Rozprawy doktorskie na temat „Targeting Mice”

Thesis advisor: Thomas N. Seyfried
It has long been posited that all cancer cells are dependent on glucose for energy, termed the "Warburg Effect". As a result of an irreversible injury to the mitochondria, cancer cells are less efficient in aerobic respiration. Therefore, calorie restriction was thought to be a natural way to attenuate tumor growth. Calorie restriction lowers blood glucose, while increasing the circulation of ketone bodies. Ketone bodies are metabolized via oxidative phosphorylation in the mitochondria. Only cells that are metabolically capable of aerobic respiration will thus be able to acquire energy from ketone bodies. To date, calorie restriction has been shown to greatly reduce tumor growth and angiogenesis in the murine CT2A, EPEN, and human U87 brain tumor models. Using the novel VM-M3 model for invasive brain cancer and systemic metastatic cancer, I found that though calorie restriction had some efficacy in reducing brain tumor invasion and primary tumor size, metastatic spread was unaffected. Using a bioluminescent-based ATP assay, I determined the viability of metastatic mouse VM-M3 tumor cells grown in vitro in serum free medium in the presence of glucose alone (25 mM), glutamine alone (4 mM), or in glucose + glutamine. The VM-M3 cells could not survive on glucose alone, but could survive in glutamine alone indicating an absolute requirement for glutamine in these metastatic tumor cells. Glutamine could also maintain viability in the absence of glucose and in the presence of the F1 ATPase inhibitor oligomycin. Glutamine could not maintain viability in the presence of the Krebs (TCA) cycle enzyme inhibitor, 3-nitropropionic acid. The data indicate that glutamine can provide ATP for viability in the metastatic VM-M3 cells through Krebs cycle substrate level phosphorylation in the absence of energy from either glycolysis or oxidative phosphorylation. I therefore developed a metabolic therapy that targeted both glucose and glutamine metabolism using calorie restriction and 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog. Primary tumor growth was about 20-fold less in DON treated mice than in untreated control mice. I also found that DON treatment administered alone or in combination with CR inhibited metastasis to liver, lung, and kidney as detected by bioluminescence imaging and histology. Although DON treatment alone did not reduce the incidence of tumor metastasis to spleen compared to the controls, DON administered together with CR significantly reduced the incidence of metastasis to the spleen, indicating a diet/drug synergy. In addition, the phagocytic capabilities of the VM-M3 tumor cells were enhanced during times of energy stress. This allowed for the digestion of engulfed material to be used in energy production. My data provide proof of concept that metabolic therapies targeting both glucose and glutamine metabolism can manage systemic metastatic cancer. Additionally, due to the phagocytic properties of the VM-M3 cell line also seen in a number of human metastatic cancers, I suggest that a unique therapy targeting metabolism and phagocytosis will be required for effective management of metastatic cancer
Thesis (PhD) — Boston College, 2010
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

Chemotherapy-induced peripheral neuropathy (CIPN) is a side-effect of chemotherapy causing pain in the hands and feet. In particular, paclitaxel causes CIPN lasting for years without effective treatment. There is a strong need for analgesics to both treat and prevent CIPN. One system containing multiple targets to treat CIPN is the endogenous cannabinoid system. This system consists of cannabinoid type 1 (CB1) and type 2 (CB2) receptors primarily expressed on presynaptic neurons and cells of the immune system, respectively. Inhibition of monoacylglycerol lipase (MAGL), which hydrolyzes the endogenous cannabinoid 2- arachidonoylglycerol (2-AG), with JZL184 or MJN110 produces antinociceptive and anti- inflammatory effects in rodent pain models. In this dissertation, we test the hypothesis that MAGL inhibitors will both reverse and prevent mouse paclitaxel-induced mechanical allodynia. JZL184 and MJN110 reversed paclitaxel allodynia in dose-dependent manners with ED50 values (95% C.L.) of 8.4 (5.2-13.6) and 1.8 (1.0-3.3) mg/kg. Using genetic and pharmacologic approaches, we demonstrate that the anti-allodynic effects of both inhibitors require both cannabinoid receptors. As CIPN treatment could require repeated dosing, we demonstrate that repeated administration of 4 mg/kg JZL184 for six days produces anti-allodynic effects in contrast to tolerance development after repeated treatment with 40 mg/kg. We also show that MJN110 attenuates paclitaxel-induced inflammation in the spinal cord and dorsal root ganglia (DRG), namely monocyte chemoattractant protein-1 (MCP-1, CCL2) and phosphorylated p38 MAPK (phospho-p38) expression. Using the conditioned place preference (CPP) paradigm, we demonstrate that MJN110 produces a CPP in paclitaxel-treated, but not in control mice. As CIPN develops during chemotherapy, we also show that JZL184 does not interfere with the anti- proliferative and anti-apoptotic effects of paclitaxel in A549 or H460 lung cancer cell lines. Lastly, we show that co-administration of MAGL inhibitors with paclitaxel prevents the development of allodynia. Co-treatment with 5 mg/kg MJN110 or 40 mg/kg JZL184 prevents allodynia up to one or two week(s), respectively, after paclitaxel cessation. Treatment with 40 mg/kg JZL184 prevents allodynia in both CB1 (+/+) and (-/-) mice, suggesting that prevention is CB1-independent. Taken together, these results suggest that MAGL is a viable target for both the treatment and prevention of paclitaxel-induced allodynia in mice.

Na$ sp+$-Pi cotransport across the brush border membrane is the rate limiting step in renal Pi reabsorption. To determine its precise role in the maintenance of Pi homeostasis, we cloned and characterized the renal-specific Na$ sp+$-Pi cotransporter/Npt2 gene and generated a gene targeting vector for the creation of a knockout mouse. The gene was cloned by screening a genomic DNA library with a rat Npt2 cDNA probe. The Npt2 gene is approximately 17kb and contains 13 exons and 12 introns. A targeting construct was generated by inserting 5$ sp prime$ and 3$ sp prime$ homologous arms of 2.1 and 2kb, respectively, into the pPNT vector and replacing 7.7kb of Npt2 with a neomycin resistance gene. The vector also contained the herpes simplex virus thymidine kinase gene for negative selection. After electroporation into embryonic stem cells, clones were picked following selection in G418 and FIAU. Of 100 doubly-resistant clones that were screened by Southern analysis, 6 positive clones were detected giving a targeting frequency of 6%.

The receptor for advanced glycation end-products (RAGE) has increasingly been demonstrated to be an important modulator of inflammation in cases of pulmonary disease. Published reports involving tobacco smoke exposure have demonstrated increased expression of RAGE, its participation in pro-inflammatory signaling and its role in irreversible pulmonary remodeling. The current research evaluated for the first time the in vivo effects of short-term tobacco smoke exposure in RAGE null and control mice compared to identical animals exposed to room air only. Quantitative real time PCR, immunoblotting, and immunohistochemistry revealed elevated RAGE expression in controls after four weeks of exposure and an anticipated absence of RAGE expression in RAGE null mice regardless of smoke exposure. Inflammatory cell behaviors were confirmed by measuring active Ras, NF-κB, and cytokine synthesis and secretion. Furthermore, bronchoalveolar lavage fluid (BALF) was procured from RAGE null and control animals after exposure for the assessment of total protein in order to indirectly measure vascular permeability, inflammatory cells and chemoattractant molecules involved in the inflammatory response. As a general theme, inflammation induced by tobacco smoke exposure was influenced by the availability of RAGE. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to tobacco smoke. Furthermore, research may demonstrate RAGE signaling as an important therapeutic target capable of ameliorating cell level inflammation in those coping with exposure.

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

A ce jour, les thérapies anti-obésité restent limitées. De récente études ont fourni des résultats prometteurs en démontrant une diminution du poids de la souris via une injection stéréotaxique d’une forme dominante négative de l’AMPK (AMPK DN) directement dans le noyau ventromédial hypothalamique (VMH). Cependant, le potentiel thérapeutique de cette thérapie génique se voit entravé par une libération non spécifique de l’AMPK suite à une injection intraveineuse, plus adaptée à une approche clinique. Nous avons donc développé une approche de « nanobiomédecine » en utilisant des exosomes - nanovésicules contenant des lipides, des protéines et des acides nucléiques - pour délivrer l’AMPK DN spécifiquement au niveau du VMH. Des cellules dendritiques immatures ont été utilisées pour produire des exosomes non-inflammatoires. Pour permettre le ciblage spécifique du VMH par les exosomes, les cellules dendritiques ont été transfectées pour exprimer Lamp2b, une protéine exosomale, fusionnée au peptide de ciblage neuronal RVG. De façon intéressante, les exosomes Lamp2b-RVG ont été localisés au niveau du cerveau suite à une injection intraveineuse. Les exosomes Lamp2b-RVG ont ensuite été chargés par l’AMPK DN sous le contrôle d’un promoteur spécifique du VMH, apportant une double spéficité tissulaire aux exosomes. Les exosomes Lamp2b-RVG chargés avec l’AMPK DN induisaient une diminution de la phosphorylation de l’acetyl-CoA carboxylase dans des cellules Neu2A in vitro. De plus, l’injection intraveineuse d’exosomes Lamp2b-RVG chargés avec l’AMPK DN induisait une perte de poids de l’animal après 6 jours de traitement, démontrant le potentiel de cette approche de « nanobiomédecine »
Actual pharmacological therapies for treating obesity are limited. Promising results on decreasing mice body weight were obtained using a ventromedial nucleus hypothalamic (VMH) stereotaxic injection of a dominant negative isoform of AMPK (AMPK DN). However, DNA-mediated therapeutic potential is hampered by inadequate tissue specific delivery following a systemic injection - more adapted to a bedside approach -. Herein, we developed a nanobiomedicine approach using exosomes - nano-scaled endogenous vesicles containing lipids, proteins and nucleic acids - to deliver DNA in a hypothalamic specific way. Immature dendritic cells were used to generate non inflammatory exosomes. Exosome neuronal targeting aptitudes were achieved by constraining the dendritic cells to express Lamp2b, an exosomal protein, fused to the neuron-specific RVG peptide. Interestingly, DID-labelled Lamp2b-RVG exosomes were found into the mice brain following an intravenous injection. Isolated Lamp2b-RVG exosomes were then loaded by transfection-mediated techniques with AMPK DN under the control of a VMH specific promoter conferring double tissue expression specificity to the exosomes. AMPK DN-loaded exosomes induced a decrease of acetyl-CoA carboxylase phosphorylation in Neu2a neuronal cells in vitro. Furthermore, intravenously injected AMPK DN loaded exosomes induced a decrease of mice body weight following 6 days of treatment, demonstrating the potential of this nanobiomedicine approach

