Made available in DSpace on 2015-07-22T20:50:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Introdução: Diversos fatores podem diminuir a viabilidade do retalho TRAM, dentre eles a nicotina que tem sido responsabilizada pela perda parcial ou total destes retalhos. Objetivo: Avaliar a ação do Fator de Crescimento do Endotélio Vascular na viabilidade do Retalho Musculofasciocutâneo Transverso do Reto do Abdome, em ratos submetidos à nicotina. Métodos: Foram utilizados 60 Ratos Wistar EPM-1, machos adultos, pesando de 230 a 300g, aleatorizados em 4 grupos de 15 animais cada: Grupo Controle composto por animais que foram submetidos ao retalho TRAM; Grupo Nicotina composto por animais que foram submetidos á nicotina e ao retalho TRAM; Grupo VEGF composto por animais submetidos à administração de VEGF plasmidial antes do retalho TRAM; e Grupo Nicotina+VEGF composto por animais que foram submetidos à nicotina, tratados com administração de VEGF e submetidos ao retalho TRAM. Para análise dos resultados foi realizado método de análise da área de necrose e de densidade vascular. Resultados: Houve diferença estatisticamente significativa na comparação entre todos os grupos, com relação às variáveis área de necrose e densidade vascular (p<0,05). Os animais do Grupo VEGF apresentaram a menor área de necrose (4.10%) e a maior densidade vascular (39%) em relação aos outros grupos do estudo. Conclusão: O Fator de Crescimento do Endotélio Vascular aumentou a viabilidade do retalho musculofasciocutâneo transverso do reto do abdome, em ratos submetidos à nicotina. / Introduction: Several factors can reduce the viability of the TRAM flap, among them the nicotine has been made responsible by the loss partial or total of these flaps. Objective: To evaluate the action of the Vascular endothelial Growth Factor in the viability of the Transverse Rectus Abdominis Musculocutaneous, in rats submitted to the nicotine. Methods: Sixty Wistar EPM-1 rats were used, adult males, weighing from 230 to 300g, randomized in 4 groups of 15 animals each: Group Control composed by animals that were submitted to the TRAM flap; Group Nicotine composed by animals that were nicotine exposed and submitted of TRAM flap; Group VEGF composed by animals submitted to the administration of VEGF plasmidial before the TRAM flap; and Group Nicotina+VEGF composed by animals that were exposed to the nicotine, trated with administration of VEGF and submitted to the TRAM flap. For analysis of the results they were done necrosis area and vascular density. Results: There was estatistic significant differentiates in the comparison among all of the groups, regarding the variables necrosis area and vascular density (p <0,05). Conclusion: The Vascular Endothelial Growth Factor increased the viability of the Transverse Rectus Abdominis Musculocutaneous, in rats submitted to the nicotine. / TEDE / BV UNIFESP: Teses e dissertações

Muitas das técnicas utilizadas para gerar animais transgênicos são caras e laboriosas. Neste contexto, a transferência gênica mediada por espermatozóides (TGME) pode ser uma alternativa para a produção em larga escala de animais transgênicos. Estudos de SMGT têm seu foco no número de cópias de DNA incorporada pelo espermatozóide. Por isso, há pouca informação disponível sobre como as moléculas de DNA se comportam durante o processo de fecundação e quais os efeitos dos protocolos de TGME sobre a célula espermática. Neste sentido, com o objetivo de avaliar a existência de sitio de integração preferencial das moléculas exógenas de DNA no genoma hospedeiro, utilizamos a hibridização in situ para acompanhar a veiculação do transgene durante o processo de fecundação. Foram avaliadas as membranas acrossomais, plasmática e potencial de membrana mitocondrial de espermatozóides submetidos a TGME. Para isso, o sêmen de três diferentes touros foram submetidos ao gradiente de Percoll 45-90%. As células viáveis foram incubadas com o vetor recombinante pCX-EGFP (0, 250, 500 ou 1000ng/106 células) seguidas ou não de eletroporação (300v, 35µF e 0,25ms). Os espermatozóides tratados foram utilizados para a produção in vitro de embriões. Os embriões foram cultivados por sete dias até o estágio de blastocisto. Espermatozóides e embriões produzidos in vitro foram submetidos ao ensaio de hibridização in situ, com metodologia descrita Whyte et al. (2000). O potencial de membrana mitocondrial (PMM), integridade de membrana acrossomal (MA) e plasmática (MP) foram avaliados por citometria de fluxo (Guava Technologies, Hayward, CA, USA) utilizando as sondas fluorescentes JC1, FITC-PSA e PI (Molecular Probes), respectivamente. Os dados foram analisados pelo teste paramétrico ANOVA (teste LSD) usando o programa estatístico SAS for Windows, com nível de significância de 5%. A hibridização in situ não foi possível em espermatozóides bovinos, pois não houve hibridação da sonda controle. Blastocistos oriundos de espermatozóides incubados com DNA exógeno apresentaram integração de forma difusa, embriões oriundos de espermatozóides eletroporados apresentaram integração pontual. As diferentes concentrações de DNA não exerceram efeitos deletérios nas MP ou PMM, a adição de 500ng de DNA causou aumento de lesão na MA (p<0,05). A eletroporação não afeta a MP e MA, mais apresenta grande efeito no PMM causando redução da função mitocondrial. Este estudo conclui que maiores esforços são necessários para elucidar o comportamento das moléculas exógenas de DNA durante o processo de fecundação e quais são os efeitos da TGME sobre a célula espermática. / Most techniques used to produce transgenic animals are laborious and expensive. In this manner, sperm mediated gene transfer (SMGT) may be a viable alternative for long-scale production of transgenic animals. Many SMGT studies have focused the DNA internalization and number of DNA copies incorporated by spermatozoa. However, limited data is available about how foreign DNA molecules behave during fertilization and the direct effects of the SMGT technique on sperm cells. Hence, in order to monitor the existence of preferential integration sites by the exogenous DNA at the host genome, in situ hybridization was used to track the transgene conveyance during in vitro fertilization. In addition, acrosome and plasmatic membrane integrity and mitochondrial membrane potential of sperm cells subjected to SMGT were assessed. Briefly, thawed semen from three different bulls was submitted to a 45- 90% Percoll gradient. Viable cells were incubated with recombinant PCX-EGFP vector (0, 250, 500 or 1000ng/106 sperm cells) or incubated and electroporated (300V, 35µF and 0.25ms). Treated sperm cells were then used for in vitro production of embryos. Embryos were in vitro cultured for 7 days until blastocyst stage. Treated spermatozoa and in vitro produced blastocysts were submitted to in situ hybridization assay, as described by Whyte et al. (2000). The mitochondrial membrane potential (MMP), acrosomal membrane (AM) and plasmatic membrane (PM) integrity were assessed by flow cytometry (Guava Technologies, Hayward, CA, USA) using JC1, FITC-PSA and PI probes (Molecular Probes), respectively. Data were analyzed by parametric ANOVA (LSD test) using SAS for Windows software, at a 5% level. The transgene was not observed at the bovine spermatozoa because the control probe could not be hybridized. In situ hybridization revealed that blastocysts produced from incubated sperm cells had a diffuse foreign DNA integration while blastocysts produced from electroporated sperm cells had a punctual DNA integration. No deleterious effects of exogenous DNA concentrations on PM or MMP were observed. However, the addition of 500ng of exogenous DNA caused sperm AM injury (P<0.05). Electroporation did not affect PM or AM integrity, but it had a great effect on MMP, which may cause a reduction of mitochondrial function. This study suggest that more efforts are needed to elucidate the behavior of exogenous DNA during fertilization and the effects of SMGT in bovine sperm cells.

