and valuable information for intending and existing growers. This publication was last revised in 1997. The information is not current and the accuracy of the information cannot be guaranteed by the State of Queensland. This information has been made available to assist users to identify issues involved in the production of potato. This information is not to be used or relied upon by users for any purpose which may expose the user or any other person to loss or damage. Users should conduct their own inquiries and rely on their own independent professional advice. While every care has been taken in preparing this publication, the State of Queensland accepts no responsibility for decisions or actions taken as a result of any data, information, statement or advice, expressed or implied, contained in this publication.

Onion Information Kit. Agrilink, your growing guide to better farming guide

Each Agrilink kit has been designed to be both comprehensive and practical. As the kits are arranged to answer questions of increasing complexity, they are useful references for both new and experienced producers of specific crops. Agrilink integrates the technology of horticultural production with the management of horticultural enterprises. REPRINT INFORMATION - PLEASE READ! For updated information please call 13 25 23 or visit the website www.deedi.qld.gov.au (Select: Queensland Industries – Agriculture link) This publication has been reprinted as a digital book without any changes to the content published in 1997. We advise readers to take particular note of the areas most likely to be out-of-date and so requiring further research: see detailed information on first page of the kit. Even with these limitations we believe this information kit provides important and valuable information for intending and existing growers. This publication was last revised in 1997. The information is not current and the accuracy of the information cannot be guaranteed by the State of Queensland. This information has been made available to assist users to identify issues involved in the production of onions. This information is not to be used or relied upon by users for any purpose which may expose the user or any other person to loss or damage. Users should conduct their own inquiries and rely on their own independent professional advice. While every care has been taken in preparing this publication, the State of Queensland accepts no responsibility for decisions or actions taken as a result of any data, information, statement or advice, expressed or implied, contained in this publication.

Lettuce Information Kit. Agrilink, your growing guide to better farming guide

Each Agrilink kit has been designed to be both comprehensive and practical. As the kits are arranged to answer questions of increasing complexity, they are useful references for both new and experienced producers of specific crops. Agrilink integrates the technology of horticultural production with the management of horticultural enterprises. REPRINT INFORMATION - PLEASE READ! For updated information please call 13 25 23 or visit the website www.deedi.qld.gov.au (Select: Queensland Industries – Agriculture link) This publication has been reprinted as a digital book without any changes to the content published in 1997. We advise readers to take particular note of the areas most likely to be out-of-date and so requiring further research: see detailed information on first page of the kit. Even with these limitations we believe this information kit provides important and valuable information for intending and existing growers. This publication was last revised in 1997. The information is not current and the accuracy of the information cannot be guaranteed by the State of Queensland. This information has been made available to assist users to identify issues involved in the production of lettuce. This information is not to be used or relied upon by users for any purpose which may expose the user or any other person to loss or damage. Users should conduct their own inquiries and rely on their own independent professional advice. While every care has been taken in preparing this publication, the State of Queensland accepts no responsibility for decisions or actions taken as a result of any data, information, statement or advice, expressed or implied, contained in this publication.