Le développement de thérapies ciblées est un enjeu majeur en santé et l’essor des nanovecteurs permet de répondre à ces besoins cliniques. Le premier axe de cette thèse est consacré à l’étude du potentiel thérapeutique de nanoparticules multifonctionnelles pour l’imagerie médicale, la thérapie photothermique et la délivrance de drogue pour le traitement du cancer. Le deuxième axe de recherche s’oriente vers le ciblage thérapeutique actif. L’entreprise NanoMedSyn a pour objectif de développer un ciblage actif du récepteur du mannose 6-phosphate, permettant un meilleur adressage des médicaments et des traitements plus efficaces. Ce type de ciblage peut avoir des retombées multiples pour la thérapie anticancéreuse mais également pour la thérapie des maladies lysosomales qui sont des maladies génétiques rares. NanoMedSyn développe des dérivés synthétiques de glycovecteurs innovants, appelés AMFA, qu’elle exploite en exclusivité. Les AMFA ont une bonne affinité pour le récepteur du mannose 6-phosphate et ont été greffés sur des nanoparticules multifonctionnelles dans le but d’améliorer l’adressage et la thérapie photodynamique biphotonique d’un cancer pédiatrique : le rhabdomyosarcome ; et sur des enzymes lysosomales pour le traitement de maladies lysosomales telle que la maladie de Pompe
The development of targeted therapies is a major health issue and the rise of nanovectors makes it possible to meet these clinical needs. The first approach of this thesis is dedicated to the study of the therapeutic potential of multifunctional nanoparticles for medical imaging, photothermal therapy and drug delivery in cancer treatment. The second line of research focuses on active therapeutic targeting. The NanoMedSyn company aims to develop an active targeting of the mannose 6-phosphate receptor, allowing a better addressing of drugs and more effective treatments. This type of targeting may have multiple benefits for cancer therapy but also for the treatment of the lysosomal diseases which are rare genetic diseases. NanoMedSyn develops innovative synthetic derivatives of glycovectors, called AMFA, which it exploits exclusively. AMFA have a good affinity for the mannose 6-phosphate receptor and have been grafted on multifunctional nanoparticles in order to improve addressing and two-photon photodynamic therapy of a pediatric cancer: the rhabdomyosarcoma; and on lysosomal enzymes for the lysosomal diseases treatment such as for Pompe disease

Si la raison d'être des aides sociales, les conditions de leur versement, l'étude des bénéficiaires sont des préoccupations anciennes et constantes de la sociologie politique de l'aide sociale, la question des modalités techniques de versement de ces aides a peu été travaillée. Parmi ces diverses modalités, des outils tels que les chèques sont restés dans l'ombre. Partant de la notion de « voucher » développée aux Etats-Unis dans les années 60 par Milton Friedman et tout particulièrement du « school voucher » ou « chèque éducation », certaines collectivités françaises ont introduit au début des années 1990 ce concept sous la forme de chèques cultures destinés au jeune public en se revendiquant d'idées libérales. Dans les années qui suivirent, on a observé un essaimage important des chèques dans les collectivités françaises sans pour autant que la filiation libérale et anglo-Saxonne ne soit revendiquée. Parallèlement, le chèque en France est arrivé sous une forme encadrée par la loi et principalement destiné au versement d'aides sociales : le Chèque Emploi Service Universel, le Chèque d'Accompagnement Personnalisé, sont désormais bien implantés dans le paysage des interventions sociales françaises. La possibilité ouverte par le législateur de payer certaines aides sociales obligatoire a permis à ces chèques d'entrer en nombre au sein des Conseils Généraux. Après avoir démontré que les chèques sociaux et culturels sont, malgré leurs modalités d'apparition différente, les descendants des « vouchers » friedmaniens, l'enquête de terrain, portant sur 6 dispositifs présents dans 3 collectivités françaises (Conseil Régional Rhône-Alpes, Conseils Généraux de la Drôme et de la Saône et Loire, a principalement consisté en la comparaison entre le discours des acteurs et les arguments traditionnellement employés dans la littérature anglo-Saxonne. Le résultat de l'enquête qualitative menée en utilisant une méthode proche de l'approche phénoménologique et priorisant donc la représentation de l'objet par l'acteur montre en effet que les 6 attributs les plus fréquemment cités dans la littérature reviennent régulièrement dans les propos des acteurs même si ces derniers ne revendiquent qu'à la marge le caractère libéral du chèque. L'enquête a pu démontrer que c'est l'action décisive de médiateurs et la promotion d'un argument absent des analyses anglo-Saxonnes qui a favorisé cette diffusion. Les acteurs citent en effet, dans les avantages du chèque, la notion de visibilité de l'aide et d'outil de communication. Il s'agit en effet de l'argument le plus répandu parmi les entreprises commercialisant ces dispositifs. Nous avons donc étudié à la fois la formation de réseaux formels et informels entre collectivités et l'apport, selon nous décisif, des entreprises ayant pu réorienter leurs « business model » afin de rendre cet outil plus attractif pour des collectivités qui auraient eu plus de difficultés à l'accepter si elles en avaient connu la vraie nature
In France, more and more social or cultural subsidies, given by local authorities to a targeted group of the population are not distributed directly in cash but under the shape of vouchers. In fact, the first local government to have proceeded like this is the Conseil Régional Rhône-Alpes in 1993 that have launched the “cultural voucher” which was given to the high school students. During the 90s, this way to proceed has become a kind of “fashion” among the French local authorities and nowadays, everything the French Region has at least one subsidy given through vouchers. This kind of tools are very wide-Spread over the world, especially in the US. As a tool of public policy it has been studied first by Milton Friedman and categorized as a pro-Choice and a libertarian tool. The most famous example of “liberal” voucher is the “school voucher” that permits the parents to choose the school of their children (it has been tested in Florida, Milwaukee and New York). The school voucher and a lot of other ones (stamp foods, medical vouchers…) has been implemented all over the world within the framework of programs leaded by institutions as the World Bank of the International Monetary Fund. The arrival of such vouchers in France seems surprising because of the political ideas of the leaders of most of the local government. It seems at least paradoxical to note that social democrat leaders have been the first to use vouchers to pay some social, cultural or other individual subsidies. This thesis tries to answer questions such “how such tools have been to spread through the French regions so easily” and “why haven't the local governments recognize that it was a pro-Choice and liberal tool that they were supposed to fight against?” Vouchers are in fact part of the French culture. France has developed in the 60s a program of luncheon vouchers that everybody knows named “Ticket restaurant”. Four firms composed the national market of the vouchers editors, three of them are the three world leaders of this global market. By using the example of the first cultural voucher created in France in 1993, they have gradually made their Business model evolved to suit these new market opportunities. One of the conclusions of the thesis is that the spreading of the vouchers among French local authorities has not only an ideological cause, but is simply the consequence commercial actions of French vouchers editor companies that have detected a new important market to conquer

Iron has been an essential element to human development and iron-oxide deposits are known to host minerals that are labelled as critical raw materials (CRMs), especially in the EU. Therefore, ensuring a sustainable supply of CRMs require access to both primary and secondary sources of their host deposits such as iron oxides. Blötberget is an old mining site in central Sweden rich in both primary and secondary iron-oxide resources (i.e. tailings) from centuries-long mining activities. Thus, this thesis focused on this site, to (1) improve the image of its iron-oxide mineralisation under the historical tailings area through the extraction and processing of 2D data from a wider and sparse 3D dataset, (2) characterise the tailings in terms of geometry delineation and geomechanical property estimation by generating P-wave velocity models of the tailings, and (3) improve the interpretation of existing results in the area through 3D visualisations and comparison. Results from this thesis work suggest possible depth and lateral extensions of the mineralisation for few hundreds of metres beyond what was known previously in the area. It is believed that about 10 Mt of primary iron-oxide resources could arguably be present under the tailing area while the tailings contain an estimated 1 Mt of secondary iron-oxide resources. Also, this thesis work findings indicate that the historical tailings are approximately 10 -12 m thick, 650 m long, and 300 m wide, and has a Vp/Vs ratio between approximately 3-4, indicating a poor geomechanical strength. Additionally, the depth to bedrock in this area was estimated to be 50 m at its deepest parts, with a morphology indicative of complex geological occurrence. Therefore, it is concluded, based on these results, that Blötberget has a good potential to ensure the supply of both iron ore and its constituent CRMs.
Järn har varit ett viktigt grundämne för mänsklig utveckling och järnoxidavlagringar är kända för att innehålla mineral som är märkta som kritiska råmaterial (KRM), särskilt inom EU. Därför kräver säkerställandet av en hållbar tillgång till KRM tillgång till både primära och sekundära källor till deras värdfyndigheter, till exempel järnoxid. Blötberget är en gammal gruvplats i mellersta Sverige som är rik på både primära och sekundära järnoxidresurser (dvs. gruvavfall) från en lång gruvverksamhet. Således fokuserade denna avhandling att (1) förbättra karaktäriseringen av järnoxidmineralisering i det historiska gruvområdet genom utvinning och bearbetning av 2D-data från ett glest 3D-dataset, (2) karakterisering av gruvavfall för avgränsning av geometri och uppskattning av geomekaniska egenskaper genom att generera P-vågshastighetsmodeller för gruvavfallsområdet, och (3) förbättra tolkningen av befintliga resultat i området genom 3D-visualiseringar. Resultat från denna avhandling tyder på möjliga djup och laterala förlängningar av mineraliseringen om några hundratals meter bortom vad som tidigare var känt i området. Det antas att cirka 10 Mt primära järnoxidresurser finnas under avfallssområdet medan gruvavfallet innehåller uppskattningsvis 1 Mt sekundära järnoxidresurser. Dessutom visar denna avhandling att det historiska gruvavfallet är cirka 10-12 m tjockt, 650 m långt och 300 m brett och har ett Vp/Vs -förhållande mellan cirka 3-4, vilket indikerar en låg geomekanisk hållfasthet. Dessutom beräknades djupet till berggrunden i detta område vara 50 m vid dess djupaste delar, med en morfologi som indikerar komplex geologisk förekomst. Därför dras slutsatsen, baserat på dessa resultat, att Blötberget har en god potential att säkerställa leveransen av både järnmalm och dess ingående KRM