Dans cette thèse, nous proposons et étudions des systèmes d’exposition en vue d’explorer les effets biologiques sanitaire et thérapeutique des ondes électromagnétiques sur le vivant. Nous proposons une antenne micro-onde pour l’ablation thermique des tumeurs cancéreuses du foie à 2.45 GHz. Son originalité réside en ses dimensions miniatures et la possibilité de l’insérer dans le foie par voie endoscopique. Pour cette antenne, un débit d’absorption spécifique (DAS) supérieur à 50 W/kg/W inc a montré une zone exposée de 1-cm de diamètre. Nous proposons ensuite une cellule transverse électromagnétique (TEM) avec une ouverture fermée par un matériau transparent conducteur l’Indium tin oxyde (ITO). Cette cellule TEM peut être utilisée pour évaluer les effets sanitaires potentiels des signaux de télécommunications sans fils. Ce système permet l’observation microscopique en temps réel du milieu biologique exposé, à travers son ouverture fermée par l’ITO. L’influence de la présence de l’ouverture et de la couche d’ITO sur le DAS dans le milieu exposé a été évaluée. Les valeurs du DAS obtenues à 1.8 GHz dans le milieu exposé dans la cellule TEM avec l'ouverture fermée ou non par l’ITO étaient de 1.1 W/kg/W inc et 23.6 W/kg/W inc, respectivement. Une excellence homogénéité du DAS a été obtenue dans le milieu en présence de l’ITO. Enfin, nous proposons plusieurs dispositifs spécifiques pour l’exposition des cellules biologiques aux champs électriques pulsés nanosecondes de haute intensité (nsPEFs). Les effets biologiques des nsPEFs sont utilisés pour des applications dans le domaine médical et en biotechnologie. Nous proposons deux dispositifs à électrodes en contact direct avec le milieu biologique et trois dispositifs à électrodes isolées. Nous démontrons l’adaptation de ces dispositifs aux impulsions courtes de durée 3-ns et la capacité de ceux à électrodes en contact à fournir des champs intenses de l’ordre de quelques MV/m. Nous illustrons aussi l’importance des dispositifs isolés pour délivrer des impulsions ultracourtes. / In this thesis, we propose and study exposure systems to explore healthy and therapeutic biological effects of EM signals. We propose a microwave antenna for thermal ablation of liver tumors at 2.45 GHz. Its original feature consists in its reduced dimensions that permits the endoscopic insertion in the zone to be treated. For this antenna, a specific absorption rate (SAR) greater than 50 W/kg/W inc showed an exposed zone of 1-cm diameter. We propose a transverse electromagnetic cell (TEM) with an aperture sealed with a transparent conducting material Indium tin oxide (ITO).This TEM cell can be used to study the potential effects of wireless communication systems on biological cells. This delivery device allows real-time observation of biological cells during exposure across the aperture sealed with ITO. The effect of the aperture and the ITO layer presence on the SAR in the exposed sample was evaluated. The SAR values obtained at 1.8 GHz in the sample exposed in the TEM cell with the sealed or non-sealed aperture of 20-mm diameter were 1.1 W/kg/W inc and 23.6 W/kg/W inc, respectively. An excellent homogeneity of SAR was achieved in the medium in the presenceof ITO. Finally, we propose several devices for the exposure of biological medium to nanosecond pulsed electric field with high intensity (nsPEFs). The biological effect of nsPEFs are used in biotechnology and medicine. We propose two devices with electrodes in direct contact with the biological medium and three devices with isolated electrodes. We demonstrate their adaptation for 3-ns duration pulses and the suitability of those with electrodes in contactwith the biological medium to provide high intensities fields in the order of several MV/m. We demonstrate the importance of the isolated devices for delivering ultrashort pulses.

Einführung: Eine entscheidende Ursache für das Aufkommen der den modernen Menschen charakterisierenden kognitiven Funktionen ist in der beachtlichen Vergrößerung des menschlichen Neocortex innerhalb der letzten 5-7 Millionen Jahre zu finden. Die Identifizierung der dieser Entwicklung zu Grunde liegenden genomischen Veränderungen ist letztlich nicht nur bedeutsam für die Beantwortung der Frage, welche evolutionären Anpassungen den Menschen kennzeichnen, sondern auch für ein besseres Verständnis einer möglicherweise besonderen Anfälligkeit gegenüber neurologischen und psychiatrischen Erkrankungen. Kürzlich konnten 15 menschenspezifische Gene, deren Expression sich vorzugsweise in neuronalen Vorläuferzellen (NPCs) des menschlichen fetalen Neokortexes nachweisen lässt, identifiziert werden (Florio et al., 2018). Drei davon (FAM72B, C und D) sind vor 3,4 – 1 Millionen Jahren im menschlichen Genom durch Genduplikationen entstanden und gehören zur Family of sequence similarity 72 (FAM72). Zielsetzung und Ansätze: Konkret wurde betrachtet, ob FAM72D durch die spezifischen Substitutionen von Aminosäuren eine sich von der Funktion des anzestralen Gens FAM72A unterscheidende Rolle in der neokortikalen Entwicklung einnimmt. Untersucht wurden deshalb die Effekte von FAM72A und D auf die Proliferationskapazität und Genexpression von NPCs nach der ektopen Expression von FAM72A oder D während der embryonalen Entwicklung des Neocortex der Maus. Methoden: Die in utero Elektroporation (IUE) embryonaler Mäusegehirne erfolgte zur Expression eines rot oder grün fluoreszierenden Proteins (RFP oder GFP) entweder gemeinsam mit einem leeren DNA pCAGGS Vektor als Kontrollbedingung oder aber einem pCAGGS-FAM72A oder pCAGGS-FAM72D Plasmid. Die in der zweiten Ergebnissektion (Results II) präsentierten IUE wurden dabei im dorsolateralen Neokortex zum Höhepunkt der Neurogenese am 14. Entwicklungstag (E 14.5) durchgeführt, im Unterschied zu den Experimenten in der dritten Sektion (Results III), die im