10 millions dans le monde, 3 millions en Europe, 250000 en France. Ces nombres représentent la prévalence des maladies inflammatoires chroniques de l'intestin (MICI), respectivement dans chaque région citée. Le plus souvent diagnostiquées entre 15 et 35 ans, les MICI regroupent la Maladie de Crohn (MC) et la Rectocolite Hémorragique (RCH), et se caractérisent par une inflammation de la paroi du tube digestif, évoluant par poussées.Le ciblage de la partie distale du tractus gastro-intestinal (TGI), ou côlon, permet d'envisager une libération locale et optimale de substance active au niveau des zones lésées, tout en diminuant les effets indésirables des traitements. Différentes stratégies sont employées pour le ciblage du côlon par voie orale, parmi lesquelles : *) l'utilisation de prodrogues, **) Les systèmes pH-dépendants, ***) Les systèmes temps-dépendants, et ****) les systèmes sensibles au microbiote intestinal.De toutes, l'approche basée sur l'activité du microbiote reste la plus fiable. Les quelques mille milliards de bactéries par gramme de selle présentes dans le côlon peuvent, via leur activité enzymatique, dégrader les polysaccharides de structure complexe. Afin de pallier les limites des approches pH- et temps-dépendantes, les systèmes à double stimuli intéressent aussi de plus en plus les équipes de recherches. En combinant plusieurs approches différentes mais complémentaires, il est possible d'améliorer significativement la libération de la substance active in situ.Notre projet a consisté à fabriquer, pelliculer, et développer des mini-comprimés de 5 mm de diamètre pour le ciblage du côlon. Au terme d'une étape de criblage de films polymériques, permettant d'identifier le mélange le plus résistant dans le haut TGI, un pelliculage associant éthylcellulose et shellac a été retenu. Différents ratios de mélanges ont été exploités. Des tests de libération in vitro ont été menés sur une durée totale de 32h, impliquant différents milieux digestifs reconstitués. Les expériences en milieu colique ont été réalisées avec et sans selles de patients sous atmosphère anaérobie, permettant de travailler au plus près des conditions physiopathologiques.Incontestablement, la protection de la substance active a été totale dans le haut TGI. Les formes galéniques pelliculées ont aussi présenté un profil de libération contrôlée dans le côlon.La formulation finale allie plusieurs propriétés :- Une prise en eau et une dissolution contrôlées grâce à l'éthylcellulose- Une dissolution pH-dépendante liée à la shellac- Une sensibilité au microbiote grâce à la présence d'un polysaccharideLes données obtenues se sont avérées encourageantes. La libération de la substance active en milieu colique peut être modulée selon la quantité de polysaccharide ajouté. Cette phase d'optimisation a été un enjeu capital
10 million people worldwide, over 1.5 million in North America and 2 million in Europe. Those are the numbers of people affected by inflammatory bowel disease (IBD) in each region quoted, respectively. Including both Crohn's disease (CD) and ulcerative colitis (UC), inflammatory bowel disease has emerged as a public health challenge worldwide in the past decades. Often diagnosed between 15 and 35 years old, IBD are characterized by moderate to severe symptoms, and have in common relapsing-remitting cycles of mucosal inflammation.To date, there is no cure for IBD. Defined as colon targeting, targeted drug delivery systems is a way to get selective and efficient delivery of pharmacologically active compounds to the predetermined targeted region in therapeutic concentrations along with minimizing side effects of the drug. Current strategies for colon targeting rely on : *) prodrugs, **) pH-dependant systems, ***) time-dependant systems, ****) microbially triggered systems.Of all approaches, microbiota sensitive systems are currently known as the best ones for colonic drug delivery. It is also possible to combine several complementary approaches (pH- and microbiota sensitive) to significantely favor localized drug release.Our project aimed to develop 5 mm mini-tablets for colon targeting. First, a comparison of different film coatings was made to highlight the most interesting drug release profiles. Then, an innovative formulation, combining synthetic and natural polymers as well as polysaccharides, was evaluated. Different blend ratios were selected as well for films as for coated mini-tablets. In vitro drug release was carried out in simulated digestive fluids for a 32 h duration, including:- 0.1 N HCl or simulated gastric fluid (2 h)- PBS 6.8 or simulated intestinal fluid (6 h)- Colonic simulated medium with and without patients' faeces (24 h).Colonic simulated medium inoculated with patients' faeces allowed for working closer to pathophysiological conditions. Relevant results were obtained and paved the way for a promising monolayer technology. None or negligible drug release occurred up to 8 h, in the upper GIT. Also, drug could be totally protected in the lower gastrointestinal tract.Ethylcellulose, as a thermoplastic polymer, prevented from premature dissolution.Shellac, as a natural resin, provided pH-dependant properties.The adjunction of a polysaccharide acted as a substrate of microbiota.Interestingly, colonic release profiles could be optimized depending on the amount of polysaccharide added into the system

La micro-tomodensitométrie à rayons X (dite micro-CT, CT = Computed Tomography), est une technique d’imagerie de haute résolution qui consiste d’une part à mesurer l’absorption des rayons X par les tissus, et d’autre part de reconstruire les images et les structures anatomiques en 3 dimensions par traitement informatique. L’agent de contraste est une substance capable d’améliorer la visibilité des structures d’un organe ou d’un liquide organique in vivo. Ce travail de thèse a eu pour objectif le développement d’agents de contraste iodés sous formes de nano-émulsions pour des applications précliniques en imagerie biomédicale. Nous nous sommes proposés d’étudier d’une part des nano-émulsions iodées afin d’avoir une longue rémanence vasculaire in vivo, une meilleure biocompatibilité et d’autre part de mettre au point une synthèse et une formulation plus simples que celles des agents de contraste nanoparticulaires commercialisés. Trois différentes huiles iodées ont été synthétisées et utilisées comme partie contrastante dans les nano-émulsions. Enfin, les nano-émulsions de l’α-tocophérol iodé nous ont permis d’atteindre l’objectif de cette thèse. Ces nano-émulsions iodées ont montré une très bonne biocompatibilité et combinent à la fois les propriétés d’un agent de contraste à longue rémanence vasculaire et un agent de contraste spécifique du foie
The X-ray microtomography (called mico-CT, CT = Computed Tomography) is a high-resolution X-ray tomography, uses X-rays to create cross-sections of a 3D-object that later can be used to recreate a virtual model without destroying the original model. The contrast agent is a substance used to enhance the contrast of structures or fluids within the body in medical imaging. The purposes of the thesis were the development of iodine-containing nano-emulsion based contrast for preclinical applications in biomedical imaging. We proposed to study blood pool contrast agents based on iodine-containing nano-emulsions and to develop simpler procedure for the preparation of these iodine-containing nano-emulsions. Three different iodinated oils were synthesized and used as the contrasting part in the nano-emulsions. Finally, nano-emulsions of iodinated α-tocopherol have been enabled us to achieve the purpose of the thesis. These iodinated nano-emulsions demonstrated very good biocompatibility and showed prolonged and significant contrast enhancement in both bloodstream and liver tissues

The present study focuses on the biological functions of the transcription factor EMA/E2F-6, a member of the E2F-family of transcription factors that play an import role in cell cycle progression, differentiation and apoptosis. EMA/E2F-6 functions as a transcriptional repressor by recruiting a large protein complex, that includes polycomb group proteins, to specific target genes in order to silence their expression. To identify the biological functions of EMA/E2F-6 mice lacking this factor were developed and subsequently analysed. EMA/E2F6-/- mice are born with the expected frequency, are fertile and develop normally up to 18 months of age. Then about 25 % of these mice develop a paralysis of the hind limbs and present with a severe primary myelination defect of the spinal cord (and in part of peripheral nerves, too) that is accompanied by a massive infiltration of macrophages. Importantly, the histological findings were also detected in EMA/E2F-6-/- mice lacking clinical symptoms albeit to a lesser extend. With respect to EMA/E2F-6 association with polycomb group (Pc-G) proteins there were no significant findings such as skeletal transformations. In addition, only a mild proliferation defect of T-lymphocytes was observed that, in a more severe form, is typical for Pc-G mutations in the mice. Surprisingly, embryonic fibroblasts from EMA/E2F-6-/- mice have no obvious cell cycle defects. Accordingly, gene expression profiles showed that classical E2F target genes were normally regulated in these cells. However, EMA/E2F-6-/- fibroblasts ubiquitously express genes like alpha-tubulin-3 and -7 that are normally expressed in a strictly testis-specific manner. All EMA/E2F-6-dependent target genes identified contain a conserved E2F-binding site in their promoters that is required both for EMA/E2F-6 binding and regulation.

碩士
國立陽明大學
放射醫學科學研究所
94
The in vitro evaluation, biodistribution and pharmacokinetics of 111In-labelled DTPA- and VNB-liposomes in tumor-bearing BALB/c mice were studied to examine their possible role of 111In-labelled liposomes as tumor targeting agents. Methods: The DTPA/VNB-liposome was synthesized with a medium size of 110 nm, conjugated with 111In-(oxine)3 or 111In-ionomycin conjugator to afford 111In-liposomes. The stability of 111In-liposomes in serum was investigated. The biodistribution, scintigraphic imaging and pharmacokinetics of 111In-liposomes after i.v. injection into CT-26 tumor-bearing BALB/c mice were studied. Radiation toxicity of 111In-liposomes after i.v. injection into BALB/c mice was also investigated. Results: The incorporation efficiency of 111In into liposomes was 95% in both methods. After incubation at 37℃ for 72 h in serum, more than 70% of radioactivity was still retained in the intact 111In-DTPA- and 111In-VNB-liposomes labeled by oxine. However, there was only 53.5% and 40.9% of radioactivity was still retained in 111In-DTPA- and 111In-VNB- liposomes labeled by ionomycin after incubation at 37℃ for 5 mins in serum. It decreased to 25.3% and 18.6% after incubation at 37℃ for 72 h in serum, respectively. The biodistribution of 111In-DTPA-liposome showed that the radioactivity in the blood decreased from 23.14 ± 8.16 %ID/g at 1 h to 0.02 ± 0.00 %ID/g at 72 h post injection (p.i.). Accumulation of radioactivity in tumors reached a maximum at 48 h p.i. The half-life of 111In-DTPA- and 111In-VNB- liposomes in blood was 10.2 h and 7.1h, respectively. Scintigraphic imaging with 111In-DTPA-liposomes showed unambiguous tumor images at 48 h p.i. Radiation toxicity investigation showed that no critical toxicity was observed in mice injected 111In-DTPA-liposomes. IV Conclusions: This study demonstrates prolonged retention of radiolabeled lightly-pegylated liposomes within the tumor after i.v. injection and confirms the capability of 111In-liposomes to target CT-26 tumors in mice.

碩士
台北醫學院
生物醫學技術研究所
89
Growth arrest-specific gene 7 and 8 were originally identified by using a gene promoter trapping strategy from growth arrested NIH3T3 cell. Gas7 was reported expression in vivo selectively in neuronal cells of the mature cerebral cortex, hippocampus, and cerebellum. There are two kinds mRNA transcription, one is Gas7 and the other is Gas7 isoform: Gas7-cb. Gas7 its open reading frame is 421 amino acid encoded 48-kDa protein and it expresses in cerebrum; Gas7-cb are 320 amino acid encoded 38-kDa protein and it expresses in cerebellum. The preliminary results are that Gas7 interact with F-actin and Gas7 over-expression in undifferentiated neuroblastoma cell cultures dramatically promotes neurite-like outgrowth. In order to understand Gas7 in vivo function, we developed Gas7 knockout mice model. Here I screened and obtained Gas7 knockout mice by performing Southern blotting analysis. The pedigree shows how many mice generations now. After repeat 10 generations backcross to get pure B6-strain background Gas7 knockout mice. At that time we will investigate Gas7 knockout mice phenotype obviously. The second, Gas8 is 489 amino acid and encodes 57kD protein that is highly expressed in testis and adnexa. Immunohistochemical analyses were shown that Gas8 is specifically expressed in mature spermatid and highly localized in the cilia of epithelial cells from pulmonary bronchi and fallopian tubes (Yeh et al., manuscript in preparation). To understand the in vivo function of Gas8 gene, we have used gene targeting approach to develop Gas8 knockout mice model. For this purpose, genomic structure of mouse Gas8 gene has been characterized. I have determined exon-intron junction of the Gas8 gene and also the restriction enzyme map. In order to construct the Gas8 targeting vector, I have subcloned two fragments included targeted region and probe specific for Gas8 knockout ES cell and mice screening. This thesis let me systematically learn all knockout mice strategy from genomic analysis to mice breeding. Future work we will focus on Gas7 knockout mice neuron primary culture, the mice behavior and Gas8 knockout mice model development.

Background and Aims: it has been demonstrated that the anti-NGF αD11 and the anti-TrkA MNAC13 counteract neuropathic pain in mice. The aim of this study was to evaluate the duration of the action of the two antibodies and the structural and morphological alterations induced in central and peripheral nervous system. Methods: Chronic Constriction Injury (CCI) of the sciatic nerve was performed in C57BL/6J mice. Mice were administered with αD11 or MNAC13 (70 or 100 µg/mouse/day) from day 3 until day 10 post-CCI. Analgesic effects were tested through Dynamic Plantar Aesthesiometer from day 3 to day 90. Spinal cords and sciatic nerves were collected at D3, D11, D24 and D90 for immunohistochemistry. Results: αD11 and MNAC13 induce significant dose- and time-dependent analgesic effects: the antiallodynic effect was still present at D90 following the highest doses of both antibodies. Immunohistochemical analysis show significant differences in inflammatory and myelination markers between treated and control animals, treated animals showing reduced glial and mast cells activation and a better nerve regeneration. Conclusions: Data obtained prove that: i) the analgesic effect of the antibodies αD11 and MNAC13 are extremely long-lasting, being observable more than two months after the end of the treatment and ii) the antiNGF and antiTrkA antibodies reduce inflammation and facilitate the regenerative processes. Therefore, our results strongly support the importance of considering the NGF system in the development of novel therapies to modulate and control neuropathy.

Isocitrate Dehydrogenase-1 has been found to be mutated in over 70% of lower grade gliomas and has become an important diagnostic tool for tumor prognosis, however its role in glioma development, and its impact on response to therapy, is still not fully understood. Unmutated IDH1 functions to convert isocitrate to alpha-ketoglutarate (a-KG) in the tricarboxylic acid (TCA) cycle. Mutated IDH1R132H changes the enzymatic equilibrium and converts a-KG to 2-hydroxyglutarate (2-HG), an oncometabolite. IDH1R132H mutated tumors show an elevated production of 2-HG and epigenetic alterations in DNA and histone methylation. This mutation is predominantly seen in the proneural glioma subtype in which oligodendrocytes progenitors (OPCs) are considered the cell of origin due to phenotypic similarities. The effect of IDH1R132H mutation in cellular transformation has not been fully established. Epigenetic modifications connect genotype to phenotype by genetic expression alterations and epigenetic modifications are necessary for OPC differentiation. Tri-methylation of lysine residue K27 on histone H3 (H3K27me3) is a repressive mark associated with cell pluripotency. H3K27me3 is trimethylated by Enhancer of Zeste 2 (EZH2) and demethylated by the a-KG-dependent demethylases UTX/KMD6A and JMJD3/KDM6B. 2-HG is a competitive inhibitor of a-KG-dependent demethylases, providing a mechanistic link between IDH mutations and increases H3K27me3 by inhibiting demethylation. In this thesis, we evaluated the epigenetic changes in mouse models of IDH mutant and wildtype glioma and genetically-transformed OPCs and tested the effects of drugs that target specific epigenetic marks. We developed a mouse model of glioma to compare IDH1R132H cells to wildtype glioma cells and found that although there was no difference in survival, IDH1R132H gliomas have increased levels of 2-HG by MALDI-IMS and metabolomic analysis. Interestingly, based on RNA-sequencing analysis our IDH1R132H model has a more OPC-restricted expression profile compared the wildtype glioma model which have higher enrichment of genes from other cell lineages, including neurons, astrocytes, myelinating oligodendrocytes and microglia. We used the EZH2 inhibitor (Tazemetostat, EPZ-6438) and found that this treatment was not cytotoxic or cytostatic to our cells although H3K27me3 was reduced. Interestingly, Tazemetostat treatment increased the expression of non-OPC genes (genes normally expressed by other lineages as assessed using the Barres transcriptomic database). To better understand how IDH1R132H influences OPC transformation, we transformed OPCs in vitro. OPCs were isolated from floxed p53 postnatal day 5 mice from and retrovirally infected with viruses to delete p53 alone or to also express IDH1R132H. OPCs that express IDH1R132H had increased levels of 2-HG by metabolomics and showed alteration in H2K27 methylation and acetylation that resembled those seen in glioma cells. Standard methods of western blot analysis consist of analyzing whole cell lysate, cytoplasmic and nuclear fractionation, or histone acid extraction. To analyze both the cytoplasmic fraction as well as histone modification, I developed a cellular extraction method in which cells were fractionated and the nuclear fraction was acid extracted. This method allows for the analysis of both cytoplasmic proteins as well as histone modifications by western blot. Using this method, we found that treating glioma cells or OPCs with synthetic cell permeable octyl-2HG, or expressing IDH1R132H, caused cells to have increased H3K27me3, while treatment with Tazemetostat caused a decrease in H3K27me3. Based on the RNA-sequencing data we found that increased H3K27me3 (ID1R132H mutation) express more OPC-like phenotype while reduced H3K27me3 (Tazemetostat treatment) induced an upregulation of genes associated with other lineages making them less restricted to the OPC transcriptional phenotype. We found that in both the glioma cells and OPCs, Tazemetostat treatment decreased H3K27me3 and increased H3K27ac. Based on the increase of H3K27ac after Tazemetostat treatment, we hypothesized that a Histone deacetylase inhibitor (HDACi) would be synergistic. We found that although the HDACi Panobinostat was less cytotoxic to IDH1R132H mutated glioma cells and OPCs, co-treatment with Tazemetostat is synergistic in mutant and wildtype models. We also saw that in IDH1R132H ex vivo slices, the co-treatment reduced tumor marker composition. These findings point to a novel therapeutic strategy for IDH1-mutated proneural gliomas that targets the specific epigenetic alteration in these tumors.

Unique among the intracellular lipid binding proteins, acyl CoA binding protein (ACBP) exclusively binds long chain fatty acyl CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not result in any live homozygous deficient (null) ACBP^(-/-) pups. Heterozygous and wild type embryos were detected at all postimplantation stages, but no homozygous ACBP null embryos were obtained– suggesting that an embryonic lethality occurred at a preimplantation stage of development, or that embryos never formed. While ACBP null embryos were not detected at any blastocyst stage, ACBP null embryos were detected at the morula (8- cell), cleavage (2-cell), and zygote (1-cell) preimplantation stages. Two other LCFACoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCPx) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins only at the earliest preimplantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.