medialen Neokortex am 18. Entwicklungstag (E 18.5) während der Spätphase der embryonalen Neurogenese realisiert wurden. Die Proliferation der NPCs wurde durch Immunfluoreszenzanalysen zweier Marker (Ki67 und phosphoryliertes Histon 3) bestimmt. Zudem wurde die Häufigkeit wichtiger Subtypen von NPCs ebenfalls durch Immunfluoreszenzanalysen eines Markers für basale intermediäre Vorläuferzellen (bIPs → Tbr2) sowie für basale und apikale radiale Gliazellen (aRGs, bRGs → Sox2) ermittelt. Die Gliogenese wurde durch Olig2 Immunfluoreszenz quantifiziert. Weitere Experimente wurden durchgeführt, um die Fähigkeit der NPCs, den Zellzyklus nach der IUE von FAM72D erneut einzuleiten, zu untersuchen. Zu diesem Zweck wurde schwangeren Mäusen 24 h nach der IUE das Thymidin-Analogon 5-Ethinyl-2'-desoxyuridin (EdU) intraperitoneal injiziert. Damit wurden alle Zellen markiert, die sich zu diesem Zeitpunkt in der S-Phase des Zellzyklus befanden und damit den Zellzyklus nach der IUE fortsetzten. Nach weiteren 24 h (48 h post-IUE) erfolgte die Auswertung: alle Ki67- und EdU-doppelt positiven Zellen wurden als solche betrachtet, die den Zellzyklus nach IUE fortführten (EdU+) und nach weiteren 24 h noch immer proliferierten (Ki67+). Zur Durchführung der Transkriptomanalyse wurden Mäuse am 13. Entwicklungstag mit pCAGGS-GFP und entweder dem leeren DNA-Vektor (pCAGGS, Kontrolle) oder einem die Expression von FAM72A (pCAGGS-FAM72A) oder FAM72D (pCAGGS-FAM72D) ermöglichenden Vektor elektroporiert. Anschließend wurden die elektroporierten dorsolateralen neokortikalen Bereiche am 14. Entwicklungstag mikroskopisch seziert und in einzelne Zellen dissoziiert. Die Isolation der elektroporierten (GFP+) Zellen erfolgte aus den Einzelzellsuspensionen durch Fluoreszenz-aktivierte Zellsortierung (FACS). Im Anschluss wurden die isolierten Zellen für die RNA-Sequenzierung vorbereitet. Die primäre Datenanalyse der Ergebnisse der RNA-Sequenzierung wurde entsprechend etablierter Protokolle durchgeführt (Florio et al., 2015). Ergebnisse: Die Analyse der Immunfluoreszenzquanitfizierungen (Results II und III) ergab keine signifikanten Veränderungen der proliferativen Parameter oder der Häufigkeit der NPCs in der ventrikulären Zone (VZ) oder subventrikulären Zone (SVZ) des sich entwickelnden Mausneokortex nach der ektopen Expression von FAM72A oder FAM72D im Vergleich zur Kontrollbedingung. Die Transkriptomanalyse (Results IV) zeigte jedoch 88 signifikant hoch- und 52 herunterregulierte Gene in Folge der FAM72A sowie 91 signifikant hoch- und 67 herunterregulierte Gene nach der FAM72D Expression im Vergleich zur Kontrolle. Es wurde festgestellt, dass nur zwei dieser differentiell exprimierten Gene in Folge der ektopen Expression sowohl von FAM72A als auch FAM72D hochreguliert wurden und ein Expressionslevel > 1 fpkm aufwiesen: Syde1 und Shisa5. Darüber hinaus wurden sechs Gene mit > 1 fpkm identifiziert, die spezifisch nach der Expression von FAM72D hochreguliert waren: Tapbp, Mtfp1, Slitrk5, Parp9, Cnp, Rbm43. Darüber hinaus zeigte die Genontologie-Analyse (Gen Ontology) eine signifikante Anreicherung von Angiogenese-assoziierten Genen (z. B. Vegfc) im Datensatz der artifiziell FAM72A exprimierenden Zellen. Interessanterweise konnte beobachtet werden, dass unter den im Vergleich zur Kontrolle differentiell exprimierten Genen mehr Gene mit typischer Expression in NPCs in Folge von FAM72D als FAM72A Expression hochreguliert und mehr NPC typische Gene nach FAM72A Expression herunterreguliert wurden. Im Falle der Gene, deren Expression eher in Neuronen zu finden ist, zeigte sich ein entgegengesetztes Bild (Results IV). Diese Befunde lassen den vorsichtigen Schluss zu, dass FAM72D stärker als FAM72A die Aufrechterhaltung von NPC-Eigenschaften positiv beeinflussen kann. Schlussfolgerungen: In einer früheren Studie erhöhte der Knockdown von Fam72a in NPCs erwachsener Mäuse die Neurogenese (Benayoun et al., 2014). Dies legt in Verbindung mit den vorliegenden Ergebnissen nahe, dass FAM72A und FAM72D nicht hinreichend, möglicherweise jedoch notwendig sind, um die Aufrechterhaltung des Vorläuferzellcharakters von NPCs zu fördern (Results II, III). Aus diesem Grund sollte das in dieser Studie verfolgte Gain of Function Design durch einen Loss of Function Ansatz ergänzt werden. Als Modellsystem bieten sich hierfür insbesondere Hirnorganoide aus Stammzellen des Schimpansen oder Menschen an. Da alle der kürzlich identifizierten menschenspezifischen Gene in den gleichen NPCs exprimiert werden, sollte auch die potenzielle synergistische Wirkung auf die NPC-Proliferation der FAM72 und der zwölf anderen humanspezifischen Gene wie etwa ARHGAP11B analysiert werden. Neben anderen möglichen Mechanismen, die auf Grundlage der Genexpressionsanalyse im Diskussionsteil dieser Arbeit (Results IV und Discussion) erörtert wurden, könnte die Hochregulierung von Slitrk5 in Folge der ektopen Expression des humanspezifischen FAM72D besonders relevant sein. Es ist bekannt, dass Slitrk5 am Recycling des TrKB-Rezeptors beteiligt ist (Song et al., 2015), der wiederum grundlegende Aspekte der Gehirnentwicklung beeinflusst. Ebenfalls konnte bereits gezeigt werden, dass FAM72A die Funktion des TrKB Rezeptors hemmt (Nehar et al., 2009). Somit ist denkbar, dass FAM72D im menschlichen Neokortex die Wiederherstellung der TrKB-Rezeptorfunktion indirekt über Slitrk5 verbessert und dadurch wesentliche Parameter wie das Überleben von Vorläuferzellen und die Neurogenese beim Menschen verlängern oder verstärken könnte. Diese Studie stellt damit die erste funktionelle Charakterisierung der evolutionär hochinteressanten, die FAM72 Gene beinhaltende Region des menschlichen Genoms während der Entwicklung in utero dar. Daraus ergeben sich zahlreiche Ansatzpunkte für zukünftige Untersuchungen, die in ihrer Gesamtheit ein umfassendes Verständnis der Evolution des menschlichen Gehirns ermöglichen werden.:1 INTRODUCTION 11 1.1 WHAT MADE US HUMAN? 