博士
國立臺灣大學
醫學檢驗暨生物技術學研究所
102
In mammals, Cullin genes constitute a family of eight proteins (CUL1, 2, 3, 4A, 4B, 5, 7, 9). Cullin 4B (CUL4B), a member of the Cullin protein family, serves as the structural scaffolds of the CUL4B-RING ligase complex, which recognizes and ubiquitinates selective substrates for protein degradation via the ubiquitin-proteasome system. Mutation of CUL4B in human results in X-linked intellectual disability (XLID) associated with impaired behavior and hypogonadism. However, the pathogenic role of CUL4B mutation in neuronal or other developmental defects is not understood and a mouse model for targeted Cul4b has not been described. Part I: To investigate the biological function of CUL4B, we here report the generation of Cul4b genetically engineered Cul4b mutant mice, in which exons 4 to 5 were deleted by gene targeting approach using Cre/loxP recombination system. We generated Cul4b mutant mice by crossing females carrying Cul4b floxed alleles with Sox2-Cre transgenic males, in which the deletion of Cul4b takes place specifically in embryo proper during embryogenesis. Firstly, Cul4b mutant mice were analyzed by behavior and neurological manners and we found mutant mice had abnormal spatial learning and memory ability and fewer parvalbumin-positive hippocampal neurons. Moreover, Cul4b mutant hippocampal neurons exhibited reduced dendritic complexity and spine density compared with control neurons. These data indicated the genetically engineered Cul4b mutant mouse as a potential model for human X-linked intellectual disability. In addition, CUL4B is strongly expressed in testes, suggesting that CUL4B- dependent protein degradation is involved in the control of the precisely timed and highly organized process of spermatogenesis. We found that Cul4b mutant male mice were infertile and displayed a progressive loss of germ cells from an initially normal germ epithelium of the tubules leading to oligoasthenospermia. Adult Cul4b mutant epididymides contained very low number of mature spermatozoa with pronounced morphological abnormalities. Mitosis of spermatogonial stem cells and meiosis of spermatocytes appeared unaffected. However, the loss-of-function allele affected the post-meiotic haploid spermatids during spermiogenesis. Decreased spermatids and an increased number of apoptotic germ cells were observed in Cul4b mutant testes. Because the most prominent defects were found during haploid differentiation, CUL4B was demonstrated to be critical for acrosome formation, chromatin remodeling and nuclear condensation which controls the cell death and sperm head shaping. In Cul4b mutant testes, spermatids with normal Golgi phase acrosome could be detected. However there were a variety of acrosome abnormalities including overly extended acrosomes and acrosomes encircled the nuclei in acrosome phase spermatids. Analysis of the first wave of spermatogenesis in Cul4b mutant mice also showed degeneration of round spermatids, amorphous acrosomes and disintegrated nuclei by day 27 and this phenomenon was consistent with adult spermatogenic cycle. We further isolated total protein from control and mutant testes at P20 and P27 to proceed with multidimensional liquid chromatography and analyzed by mass spectrometry. Quantification of identified proteins and relative expression changes were compared using an ANOVA statistical measurement to present the proteomic dataset. To obtain a global view of the molecular pathways and process networks, differential proteins were determined by MetaCore database. The pathways and networks with the higher representation were related with cytoskeleton rearrangement, cell adhesion, apoptosis, ubiquitin-proteasomal proteolysis and protein folding. Taken together, these collective data indicated that perturbed CUL4B function, as evidenced in the Cul4b mutant mice, results in disrupted haploid spermatid differentiation and male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility.Part II:In part I, mutation of Cul4b gene in mice causes abnormal spatial learning ability and male infertility. However, the epiblast-specific Cul4b knockout mice could not present the early embryogenesis and placentation. We here report the generation of Cul4b knockout mice, in which exons 4 to 5 were deleted by gene targeting approach using Cre/loxP recombination system. Cul4b conditional knockout mice were mated with Prm1-cre and Zp3-cre transgenic mice as deletion of Cul4b was exclusively occurred in spermatid and oocyte. We found that Cul4b null embryos exhibit arrested development and lethality around embryonic day 7.5. (E7.5). Cul4b heterozygotes had different phenotypes due to parent-of-origin mutant allele. Cul4b+/Δ heterozygotes were viable, fertile, normal in size and did not display any gross physical abnormalities. However, Cul4bΔ/+ exhibited a severe developmental delay from E11.5 and mostly suffered prenatal death due to the paternal X chromosome is preferentially inactivated in the placenta and resulted in Cul4b null placentas in Cul4bΔ/+ heterozygotes. Cul4bΔ/+ placentas exhibited deficiency of lower count of trophoblast giant cells at E8.5, decreased size in spongiotrophoblast layer from E11.5, disorganized labyrinth layer and impaired vascularization during E11.5-E18.5. The blood spaces within the labyrinthine layer were disrupted and the fetal blood vessels and the maternal sinusoids were considerably larger, leading to a reduction in the surface area available for nutrient and gas exchange. Although Cul4b null embryos exhibited more pronounced phenotypes than Cul4bΔ/+ heterozygotes, the lethality could be rescued by epiblast-specific deletion (Sox2-cre) of Cul4b and gave rise to viable Cul4b null mice and Cul4bΔ/+ heterozygotes. Together, our results showed that CUL4B is required in extra-embryonic tissues for placental development but indispensable for embryonic development in the mouse.

博士
國立臺灣大學
分子醫學研究所
95
Brain function needs the precise anatomical and histological connections generated by exquisitely specific axonal guidance system during development. Neuronal outgrowth is directed by attracting by attracting and repelling signaling molecules. Well known guidance cues are semaphorins/collapsins. Semaphorin 3A, also known as Collapsin-1 is thought to be an initiator of signaling involved in neural growth cone outgrowth. Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that mediate the signal from Semaphorin 3A. Collapsing response mediator protein-1 (CRMP-1) was initially identified in brain and has been implicated in plexin-dependent neuronal function. The high amino acid sequence identity among the five CRMPs has hindered determination of the functions of each individual CRMP. In 2001, CRMP-1 had also been characterized as a tumor metastasis suppressor gene dependent on its role in lung cancer metastasis and clinical outcome. In order to study the physiological function of CRMP-1 in vivo, we generated viable and fertile CRMP-1 knockout (CRMP-1-/-) mice with no evidence of gross abnormality in the major organs and difference in hematological and biochemical analysis between wild-type mice. According to high CRMP-1 expression in the hippocampus, we analyzed the formation of axon and dendrite there by staining with neural markers. CRMP-1-/- mice exhibited intense MAP2 staining in the proximal portion of the dendrites, but reduced and disorganized MAP2 staining in the distal dendrites of hippocampal CA1 pyramidal cells. Immunoreactivity to GAP-43 and PSD95 (a postsynaptic membrane adherent cytoskeletal protein) was also decreased in the CA1 region of the knockout mice. These changes were consistent with the mutant mice showing a reduction in long-term potentiation (LTP) in the CA1 region and impaired performance in hippocampal-dependent spatial learning and memory tests. CRMP-1-/- mice showed a normal synapsin I labeling pattern in CA1 and normal paired pulse facilitation. In addition, CRMP-1-/- also showed more depressed in forced swimming test suggested that CRMP-1 may involved in emotion control. These findings provide the first evidence suggesting that CRMP-1 may be involved in proper neurite outgrowth in the adult hippocampus and that loss of CRMP-1 may affect LTP maintenance and spatial learning and memory. Furthermore, CRMP-1-/- mice also exhibit more depressed in forced swimming test compared with wild-type mice suggested that CRMP-1 may regulate emotion response. On the other hand, CRMP-1-/- mice also show defect in cerebellum development. Mice deficient in CRMP-1 not only showed reduced weight in cerebellum but also lack the normal organic structure in VIb lobe suggested that CRMP-1 also involved in cerebellum development.