11 1.2 THE NEOCORTEX 12 1.2.1 Origin and structure 12 1.2.2 Neurogenesis in the developing neocortex 14 1.2.3 How to increase the neuronal output 18 1.3 EVOLUTION AND GENE DUPLICATION 19 1.3.1 Gene duplication and evolutionary novelty 19 1.3.2 Mechanisms of replication 21 1.3.3 Fates of duplicated genes 22 1.3.4 Which genes tend to duplicate? 24 1.3.5 Human adaptation and gene duplication 24 1.4 HUMAN-SPECIFIC SIGNATURES OF NEOCORTICAL EXPANSION 25 1.5 IDENTIFICATION OF HUMAN-SPECIFIC GENES EXPRESSED IN THE DEVELOPING NEOCORTEX.. 25 1.6 FAMILY WITH SEQUENCE SIMILARITY 72 (FAM72) 26 1.6.1 Evolutionary origin 26 1.6.2 Subcellular localization 27 1.6.3 Cell cycle regulation 28 1.6.4 NPC maintenance 28 2 AIMS & APPROACHES 30 3 RESULTS I 31 3.1 FROM GENES TO PROTEINS: 1 FAMILY – 4 PARALOGUES 31 3.2 FAM72 MRNA EXPRESSION LEVELS IN THE DEVELOPING MOUSE AND HUMAN NEOCORTEX 32 3.3 COMPUTATIONAL ANALYSES 34 3.3.1 Proportion of cysteines 34 3.3.2 Transmembrane domain 34 3.4 AMPLIFICATION, SUBCLONING AND MUTAGENESIS 36 3.4.1 Amplification from human cDNA 36 3.4.2 Verification of the pCAGGs vectors 36 4 RESULTS II 38 4.1 ECTOPIC EXPRESSION OF FAM72A AND FAM72D IN THE MOUSE DORSOLATERAL NEOCORTEX AT MID-NEUROGENESIS 38 4.2 NPC PROLIFERATION 39 4.2.1 Assessment of NPC proliferation using Ki67 immunofluorescence 39 4.2.2 Cell cycle reentry 41 4.2.3 Assessment of mitosis using PH3 immunofluorescence 43 4.2.4 Conclusion 45 4.3 NPC ABUNDANCE 46 4.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 46 4.3.2 Conclusion 49 5 RESULTS III 50 5.1 ECTOPIC EXPRESSION OF FAM72A and FAM72D IN THE MOUSE MEDIAL CORTEX AT LATE-NEUROGENESIS 50 5.2 NPC PROLIFERATION 51 5.2.1 Assessment of the NPC proliferation using Ki67 immunofluorescence 51 5.3 NPC ABUNDANCE 52 5.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 52 5.4 GLIOGENESIS 53 5.4.1 Assessment of gliogenesis using Olig2 immunofluorescence 53 5.5 CONCLUSION 54 6 RESULTS IV 55 6.1 DIFFERENCES IN GENE EXPRESSION UPON ANCESTRAL FAM72A AND HUMAN-SPECIFIC FAM72D EXPRESSION AT MID-NEUROGENESIS 55 6.1.1 Rationale and experimental setup 55 6.2 DIFFERENTIALLY EXPRESSED GENES UPON ECTOPIC FAM72A AND FAM72D EXPRESSION IN THE DEVELOPING MOUSE DORSOLATERAL NEOCORTEX 56 6.3 UPREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 57 6.3.1 Upregulated genes upon the ectopic FAM72A and FAM72D expression 57 6.3.2 Upregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 58 6.3.3 Upregulated genes upon the ectopic FAM72A and D expression – cut off: fpkm >1 59 6.4 UPREGULATED GENES SPECIFICALLY UPON THE ECTOPIC FAM72D EXPRESSION – CUT OFF: FPKM >1 60 6.4.1 Tapbp (TAP binding protein, Tapasin) 60 6.4.2 Mtfp1 (mitochondrial fission protein 1, Mtp18) 61 6.4.3 Slitrk5 (Slit and Ntrk-like protein 5) 61 6.4.4 Parp9 (Poly(ADP-ribose) polymerase 9) 63 6.4.5 Cnp (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) 63 6.4.6 Rbm43 (RNA binding motif protein 43) 65 6.5 DOWNREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 66 6.5.1 Downregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 66 6.5.2 Downregulated genes upon ectopic FAM72A expression – cut off: fpkm >1 66 6.5.3 Downregulated genes upon ectopic FAM72D expression – cut off: fpkm >1 67 6.6 GENES PREVIOUSLY SHOWN TO BE DIFFERENTIALLY EXPRESSED UPON FORCED FAM72A EXPRESSION 69 6.6.1 Cell cycle regulators 69 6.6.2 Tumor suppressor genes 69 6.6.3 PROTEINS PREVIOUSLY OBSERVED TO INTERACT WITH FAM72A 70 6.7 EFFECT OF ECTOPIC FAM72A AND FAM72D EXPRESSION ON GENES IMPLICATED IN NEURAL LINEAGE FATE DECISION 70 6.7.1 Upregulated and NPC-enriched genes 71 6.7.2 Downregulated and NPC-enriched genes 73 6.7.3 Upregulated and neuron-enriched genes 74 6.7.4 Downregulated and neuron-enriched genes 75 6.8 GO ENRICHMENT ANALYSIS 75 6.9 CONCLUSION 75 7 DISCUSSION 78 7.1 WHAT MAKES US HUMAN? 78 7.2 IN UTERO ELECTROPORATION OF A HUMAN-SPECIFIC GENE IN THE DEVELOPING MOUSE NEOCORTEX 81 7.2.1 Opportunities and limitations of the approach 81 7.3 THE FAMILY OF SEQUENCE SIMILARITY 72 AND HUMAN UNIQUENESS 83 7.3.1 Cell cycle regulation and NPC maintenance 83 7.3.2 Cell death 84 7.3.3 Neurogenic period 85 7.3.4 TrkB signaling 85 7.3.5 Mitochondria 86 7.3.6 Angiogenesis 88 7.3.7 An evolutionary immunological adaptation in the brain? 89 7.3.8 FAM72 and SRGAP2 90 7.3.9 FAM72, Neanderthals, and lncRNAs 91 7.4 FUTURE DIRECTIONS 92 7.4.1 Loss of function 92 7.4.2 Gain of function 92 8 SUMMARY / ZUSAMMENFASSUNG 95 8.1 SUMMARY 95 8.2 ZUSAMMENFASSUNG 98 9 MATERIALS AND METHODS 101 9.1 CHART OF ALL EXPERIMENTS 101 9.2 COMPUTATIONAL ANALYSIS 101 9.2.1 Reference sequences and multiple sequence alignments 101 9.2.2 Transmembrane domain prediction 102 9.3 AMPLIFICATION, SUBCLONING, MUTAGENESIS 102 9.3.1 Amplification from human brain cDNA 102 9.3.2 Subcloning 103 9.2.3 Mutagenesis 103 9.4 PLASMID VERIFICATION 104 9.4.1 Transfection of Cos7 cells 104 9.4.2 Immunoblots 104 9.4.3 In situ hybridization (ISH) 105 9.5 MICE 105 9.6 IN UTERO ELECTROPORATION 105 9.7 FIXATION AND CRYOSECTIONS 106 9.8 IMMUNOFLUORESCENCE AND ANTIBODIES 106 9.9 EDU DETECTION 107 9.10 IMAGE ACQUISITION 108 9.11 STATISTICS 108 9.12 MICRODISSECTION AND SINGLE CELL SUSPENSION 108 9.13 FACS 109 9.14 RNA SEQUENCING 109 9.15 TRANSCRIPTOME ANALYSIS 110 10 REFERENCES 111 11 APPENDIX 145 11.1 CONFERENCE PRESENTATION 145 V. ACKNOWLEDGMENTS 146 / Introduction: The higher cognitive functions that characterize modern humans can be attributed to the cerebral neocortex and its remarkable expansion in size during the last 5 – 7 million years of human evolution. The identification of the underlying genomic changes will be not only of importance to better understand the unique complexity of the human brain, but also its susceptibility to neurological and psychiatric diseases. Recently, 15 human-specific genes