The aim of the present study was to design different dosage forms as carrier systems to deliver sorafenib to the lung of BXB-23 transgenic mice using different routes of administration. Three dosage forms were used one of them was an oil-in-water emulsion and the oral route was chosen for this experiment. The other delivery system was a liposome preparation for intratracheal instillation. In this case the oral route was considered as a control experiment. The last dosage form was PLGA microspheres. Before sorafenib administration it was important to develop a HPLC method to assess sorafenib absorption after its administration and to determine its concentrations in mouse serum. The HPLC method allowed sorafenib quantification in small volumes (30 µl) of mouse serum and tissues. The developed HPLC method was validated resulting in satisfactory selectivity, good linearity, good accuracy and precision over the concentration range examined. Sorafenib was successfully incorporated in a fat emulsion (o/w) using a traditional method resulting in a white homogenous emulsion and no particle aggregation was observed. Sorafenib exhibited antitumor activity on the lung adenoma in BXB-23 transgenic mice when administered orally (2 mg sorafenib per mouse) in the emulsion preparation. The determined effect was an approximately 29 % reduction in the tumor area of the adenoma foci and a proliferation reduction. In order to improve the pharmacological effects of sorafenib on the lung adenoma in BXB-23 mice, the targeting of sorafenib directly to the site of action (the lung) was an attractive concept. For this purpose the intratracheal route was used. Since sorafenib administration by instillation required incorporation of sorafenib in a dosage form suitable for its lipophilic nature, a liposome suspension was the second dosage form used. A lyophilization method was employed for sorafenib liposome preparation utilizing dilauroylphosphatidylcholine (DLPC) which is safe and tolerable for the lung. Incorporation of sorafenib in the liposomes did not influence the particle size and its distribution. The sorafenib liposomes showed high encapsulation efficiency, good stability at 4 °C for one month and satisfactory in vitro release properties and inhibited Raf-1 mediated activation of ERK in cell culture assay. In a pharmacokinetic experiment sorafenib loaded liposomes were instilled directly into the lung. The results revealed that a significant level of sorafenib was achieved in the lung tissues after 2 hours and then reduced after 48 h and remained nearly constant for one week. On the other hand, only traces of sorafenib were found in the mice serum up to 48 h. Subsequently, the pharmacological activity of sorafenib (1 mg per mouse) was studied when delivered in a liposomal suspension intratracheally to treat the lung adenoma of BXB-23 mice. The data of this experiment demonstrated that sorafenib intratracheal instillation resulted in a reduction of tumor area of adenoma foci (67 %) and an elevation of the percent of apoptotic cells. In contrast, prolongation of the treatment period did not further enhance sorafenib activity on the lung adenoma. This previous finding suggested a development of multidrug resistance (MDR) by the adenoma foci cells against sorafenib instillation, which was examined by immunohistochemistry staining. The percent of MDR positive cells was higher after two and three weeks sorafenib liposome instillation treatment than that after one week treatment. The last dosage form used for sorafenib was microspheres, which were prepared by emulsion-diffusion-evaporation method using biodegradable PLGA 50:50 resulting in a white lyophilized powder. The system was characterized physicochemically and revealed a good microspheres yield, high encapsulation efficiency, a homogenous particle size distribution and slow in vitro release of sorafenib. The other strategy studied in the present research project was gene delivery to target the lung bearing tumor of BXB-23 mice using a non-viral vector (polyethylenimine). Polyethylenimine (PEI) was used to investigate its efficiency in transfecting lung bearing tumor of BXB-23 mice model and its ability to transfect the adenoma foci cells. LacZ, which encodes Beta-galactosidase was used in the present study as a reporter gene and was complexed with PEI before delivered intravenously. A high LacZ expression in the alveolar region with some expression in the adenoma foci was observed. On contrary, a low LacZ expression in the alveoli and in the adenoma foci was achieved after instillation of the same polyplex intratracheally
Das Ziel der vorliegenden Dissertation war es, verschiedene galenische Darreichungsformen als Trägersystem für Sorafenib zu entwickeln, um den direkten Transport des Arzneistoffes zum Zielorgan Lunge von BXB-23 transgenen Mäusen zu ermöglichen. Für die verschiedenen Applikationswege wurden drei Darreichungsformen gewählt. Eine Öl-in-Wasser-Emulsion sollte oral verabreicht werden. Für die intratracheale Instillation wurde ein liposomales Präparat gewählt. Die letzte Darreichungsform stellten PLGA Mikrosphären dar. Um die Absorption von Sorafenib nach Administration bestimmen zu können, wurde die Konzentration des Arzneistoffes im Mäuseserum gemessen. Zur Quantifizierung von Sorafenib in einem geringen Volumen Serum und in Gewebe wurde eine HPLC-Methode entwickelt und validiert. Sorafenib wurde erfolgreich in eine Fettemulsion (o/w) mittels einer traditionellen Methode eingearbeitet. Nach oraler Verabreichung der Emulsion (2 mg/Maus) zeigte Sorafenib auf Lungenadenome eine Antitumor-Aktivität, wobei eine Reduktion der Tumorfläche der Adenomfoci um etwa 29 % und eine Reduktion der Proliferation verzeichnet werden konnte. Zur Verbesserung der pharmakologischen Effekte von Sorafenib auf die Lungenadenome in BXB-23 Mäusen zu verbessern, sollte Sorafenib direkt dem Zielorgan Lunge zugeführt werden. Zu diesem Zweck wurde der intratracheale Administrationsweg gewählt. Da die Instillation von Sorafenibaufgrund seiner lipophilen Natur nur durch Einschluß in eine andere Darreichungsform zu erreichen ist, wurde für die zweite Darreichungsform eine Liposomen-Suspension verwendet. Für die Zubereitung von Sorafenib in Liposomen wurde eine Lyophilisierungsmethode unter Verwendung von DPLC erarbeitet. Die Einschluss-Effektivität der Sorafenib-beladenen Liposomen war hoch und zeigte bei 4°C eine gute Stabilität für einen Monat. Die erzielten Effekte bei der in vitro Freisetzung und die Hemmung der von Raf1-induzierten Aktivierung von ERK in Zellkulturexperimenten lieferten zufrieden stellende Ergebnisse. In einem pharmakokinetischen Experiment wurden mit Sorafenib beladenen Liposomen direkt in die Lunge appliziert. Die Ergebnisse zeigten, dass nach 2 h eine signifikante Konzentration von Sorafenib im Lungengewebe erreicht wurde. Nach 48 h nahm diese Konzentration ab und blieb dann für eine Woche fast konstant. Andererseits wurden bis zu 48 h nach Gabe des Arzneistoffes nur Spuren von Sorafenib im Mäuseserum gefunden. Folglich wurde die pharmakologische Aktivität von Sorafenib (1 mg/Maus) bei intratrachealer Verabreichung in einer liposomalen Suspension untersucht. Die Ergebnisse zeigten, dass die intratracheale Gabe von Sorafenib eine Reduktion der Tumorfläche der Adenomfoci um 67 % bewirkte, sowie eine Erhöhung des prozentualen Anteils apoptotischer Zellen. Eine Verlängerung der Behandlungszeit zeigte keine zusätzliche Verbesserung der Effekte. Dies lies vermuten, dass hier eine Entwicklung von Multidrug-Resistenz in den Adenomfocizellen gegenüber der Instillation von Sorafenib erfolgte. Dies wurde in immunochemischen Anfärbe-Experimenten untersucht. Die Prozentzahl von MDR-positiven Zellen war nach zwei und drei Wochen Instillation von Sorafenib-Liposomen höher als nach einer Woche. Die letzte verwendete Darreichungsform für Sorafenib waren Mikrosphären, die durch Emulsions-Diffusions-Evaporations-Methoden in biologisch abbaubarem PLGA 50:50 hergestellt wurden. Dies ergab ein weißes, lyophilisiertes Pulver. Das System wurde physiochemisch charakterisiert und ergab ein gutes Mikrosphären-Ergebnis, hohe Einschluss-Effektivität, eine homogene Verteilung der Partikelgrößen und eine langsame in vitro Freisetzung von Sorafenib. Die andere untersuchte Strategie war Gen-Delivery, um den Lungentumor von BXB-23 Mäusen mittels eines nicht-viralen Vektors (Polyethylenimin, PEI) anzuzielen. PEI wurde verwendet, um die Effektivität der Transfektion des Lungentumors zu untersuchen und seine Fähigkeit, die Adenomfocizellen zu transfizieren. LacZ, das Beta-Galactosidase codiert, diente bei diesem Experiment als Reportergen und wurde vor intravenöser Gabe mit PEI komplexiert. Eine hohe LacZ-Expression in der alveolaren Region, aber nur eine geringe Expression in den Adenomfoci wurde beobachtet. Im Gegensatz dazu wurde eine geringe Expression von LacZ in den Alveolen und den Adenomfoci nach intratrachealer Instillation des gleichen Polyplex erreicht

Lee Yiu Fai.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 178-183).
Abstracts in English and Chinese.
Acknowledgements --- p.ii
Abstract --- p.iii
Abstract (Chinese version) --- p.vi
Table of contents --- p.viii
List of Abbreviations --- p.xvi
List of Figures --- p.xviii
List of Tables --- p.xxiv
Chapter Chapter 1 --- INTRODUCTON
Chapter 1.1 --- Cytochrome P450 (P450) --- p.1
Chapter 1.1.1 --- Cytochrome P450 family --- p.1
Chapter 1.1.2 --- Role in metabolism --- p.4
Chapter 1.1.3 --- P450 catalytic cycle --- p.6
Chapter 1.2 --- NADPH-cytochrome P450 reductase (RED) --- p.6
Chapter 1.2.1 --- Characterization and distribution --- p.6
Chapter 1.2.2 --- Structural and functional domains --- p.8
Chapter 1.2.3 --- Role in P450 catalytic cycle --- p.10
Chapter 1.3 --- Drug metabolism --- p.10
Chapter 1.3.1 --- Understanding of drug metabolism is important for drug development --- p.10
Chapter 1.3.2 --- Role of P450 in drug metabolism --- p.12
Chapter 1.4 --- Production of RED knockout in vivo mouse model for screening of P450-dependent new drugs --- p.13
Chapter 1.4.1 --- Background --- p.13
Chapter 1.4.2 --- Gene targeting --- p.13
Chapter 1.4.3 --- Gene targeting vector --- p.15
Chapter 1.4.3.1 --- Classical knockout --- p.15
Chapter 1.4.3.2 --- Conditional knockout --- p.19
Chapter 1.4.4 --- Gene knockout mice and its use --- p.21
Chapter Chapter 2 --- OBJECTIVES --- p.22
Chapter Chapter 3 --- MATERIALS AND METHODS --- p.24
Chapter 3.1 --- Preparation of RED cDNA by RT-PCR --- p.24
Chapter 3.1.1 --- Total RNA isolation --- p.24
Chapter 3.1.1.1 --- Materials --- p.24
Chapter 3.1.1.2 --- Methods --- p.24
Chapter 3.1.2 --- Reverse transcription- polymerase chain reaction (RT-PCR) --- p.25
Chapter 3.1.2.1 --- Materials --- p.25
Chapter 3.1.2.2 --- Methods --- p.25
Chapter 3.1.3 --- T/A cloning of RED cDNA --- p.28
Chapter 3.1.3.1 --- Materials --- p.28
Chapter 3.1.3.2 --- Methods --- p.28
Chapter 3.1.4 --- Midi-preparation of RED cDNA clone --- p.32
Chapter 3.1.4.1 --- Materials --- p.33
Chapter 3.1.4.2 --- Methods --- p.33
Chapter 3.1.5 --- Confirmation of RED cDNA clone --- p.34
Chapter 3.1.5.1 --- Restriction enzyme mapping --- p.34
Chapter 3.1.5.1.1 --- Materials --- p.34
Chapter 3.1.5.1.2 --- Methods --- p.34
Chapter 3.1.5.2 --- DNA sequencing of RED cDNA sequence --- p.35
Chapter 3.1.5.2.1 --- Materials --- p.35
Chapter 3.1.5.2.2 --- Methods --- p.35
Chapter 3.1.6 --- Preparation and purification of RED cDNA for probe labeling --- p.38
Chapter 3.1.6.1 --- Materials --- p.38
Chapter 3.1.6.2 --- Methods --- p.38
Chapter 3.1.7 --- Non-radioactive random-primed labeling of RED cDNA --- p.39
Chapter 3.1.7.1 --- Materials --- p.39
Chapter 3.1.7.2 --- Methods --- p.39
Chapter 3.2 --- Isolation of RED gene by genomic library screening --- p.40
Chapter 3.2.1 --- Titering of genomic library --- p.41
Chapter 3.2.1.1 --- Materials --- p.41
Chapter 3.2.1.2 --- Methods --- p.41
Chapter 3.2.2 --- Primary screening of genomic library by RED cDNA probe --- p.42
Chapter 3.2.2.1 --- Plaque lift --- p.42
Chapter 3.2.2.1.1 --- Materials --- p.42
Chapter 3.2.2.1.2 --- Methods --- p.42
Chapter 3.2.2.2 --- Proteinase K treatment --- p.43
Chapter 3.2.2.2.1 --- Materials --- p.43
Chapter 3.2.2.2.2 --- Methods --- p.43
Chapter 3.2.2.3 --- "Pre-hybridization, hybridization and detection" --- p.44
Chapter 3.2.2.3.1 --- Materials --- p.44
Chapter 3.2.2.3.2 --- Methods --- p.44
Chapter 3.3 --- Isolation of RED by hybridization screening by Genome System Inc. --- p.45
Chapter 3.4 --- Characterization of BAC clones containing RED genomic DNA fragments commercially obtained from Genome System Inc. --- p.45
Chapter 3.4.1 --- Large scale preparation of BAC DNA --- p.45
Chapter 3.4.1.1 --- Materials --- p.47
Chapter 3.4.1.2 --- Methods --- p.47
Chapter 3.4.2 --- Restriction enzyme mappings and Southern blotting analysis of BAC DNA fragments --- p.47
Chapter 3.4.2.1 --- Materials --- p.48
Chapter 3.4.2.2 --- Methods --- p.48
Chapter 3.4.3 --- Shot-gun sub-cloning of RED genomic DNA fragments from BAC clone in pGEM®-3Z vector --- p.49
Chapter 3.4.3.1 --- Preparation of cloning vector and DNA insert for ligation --- p.50
Chapter 3.4.3.1.1 --- Materials --- p.50
Chapter 3.4.3.1.2 --- Methods --- p.50
Chapter 3.4.3.1.2.1 --- Cloning vectors --- p.50
Chapter 3.4.3.1.2.2 --- DNA inserts --- p.52
Chapter 3.4.3.2 --- Preparation of competent cells and transformation --- p.52
Chapter 3.4.3.2.1 --- Materials --- p.52
Chapter 3.4.3.2.2 --- Methods --- p.53
Chapter 3.4.3.3 --- Screening for positive recombinant clones --- p.54
Chapter 3.4.3.3.1 --- Picking of colonies randomly from the agar plates (method 1) --- p.54
Chapter 3.4.3.3.1.1 --- Materials --- p.54
Chapter 3.4.3.3.1.2 --- Methods --- p.54
Chapter 3.4.3.3.2 --- Colony lifts and hybridization with RED cDNA probes (method 2) --- p.55
Chapter 3.4.3.3.2.1 --- Materials --- p.55
Chapter 3.4.3.3.2.2 --- Methods --- p.55
Chapter 3.5 --- Restriction enzyme mappings and Southern blotting analysis of RED gene subcloned in pGEM®-3Z vector --- p.56
Chapter 3.5.1 --- Materials --- p.56
Chapter 3.5.2 --- Methods --- p.56
Chapter 3.6 --- Exon mappings of the RED genomic DNA fragments by PCR --- p.57
Chapter 3.6.1 --- Materials --- p.57
Chapter 3.6.2 --- Methods --- p.57
Chapter 3.7 --- Construction of gene targeting vector --- p.57
Chapter 3.7.1 --- Gene targeting vectors la and lb derived from clone H (strategy 1) --- p.60
Chapter 3.7.1.1 --- Sub-cloning 3.65 kb Hind Ill/Hind III RED gene fragment to pGEM®-3Z vector --- p.60
Chapter 3.7.1.1.1 --- Materials --- p.62
Chapter 3.7.1.1.2 --- Methods --- p.62
Chapter 3.7.1.2 --- Deletion of exonic sequence of RED gene and modification of the digested restriction end to Xho I site --- p.62
Chapter 3.7.1.2.1 --- Materials --- p.63
Chapter 3.7.1.2.2 --- Methods --- p.63
Chapter 3.7.1.3 --- Preparation of neo cassette --- p.63
Chapter 3.7.1.3.1 --- Materials --- p.64
Chapter 3.7.1.3.2 --- Methods --- p.64
Chapter 3.7.1.4 --- Cloning of neo cassette --- p.66
Chapter 3.7.1.4.1 --- Methods --- p.66
Chapter 3.7.1.5 --- Sub-cloning the neo cassette containing RED genomic fragment to pMCI-Thymidine kinase (TK) Poly A vector --- p.67
Chapter 3.7.1.5.1 --- Materials --- p.67
Chapter 3.7.1.5.2 --- Methods --- p.67
Chapter 3.7.2 --- "Gene targeting vectors 2a/2b, 3a/3b and 4a derived from clone X8 (strategy 2,3 and 4 respectively)" --- p.67
Chapter 3.8 --- Preparation and testing the genomic probes for screening recombinant embryonic stem (ES) cells --- p.73
Chapter 3.8.1 --- Cloning of genomic probes --- p.73
Chapter 3.8.1.1 --- Materials --- p.73
Chapter 3.8.1.2 --- Methods --- p.73
Chapter 3.8.2 --- Purification of DNA for labeling --- p.78
Chapter 3.8.2.1 --- Materials --- p.78
Chapter 3.8.2.2 --- Methods --- p.78
Chapter 3.8.3 --- ECF random prime labeling of genomic probes --- p.79
Chapter 3.8.3.1 --- Materials --- p.79
Chapter 3.8.3.2 --- Methods --- p.79
Chapter 3.8.4 --- Restriction enzyme digestion of genomic DNA and Southern blotting --- p.80
Chapter 3.8.4.1 --- Materials --- p.80
Chapter 3.8.4.2 --- Methods --- p.80
Chapter 3.8.5 --- Testing the specificity of genomic probes --- p.80
Chapter 3.8.5.1 --- Materials --- p.80
Chapter 3.8.5.2 --- Methods --- p.80
Chapter Chapter 4 --- RESULTS --- p.86
Chapter 4.1 --- Total RNA isolation and RT-PCR of RED cDNAs --- p.86
Chapter 4.2 --- Confirmation of the RT-PCR RED cDNA clone --- p.86
Chapter 4.2.1 --- Restriction enzyme mapping --- p.86
Chapter 4.2.2 --- DNA sequencing --- p.86
Chapter 4.3 --- Genomic library screening of RED gene --- p.90
Chapter 4 4 --- Restriction enzyme mappings and Southern blotting analysis of RED Gene containing BAC clone from Genome System Inc. --- p.90
Chapter 4.5 --- Shot-gun sub-cloning of RED gene containing genomic DNA fragments to pGEM®-3Z vectors --- p.93
Chapter 4.5.1 --- Cloning of Hind III cut RED gene fragment --- p.93
Chapter 4.5.2 --- Cloning of Xba I cut RED gene fragment --- p.93
Chapter 4.5.3 --- Cloning of EcoR I cut RED gene fragment --- p.95
Chapter 4.6 --- Identification of RED exons in the shot-gun sub-cloning clones by PCR --- p.95
Chapter 4.7 --- Construction of restriction enzyme maps of the RED gene containing clones --- p.100
Chapter 4.7.1 --- Clone H --- p.100
Chapter 4.7.1.1 --- Single restriction enzyme digestions and Southern blotting --- p.100
Chapter 4.7.1.2 --- Double restriction enzyme digestions and Southern blotting --- p.100
Chapter 4.7.1.3 --- Restriction enzyme map --- p.101
Chapter 4.7.2 --- Clone X8 --- p.101
Chapter 4.7.2.1 --- Single restriction enzyme digestions and Southern blotting --- p.101
Chapter 4.7.2.2 --- Double restriction enzyme digestion and Southern blotting --- p.104
Chapter 4.7.2.3 --- Restriction enzyme map --- p.104
Chapter 4.7.3 --- Clone El4 --- p.105
Chapter 4.7.3.1 --- Single restriction enzyme digestions and Southern blotting --- p.105
Chapter 4.7.3.2 --- Double restriction enzyme digestion and Southern blotting --- p.108
Chapter 4.7.3.3 --- Restriction enzyme map --- p.108
Chapter 4.8 --- Construction of gene targeting vector --- p.108
Chapter 4.8.1 --- Gene targeting vector based on the clone H (strategy 1) with deletion of RED exon 16 --- p.113
Chapter 4.8.1.1 --- Cloning a smaller RED genomic DNA into pGEM®-3Z vectors --- p.113
Chapter 4.8.1.2 --- Replacement of exon of RED gene by neo cassette --- p.113
Chapter 4.8.1.3 --- Cloning to TK vector --- p.113
Chapter 4.8.2 --- Targeting vector based on the clone X8 --- p.124
Chapter 4.8.2.1 --- Strategy 2 (deletion of RED exon 4) --- p.124
Chapter 4.8.2.1.1 --- Cloning 3.9 kb Kpn I/Hinc II RED genomic DNA into pGEM®-3Z vectors --- p.124
Chapter 4.8.2.1.2 --- Replacement of exon of RED gene by neo cassette --- p.124
Chapter 4.8.2.1.3 --- Cloning to TK vector --- p.124
Chapter 4.8.2.2 --- Strategy 3 (deletion of RED exon 5-8) --- p.136
Chapter 4.8.2.2.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136
Chapter 4.8.2.2.2 --- Replacement of exon of RED gene by neo cassette --- p.136
Chapter 4.8.2.2.3 --- Cloning to TK vector --- p.136
Chapter 4.8.2.3 --- Strategy 4 (deletion of RED exon 7-10) --- p.136
Chapter 4.8.2.3.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136
Chapter 4.8.2.3.2 --- Replacement of exon of RED gene by neo cassette --- p.152
Chapter 4.8.2.3.3 --- Cloning to TK vector --- p.152
Chapter 4.9 --- Testing for the specificity of genomic DNA probes --- p.152
Chapter 4.9.1 --- Preparation of restriction enzyme digested genomic DNA --- p.152
Chapter 4.9.2 --- Hybridization of the probes to genomic DNA --- p.163
Chapter Chapter 5 --- DISCUSSION --- p.167
Chapter 5.1 --- Proposed significant of RED knockout mice for new drug screening --- p.167
Chapter 5.2 --- Experimental problems --- p.168
Chapter 5.2.1 --- Genomic library screening --- p.168
Chapter 5.2.2 --- Cloning --- p.168
Chapter 5.3 --- RED gene targeting vector construction
Chapter 5.3.1 --- Isolation of RED gene for gene targeting vectors construction --- p.169
Chapter 5.3.2 --- Deletion of different exons in different RED gene targeting vectors --- p.169
Chapter 5.3.3 --- Components in the targeting vectors --- p.170
Chapter 5.3.4 --- Enhancements of homologous recombination --- p.171
Chapter Chapter 6 --- CONCLUSIONS --- p.173
Chapter Chapter 7 --- FUTURE STUDIES --- p.175
Chapter 7.1 --- Identification of the sizes of RED gene introns --- p.175
Chapter 7.2 --- Production of RED knockout mice --- p.175
Chapter 7.3 --- Characterization of RED knockout mice --- p.175
Chapter 7.4 --- Conditional gene knockout for RED gene --- p.177
REFERENCES --- p.178
APPENDIX --- p.184