preferentially expressed in neural progenitor cells (NPCs) of the human fetal neocortex were identified (Florio et al., 2018). Three of them, FAM72B, C and D belong to the Family of sequence similarity 72 (FAM72) and occurred in the human genome by gene duplication 3.4 – 1 mya. Aims & Approaches: Specifically, it was asked whether FAM72D plays a diverse role compared to the ancestral FAM72A (Results II, III, IV) due to the specific sets of amino acid substitutions it acquired (Results I). Effects of FAM72A and FAM72D on the proliferative capacity and gene expressions of embryonic mouse NPCs were analyzed upon ectopic expression either of FAM72A or FAM72D during embryonic mouse neocortical development. Methods: In utero electroporation (IUE) of embryonic mouse brains was performed to drive the expression of a red or green fluorescent protein (RFP or GFP) either plus empty DNA vector (pCAGGS; control), pCAGGS-FAM72A or pCAGGS-FAM72D plasmids in the dorsolateral neocortex at mid-neurogenesis (embryonic day 13.5, E13.5; Results II) or in the medial neocortex at late-neurogenesis (E15.5; Results III). NPC proliferation was evaluated by immunofluorescence of Ki67 (immunohistochemistry, IHC), a cell proliferation marker, and phosphorylated Histone H3 (PH3), a marker of cell mitosis. Moreover, the abundance of NPCs using immunofluorescence of basal intermediate progenitor (Tbr2) and apical and basal radial glia (Sox2) markers, and the gliogenesis by Olig2 immunofluorescence was analyzed. Additional experiments were carried out to study the capacity of NPCs to reenter the cell cycle upon IUE of FAM72D. To this end, pregnant mice were intraperitoneally injected with the thymidine analog 5-Ethynyl-2´-deoxyuridine (EdU) 24 h post-IUE, to label all cells undergoing S-phase of the cell cycle (i.e., all cells that reentered the cell cycle after IUE) in the developing mouse brains. Embryonic brains were collected 24 h after EdU injection and co-stained with Ki67. Ki67 and EdU double positive cells were considered as cells that reentered the cell cycle. To execute the transcriptome analysis E13.5 mice were electroporated with pCAGGS-GFP either plus an empty DNA vector (pCAGGS, control), a vector driving expression of FAM72A (pCAGGS-FAM72A) or FAM72D (pCAGGS-FAM72D). Subsequently, the electroporated dorsolateral neocortical areas were microdissected at E14.5 and dissociated into single cells. The electroporated (GFP+) cells were isolated from the single cell suspensions by the fluorescence-activated cell sorting (FACS). The isolated cells were processed for RNA sequencing. Data analysis was performed as previously reported (Florio et al., 2015). Results: By immunohistochemistry, no significant changes in any of the proliferative parameters or in the abundance of progenitors in the ventricular zone (VZ) and subventricular zone (SVZ) of the developing mouse neocortex upon ectopic expression of FAM72D compared to FAM72A and control samples were detected (Results II, III). However, the transcriptome analysis (Results IV) showed 88 significantly up- and 52 down-regulated genes upon FAM72A and 91 significantly up- and 67 downregulated genes upon FAM72D expression compared to the control. Only two of these differentially expressed genes were found to be upregulated upon FAM72A and FAM72D with an expression >1 fpkm: Syde1 and Shisa5. Besides, six genes specifically upregulated upon ectopic expression of FAM72D exhibiting fpkm > 1 were identified and characterized using the existing literature: Tapbp, Mtfp1, Slitrk5, Parp9, Cnp, Rbm43. Beyond that, gene ontology analysis showed significant enrichment of angiogenesis-related genes (e.g., Vegfc) upon FAM72A expression. Interestingly, there were more genes found to be enriched in NPCs that were upregulated compared to control upon FAM72D than FAM72A expression, but more NPC enriched genes downregulated upon FAM72A compared to FAM72D expression. In the case of differentially expressed neuron-enriched genes, the data was were inverse, which slightly supports the idea that FAM72D rather than FAM72A could positively affect the maintenance of NPC characteristics. Conclusions: In a previous study knockdown of Fam72a in adult mouse NPCs increased neurogenesis (Benayoun et al., 2014). This suggests, in conjunction with the present results, that FAM72A and FAM72D are not sufficient, but may be required, to promote NPC maintenance (Results II, III). This is why the gain of function experiments conducted in this study should be complemented by a loss of function approach in the developing mouse neocortex, in chimpanzee or human-derived brain organoids. Because of their expression in the NPCs of the developing human neocortex, it might be productive to analyze the potential synergistic effect on NPC proliferation of the FAM72s and the 12 other human-specific genes such as ARHGAP11B. Among other mechanisms discussed based on the gene expression analysis in this thesis (Results IV and Discussion), the upregulation of Slitrk5 upon ectopic expression of the human-specific FAM72D could be particularly remarkable. Slitrk5 is known to be involved in the recycling of the TrKB receptor (Song et al., 2015), which affects fundamental aspects of brain development. While FAM72A was found to inhibit the TrKB receptor (Nehar et al., 2009), the occurrence of FAM72D could indirectly rescue the TrKB receptor function via Slitrk5 and thereby prolonging or enhancing essential features such as precursor cell survival and neurogenesis in humans. Therefore, this study provides the first functional characterization of the evolutionary highly interesting region in the human genome comprising the FAM72 genes during embryonic neocortical development in vivo and offers numerous starting points for further investigations, that will collectively facilitate a comprehensive understanding of the genomic adaptations underlying the astonishing evolution of the human brain.:1 INTRODUCTION 11 1.1 WHAT MADE US HUMAN? 11 1.2 THE NEOCORTEX 12 1.2.1 Origin and structure 12 1.2.2 Neurogenesis in the developing neocortex 14 1.2.3 How to increase the neuronal output 18 1.3 EVOLUTION AND GENE DUPLICATION 19 1.3.1 Gene duplication and evolutionary novelty 19 1.3.2 Mechanisms of replication 21 1.3.3 Fates of duplicated genes 22 1.3.4 Which genes tend to duplicate? 24 1.3.5 Human adaptation and gene duplication 24 1.4 HUMAN-SPECIFIC SIGNATURES OF NEOCORTICAL EXPANSION 25 1.5 IDENTIFICATION OF HUMAN-SPECIFIC GENES EXPRESSED IN THE DEVELOPING NEOCORTEX.. 25 1.6 FAMILY WITH SEQUENCE SIMILARITY 72 (FAM72) 26 1.6.1 Evolutionary origin 26 1.6.2 Subcellular localization 27 1.6.3 Cell cycle regulation 28 1.6.4 NPC maintenance 28 2 AIMS & APPROACHES 30 3 RESULTS I 31 3.1 FROM GENES TO PROTEINS: 1 FAMILY – 4 PARALOGUES 31 3.2 FAM72 MRNA EXPRESSION LEVELS IN THE DEVELOPING MOUSE AND HUMAN NEOCORTEX 32 3.3 COMPUTATIONAL ANALYSES 34 3.3.1 Proportion of cysteines 34 3.3.2 Transmembrane domain 34 3.4 AMPLIFICATION, SUBCLONING AND MUTAGENESIS 36 3.4.1 Amplification from human cDNA 36 3.4.2 Verification of the pCAGGs vectors 36 4 RESULTS II 38 4.1 ECTOPIC EXPRESSION OF FAM72A AND FAM72D IN THE MOUSE DORSOLATERAL NEOCORTEX AT MID-NEUROGENESIS 38 4.2 NPC PROLIFERATION 39 4.2.1 Assessment of NPC proliferation using Ki67 immunofluorescence 39 4.2.2 Cell cycle reentry 41 4.2.3 Assessment of mitosis using PH3 immunofluorescence 43 4.2.4 Conclusion 45 4.3 NPC ABUNDANCE 46 4.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 46 4.3.2 Conclusion 49 5 RESULTS III 50 5.1 ECTOPIC EXPRESSION OF FAM72A and FAM72D IN THE MOUSE MEDIAL CORTEX AT LATE-NEUROGENESIS 50 5.2 NPC PROLIFERATION 51 5.2.1 Assessment of the NPC proliferation using Ki67 immunofluorescence 51 5.3 NPC ABUNDANCE 52 5.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 52 5.4 GLIOGENESIS 53 5.4.1 Assessment of gliogenesis using Olig2 immunofluorescence 53 5.5 CONCLUSION 54 6 RESULTS IV 55 6.1 DIFFERENCES IN GENE EXPRESSION UPON ANCESTRAL FAM72A AND HUMAN-SPECIFIC FAM72D EXPRESSION AT MID-NEUROGENESIS 55 6.1.1 Rationale and experimental setup 55 6.2 DIFFERENTIALLY EXPRESSED GENES UPON ECTOPIC FAM72A AND FAM72D EXPRESSION IN THE DEVELOPING MOUSE DORSOLATERAL NEOCORTEX 56 6.3 UPREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 57 6.3.1 Upregulated genes upon the ectopic FAM72A and FAM72D expression 57 6.3.2 Upregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 58 6.3.3 Upregulated genes upon the ectopic FAM72A and D expression – cut off: fpkm >1 59 6.4 UPREGULATED GENES SPECIFICALLY UPON THE ECTOPIC FAM72D EXPRESSION – CUT OFF: FPKM >1 60 6.4.1 Tapbp (TAP binding protein, Tapasin) 60 6.4.2 Mtfp1 (mitochondrial fission protein 1, Mtp18) 61 6.4.3 Slitrk5 (Slit and Ntrk-like protein 5) 61 6.4.4 Parp9 (Poly(ADP-ribose) polymerase 9) 63 6.4.5 Cnp (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) 63 6.4.6 Rbm43 (RNA binding motif protein 43) 65 6.5 DOWNREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 66 6.5.1 Downregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 66 6.5.2 Downregulated genes upon ectopic FAM72A expression – cut off: fpkm >1 66 6.5.3 Downregulated genes upon ectopic FAM72D expression – cut off: fpkm >1 67 6.6 GENES PREVIOUSLY SHOWN TO BE DIFFERENTIALLY EXPRESSED UPON FORCED FAM72A EXPRESSION 69 6.6.1 Cell cycle regulators 69 6.6.2 Tumor suppressor genes 69 6.6.3 PROTEINS PREVIOUSLY OBSERVED TO INTERACT WITH FAM72A 70 6.7 EFFECT OF ECTOPIC FAM72A AND FAM72D EXPRESSION ON GENES IMPLICATED IN NEURAL LINEAGE FATE DECISION 70 6.7.1 Upregulated and NPC-enriched genes 71 6.7.2 Downregulated and NPC-enriched genes 73 6.7.3 Upregulated and neuron-enriched genes 74 6.7.4 Downregulated and neuron-enriched genes 75 6.8 GO ENRICHMENT ANALYSIS 75 6.9 CONCLUSION 75 7 DISCUSSION 78 7.1 WHAT MAKES US HUMAN? 78 7.2 IN UTERO ELECTROPORATION OF A HUMAN-SPECIFIC GENE IN THE DEVELOPING MOUSE NEOCORTEX 81 7.2.1 Opportunities and limitations of the approach 81 7.3 THE FAMILY OF SEQUENCE SIMILARITY 72 AND HUMAN UNIQUENESS 83 7.3.1 Cell cycle regulation and NPC maintenance 83 7.3.2 Cell death 84 7.3.3 Neurogenic period 85 7.3.4 TrkB signaling 85 7.3.5 Mitochondria 86 7.3.6 Angiogenesis 88 7.3.7 An evolutionary immunological adaptation in the brain? 89 7.3.8 FAM72 and SRGAP2 90 7.3.9 FAM72, Neanderthals, and lncRNAs 91 7.4 FUTURE DIRECTIONS 92 7.4.1 Loss of function 92 7.4.2 Gain of function 92 8 SUMMARY / ZUSAMMENFASSUNG 95 8.1 SUMMARY 95 8.2 ZUSAMMENFASSUNG 98 9 MATERIALS AND METHODS 101 9.1 CHART OF ALL EXPERIMENTS 101 9.2 COMPUTATIONAL ANALYSIS 101 9.2.1 Reference sequences and multiple sequence alignments 101 9.2.2 Transmembrane domain prediction 102 9.3 AMPLIFICATION, SUBCLONING, MUTAGENESIS 102 9.3.1 Amplification from human brain cDNA 102 9.3.2 Subcloning 103 9.2.3 Mutagenesis 103 9.4 PLASMID VERIFICATION 104 9.4.1 Transfection of Cos7 cells 104 9.4.2 Immunoblots 104 9.4.3 In situ hybridization (ISH) 105 9.5 MICE 105 9.6 IN UTERO ELECTROPORATION 105 9.7 FIXATION AND CRYOSECTIONS 106 9.8 IMMUNOFLUORESCENCE AND ANTIBODIES 106 9.9 EDU DETECTION 107 9.10 IMAGE ACQUISITION 108 9.11 STATISTICS 108 9.12 MICRODISSECTION AND SINGLE CELL SUSPENSION 108 9.13 FACS 109 9.14 RNA SEQUENCING 109 9.15 TRANSCRIPTOME ANALYSIS 110 10 REFERENCES 111 11 APPENDIX 145 11.1 CONFERENCE PRESENTATION 145 V. ACKNOWLEDGMENTS 146