La displasia fibrosa (DF) è una malattia genetica dell’osso e del midollo osseo causata da mutazioni missenso attivanti nel gene codificante per la subunità α della proteina G stimolatoria, Gs (Gsα). Fratture patologiche, deformità e dolori ossei rappresentano comunemente l’espressione clinica della malattia, correlata a sostituzione di osso normale e midollo osseo con tessuto abnorme, a carente mineralizzazione ed instabilità dell’osso, a midollo osseo fibrotico e non ematopoietico. Tali anomalie dipendono dalla disfunzione delle cellule che formano l’osso (osteoblasti), causata dalla presenza della mutazione nelle cellule stesse e nei loro progenitori, le cellule stromali del midollo osseo (BMSC). Ad oggi, non sono note le modificazioni molecolari generate dalla mutazione, né quale sia il contributo dei diversi tipi cellulari al fenotipo malattia, né è disponibile una cura efficace per la DF. Per definire esaustivamente a livello molecolare, cellulare ed organismico gli eventi fisiopatologici della DF, e per identificare nuove strategie terapeutiche, abbiamo generato e studiato modelli di DF in vitro e in vivo. Per analizzare la modulazione trascrizionale indotta dalla mutazione attivante di Gsα (R201C), abbiamo esaminato con i microarray il profilo di espressione di BMSC umane, ingegnerizzate per esprimere stabilmente la mutazione GsαR201C. L’analisi interpretativa dei dati di microarray ha evidenziato la modulazione di geni che sottendono i fondamentali cambiamenti tissutali osservati nella DF, tra cui MGP (artefice della sotto-mineralizzazione dell’osso) e RANKL (responsabile dell’eccessivo riassorbimento osseo), entrambi possibili bersagli terapeutici. D’altra parte, per valutare il contributo di specifiche popolazioni cellulari al fenotipo malattia abbiamo generato modelli murini che consentono l’espressione tessuto-specifica di GsαR201C. In particolare, abbiamo prodotto topi con l’espressione della GsαR201C confinata alle cellule murali, ossia i progenitori scheletrici intesi come cellule microvascolari. L’analisi ai raggi X di questi animali ha messo in luce un’alterazione radiografica delle ossa femorali dei topi, suggerendo che l’espressione della Gsα mutata nelle cellule murali causi anormalità del tessuto scheletrico. Inoltre, per ottenere nuovi modelli murini tessuto-specifici, abbiamo prodotto topi condizionali per l’espressione tessuto-specifica della GsαR201C (Lox-Stop-Lox-GsαR201C). Lo studio radiografico di questi animali ha confermato l’assenza di anomalie ossee. Questi animali potranno essere incrociati con topi che esprimano la ricombinasi Cre nei diversi tessuti di interesse, per ottenere un’ ampia gamma di topi GsαR201C tessuto-specifici. Nel suo insieme, questo lavoro è stato importante per l’identificazione di possibili bersagli terapeutici, ha contribuito a definire l’istopatogenesi molecolare della DF, e, in particolare, dall’uso/analisi di topi GsαR201C tessuto-specifici, potrà derivare un’ulteriore caratterizzazione della patologia.

Fused in sarcoma (FUS) is an RNA binding protein involved in DNA repair and RNA metabolism, including mRNA transcription, splicing, transport and translation. FUS is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which pathogenic mutations in FUS cause neurodegeneration in ALS-FUS, we generated a series of FUS knock-in mouse lines that express the equivalent of the ALS-associated mutant proteins FUSP525L and FUSΔEX14 at physiological levels from the FUS locus. We demonstrate that heterozygous mutant FUS mice show progressive, age-dependent loss of vulnerable subpopulations of spinal motor neurons. While ALS-associated mutations in FUS lead to partial loss of function, we provide genetic evidence that the motor neuron phenotype observed is a consequence of a dose-dependent gain of function, associated with the insolubility of FUS and related RNA binding proteins (RBPs). Furthermore, we show that motor neuron degeneration is driven by cell autonomous mechanisms, associated with mutant FUS-independent inflammatory changes. In this faithful mouse model of ALS-FUS, we demonstrate that an antisense oligonucleotide (ASO) targeting the FUS transcript (ION363) results in the efficient silencing of both wild type and mutant FUS alleles, and that postnatal reduction of FUS protein levels in the brain and spinal cord delays disease onset in this mouse model of ALS-FUS. In a first-in-human trial of ION363, we demonstrate that repeated, intrathecal injections of this candidate therapeutic in an ALS patient with a FUSP525L mutation leads to the efficient silencing of both wild type and mutant FUS in the central nervous system, and a reduction in the burden of FUS aggregates that are a pathological hallmark of ALS-FUS. In mouse genetic and human clinical studies, we provide evidence in support of a therapeutic strategy by which silencing of the FUS gene may be used to prevent or delay disease onset in pre-symptomatic carriers of pathogenic FUS mutations, or to slow disease progression in symptomatic ALS- and FTD-FUS patients. In addition, we use this newly generated model to investigate the role of potential modifiers of FUS toxicity, including hnRNP U and UPF1, and study the role of chronic neuroinflammation in the disease progression that could lead to the development of novel therapeutics to provide immediate clinical benefit to patients with ALS-FUS.

Indiana University-Purdue University Indianapolis (IUPUI)
Lafora disease is a neurodegenerative disorder caused by mutations in either the EPM2A or the EPM2B gene that encode a glycogen phosphatase, laforin and an E3 ubiquitin ligase, malin, respectively. A hallmark of the disease is accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle and heart. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein degradation/quality control. We evaluated three arms of protein quality control (the autophagolysosomal pathway, the ubiquitin-proteasomal pathway, and ER stress response) in embryonic fibroblasts from Epm2a-/-, Epm2b-/- and Epm2a-/- Epm2b-/- mice. There was an mTOR-dependent impairment in autophagy, decreased proteasomal activity but an uncompromised ER stress response in the knockout cells. These defects may be secondary to the glycogen overaccumulation. The absence of malin, but not laforin, decreased the level of LAMP1, a marker of lysosomes, suggesting a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal. To understand the physiological role of malin, an unbiased diGly proteomics approach was developed to search for malin substrates. Ubiquitin forms an isopeptide bond with lysine of the protein upon ubiquitination. Proteolysis by trypsin cleaves the C-terminal Arg-Gly-Gly residues in ubiquitin and yields a diGly remnant on the peptides. These diGly peptides were immunoaffinity purified using anti-diGly antibody and then analyzed by mass spectrometry. The mouse skeletal muscle ubiquitylome was studied using diGly proteomics and we identified 244 nonredundant ubiquitination sites in 142 proteins. An approach for differential dimethyl labeling of proteins with diGly immunoaffinity purification was also developed. diGly peptides from skeletal muscle of wild type and Epm2b-/- mice were immunoaffinity purified followed by differential dimethyl labeling and analyzed by mass spectrometry. About 70 proteins were identified that were present in the wild type and absent in the Epm2b-/- muscle tissue. The initial results identified 14 proteins as potential malin substrates, which would need validation in future studies.

Partout dans le monde, l’identification des personnes ou ménages bénéficiaires d’interventions sociales demeure un défi. Dans les pays où la majorité de la population travaille dans le secteur informel, vit d’une agriculture de subsistance et/ou vit sous le seuil de la pauvreté, le ciblage des personnes devant bénéficier d’une intervention fait appel à des méthodes différentes dont la vérification du revenu et la classification de la pauvreté sur base monétaire. En 2014, un projet-pilote intitulé Cadre commun des filets sociaux au Nord Mali (CCFS) a été mis en place au Mali. L’objectif de ce projet est d’identifier les populations en insécurité alimentaire et nutritionnelle et leur fournir une assistance (transferts monétaires, assistance alimentaire et prévention de la malnutrition pour les femmes enceintes et les enfants), particulièrement en périodes de soudure pastorale et agricole. La méthode de ciblage Household Economy Approach (HEA) est une des méthodes utilisées pour sélectionner les ménages bénéficiaires des transferts monétaires au Nord Mali. L’objectif de cette recherche est d’identifier les facteurs et le contexte qui influencent la mise en œuvre de la méthode HEA. Deux villages dans une commune agricole et deux sites de fraction dans une commune pastorale ont été choisis comme sites. Des entretiens (48 entretiens (12 effectués par l’étudiante et 36 par l’ONG de recherche)) et une collecte de 15 documents ont été réalisés. À l’aide des 23 facteurs du cadre d’analyse de la mise en œuvre de Durlak et Dupré (2008), une analyse thématique a été effectuée à l’aide du logiciel © QDA Miner. Les résultats démontrent que l’identification des ménages bénéficiaires des transferts monétaires au Nord Mali repose essentiellement sur le ciblage géographique et communautaire. Les facteurs qui influencent le processus de ciblage sont liés à la faible connaissance de la méthode HEA, à la lassitude et la faible motivation des personnes impliquées, à la gestion top down et au manque de transparence dans les processus décisionnels au niveau des structures organisationnelles, aux logiques de domination et relations de pouvoir au sein des communautés ainsi qu’aux enjeux liés au financement et aux rapports hégémoniques existants dans le monde de l’aide humanitaire et de l’aide publique au développement. La difficile coordination multisectorielle des acteurs de la protection sociale vient appuyer le besoin en recherches nouvelles sur la mise en place du régime social unifié (RSU) au Mali.
Around the world, the identification of people or households benefiting from social interventions remains a challenge. In countries where the majority of the population works in the informal sector, lives on subsistence agriculture and/or lives below the poverty line, targeting people who have to benefit from an intervention requires methods that are different from income verification and from the classification of poverty on a monetary basis. In 2014, an experimental project entitled Cadre commun des filets sociaux au Nord Mali (CCFS) was implemented in Mali. The objective of this project is to identify the populations who suffer from food and nutritional insecurity and provide them with assistance (cash transfers, food assistance and prevention of malnutrition for pregnant women and children), particularly during pastoral and agricultural lean periods. The Household Economy Approach (HEA) targeting method is one of the methods used to select beneficiary households for cash transfers in Northern Mali. The purpose of this research is to identify the factors and the context that influence the implementation of the HEA method. Two villages in an agricultural commune and two fractional sites in a pastoral commune were chosen as sites. Interviews (48 interviews (12 carried out by the student and 36 by research NGOs)) and a collection of 15 documents were conducted. Using the 23 factors in the Durlak and Dupré Implementation Analysis Framework (2008), a thematic analysis was conducted using the software © QDA Miner. The results show that the identification of households receiving cash transfers in Northern Mali is mainly based on geographical and community targeting. The factors that influence the targeting process are related to the low knowledge of the HEA method, to the weariness and low motivation of the people involved, to top down management and lack of transparency in the decision-making processes at the level of organizational structures, to the logic of domination and power relations within communities, and finally to the issues of funding and hegemonic relationships in the world of humanitarian aid and development cooperation. The difficult multisectoral coordination of social protection actors comes to support the need for new research on the establishment of a household registration system in Mali.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.