La compréhension des mécanismes immunologiques précoces mis en place suite à l’administration de vaccins est encore de nos jours largement méconnue. Pourtant de plus en plus d’études démontrent l’importance de ces mécanismes très précoces faisant intervenir les acteurs de l’immunité innée dans la génération d’une réponse spécifique efficace après vaccination. La peau est un organe intéressant pour l'administration de vaccins du fait de sa richesse en cellules présentatrices d'antigènes (APC), qui sont des cellules essentielles dans la mise en place de la réponse immunitaire. L'administration par voie intradermique du vaccin ADN de type auxoGTU induit des réponses immunitaires fortes et persistantes, en particulier en association avec une électroporation (EP) locale chez le macaque cynomolgus. Le but de ce travail de thèse fut de caractériser les réponses immunitaires locales précocement mises en place suite à l’administration par voie intradermique du vaccin ADN auxo-GTU en association avec une EP. Dans un premier temps, nous avons décrit les populations de cellules immunitaires présentes dans la peau normale chez le macaque cynomolgus.L'épiderme contient des cellules de Langerhans (LC) qui sont : CD1a+ CD1c- et des lymphocytes T caractérisés par l’expression du CD3. Le derme contient des cellules CD1a+CD1c-, qui présentent des similitudes avec les LC et correspondent donc probablement à des LC en migration à travers le derme. Il contient également des cellules dendritiques dermales (DDC) CD1a+CD1c+, des macrophages résidants CD163highCD11b+ et les lymphocytes T CD3+. Chez certains animaux, nous avons mis en évidence la présence de granulocytes CD66+ dans le derme sain. Les populations de cellules immunitaires identifiées chez le macaque sont similaires à celles identifiées chez l’homme malgré de légères différences phénotypiques. Cette caractérisation nous a ensuite permis d'étudier l’impact de la vaccination sur les populations immunitaires de la peau.Nous avons démontré que la vaccination induit le recrutement de granulocytes et de monocytes/macrophages inflammatoires dans l'épiderme et dans le derme, ainsi qu’un recrutement plus tardif de cellules dendritiques inflammatoires épithéliales (IDEC) dans l'épiderme. Dans l'épiderme, 24h après immunisation, nous avons observé une augmentation transitoire des LC accompagnée d’une surexpression de HLA-DR, de CD86 et de CD83, ce qui démontre leur maturation. Entre 24h et 72h, le nombre de LC diminue, ce qui suggère que les LC matures quittent l’épiderme pour migrer vers les nœuds lymphatiques. Ces événements cellulaires sont majoritairement dus à l’EP, indépendamment de la présence du vaccin à ADN. L’analyse du microenvironnement mis en place dans la peau suite à la vaccination révèle une libération de facteurs solubles pro-inflammatoires, comme MCP-1, IL-18 , IL-15, IL-8 et de facteurs solubles anti- inflammatoires comme IL-1RA et sCD40L dès 24h, dont certains sont considérablement augmentés par la présence de l’ADN vaccinal. Nos résultats suggèrent que l’EP, indépendamment de la présence de l'ADN, est suffisante pour induire la mobilisation des cellules et la maturation des DC au niveau du site de vaccination, ce qui montre un important rôle adjuvant de l’EP. Cependant, il semble que l'ADN soit nécessaire pour générer un microenvironnement favorable à l'activation optimale des APC. Ce travail fournit des éléments importants sur les mécanismes de l'inflammation locale et ouvre de nouvelles possibilités pour les stratégies vaccinales. / Mechanisms involved in early vaccine response are poorly understood. However, more and more studies show the importance of innate immunity in the very early times following vaccine administration in the generation of an optimal specific immune response. Skin is an interesting target for vaccine delivery because of its richness in antigen presenting cells (APC) which are essential cells in immune responses. The intradermal delivery of auxoGTU DNA vaccine was shown to induce strong and persistent immune responses, especially in association with electroporation in cynomolgus macaque. The aim of this work was to characterize the early local immune responses followed intradermal auxoGTU DNA vaccination in association with EP in cynomolgus macaque. In a first step, we have described immune cell populations present in the normal skin in the cynomolgus macaques. The epidermis contains CD1a+CD1c- Langerhans cells (LCs), and CD3+ T cells. The dermis contains CD1a+CD1c- cells, which present similarities with LCs and probably correspond to LC in migration through dermis. It also contains CD1a+CD1c+ dermal dendritic cells (DDCs), CD163highCD11b+ resident macrophages, and CD3+ T cells. We found CD66+ polymorphonuclear cells in healthy dermis in some of the animals. Immune cell populations in the macaque are similar to those in humans despite moderate differences in phenotype. This characterization has allowed us to study the impact of vaccination on immune populations of the skin. We have demonstrated a recruitment of granulocytes and inflammatory monocytes/macrophages in epidermis and dermis, as well as a population of inflammatory dendritic epithelial cell (IDEC) in epidermis after vaccination. In epidermis, 24h after treatment, we have observed an initial increase of LC with an up-regulation of HLA-DR, CD86 and CD83, demonstrating their maturation. Between 24h and 72h, LC number decreased, suggesting that mature LC has leaved epidermis to migrate to skin draining lymph node. All these cellular events were almost due to EP process, independently of DNA vaccine presence. The skin microenvironment reveals a release of pro-inflammatory soluble factors, as MCP-1, IL-18, IL-15, IL-8 and anti-inflammatory mediators as IL-1RA and sCD40L by 24h, all considerably enhanced in the presence of DNA.Our results suggest that EP, independently of the presence of DNA, is sufficient to induce cells mobilization and DC maturation at the vaccinated site, suggesting an important adjuvant effect of EP. However, it seems that DNA is required to generate a favorable microenvironment essential for correct APC activation. This work provides important clues to local inflammation mechanisms and opens up new possibilities for vaccine strategies.

The aim of this work was to obtain better insight into the principles of cell structures and organs in the chicken embryo development. To reach this goal special methods of micromanipulations and visualization in vitro, ex vivo, ex ovo and in ovo were implemented and adjusted. These methods were used to study gene expression in neural crest development and eye development. In the course of long term research in our laboratory we observed that oncoprotein v-Myb influences the development of the neural crest and has the capacity to change natural cell fate. We performed a series of experiments to investigate v-Myb protein influence on neural crest cells differentiation, especially melanocyte lineage development, and its influence on gene expression in the neural crest. Therefore we focused on Gremlin 2 (PRDC), the gene upregulated by v-Myb in the neural crest. The established procedure of electroporation in ovo was adjusted to transfect cells of the developing eye and used to study gene expression during lens induction. The results obtained from chicken embryo experiments endorsed the study performed on mouse embryos. Futhermore, the electroporation technique was slightly modified for manipulations of the neural retina in the developing eye in ovo. Thereafter, the retinas were processed ex vivo and...

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<p>Mesenchymal stem cells have shown potential for success in gene therapy due to their ability to differentiate and their immunomodulatory properties <em>in vivo</em>. Although they have many inherent characteristics that are suitable for use within gene therapy, genetic modification of these cells is more difficult. Since MSCs are available in limited quantities and cannot be expanded indefinitely, the modification technique must ensure efficient expression of the transgene, a high cell survival rate and an intact ability to differentiate to various cell lineages. We optimized electroporation conditions for the genetic engineering of bone marrow-derived and umbilical cord blood-derived mesenchymal stem cells. MSCs engineered using electroporation conditions produced more transgene expression than cells engineered with cationic lipids in bone marrow-derived mesenchymal stem cells, but produced similar amounts in umbilical cord blood-derived mesenchymal stem cells. Optimal electroporation conditions also expressed more transgene than polymer based transfection reagent in umbilical cord blood-derived mesenchymal stem cells. Cell survival after optimal electroporation conditions was 67% in umbilical cord blood-derived mesenchymal stem cells. Most importantly, cells maintained their ability to differentiate into osteogenic, chondrogenic and adipogenic cell lineages. Electroporating umbilical cord blood-derived mesenchymal stem cells with a Factor IX containing plasmid lead to the FIX protein being expressed for over 12 days <em>in vitro</em>. This optimized electroporation protocol has created a fast, easy, economic and efficient method for genetically modifying mesenchymal stem cells without altering their ability to differentiate.</p> / Master of Applied Science (MASc)

The lymphatic and blood vascular systems are two important vessel networks that serve different roles in healthy states and in cancer. In breast cancer the most common cancer amongst women, mortality remains high despite increased treatment response due to metastatic spread, preferentially through the lymphatics. One aggressive subtype, triple negative breast cancer (TNBC) contributing to 15 to 30 percent of cases and is characterized by the absence of expression of three therapeutic biomarkers. As targeted therapy is limited, treatment relies on standard of care via surgery, radiotherapy, and chemotherapy with limited efficacy and increase in survival. Chemotherapies negatively alter the lymphatic vasculature benefiting the tumor, through lymphangiogenesis. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics, like carboplatin, and a novel 2nd generation ablative therapy called High Frequency Irreversible Electroporation (H-FIRE), which utilizes electric pulses to ablate tumor cells, affect the lymphatic and blood microvasculature in the tumor, surrounding fat pad, tumor draining lymph node (TDLN) using multiple analysis methods. This occurred through three main methods 1) identification of oxidative stress effects of chemotherapeutic application of carboplatin on lymphatic endothelial cells in vitro, 2) characterization of lymphatic and blood microvascular dynamics in a 4T1 breast cancer mouse model treated with sub-ablative H-FIRE, 3) through the development of a novel habitat imaging method to identify treatment specific changes in the tumor draining lymph node, and the development of a hybrid agent-based model (ABM) to test cancer cell flow mediated invasion in brain cancer. Herin the work showed that carboplatin induced lymphatic phenotypic changes occurred through generation of reactive oxygen species dependent on VEGFR3 and was reversed through treatment with the antioxidant N-acetylcysteine. In the 4T1 model, sub ablation with H-FIRE induced temporal remodeling of the lymphatic and blood vasculature within the viable tumor, in the surrounding fat pad, and in the tumor draining lymph node over seven days, suggesting an optimal time of application of adjuvant therapy. The development of a habitat imaging analysis method to identify TDLN vascular habitats and the perturbation to treatment in a retrospective analysis of prior work. Lastly, the development of a hybrid ABM through the incorporation of experimentally measured fluid flow fields from dynamic contrast enhanced MRI imaging building upon existing work, and showing the usefulness in comparing mechanisms of cancer cell invasion mediated fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within various treatments contexts and new methods of quantifying changes due to treatment. Hopefully, these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer. / Doctor of Philosophy / The lymphatic and blood vascular systems are two important vessel networks that serve different purposes in healthy states and in the disease called cancer. In breast cancer , a common form of cancer in women , spread of this cancer tends towards the lymphatic vasculature and eventually to other parts of the body. Triple negative breast cancer (TNBC) a less common, but more aggressive form, relies on clinical standard treatments with anti-tumor drugs called chemotherapies. These chemotherapies negatively alter the lymphatic vasculature to the tumors benefit, leaving a lack new methods of treatment. This dissertation seeks to understand how the mechanisms of commonly used chemotherapeutics and a new promising pulsed electric field therapy , High frequency Irreversible Electroporation (H-FIRE), change the lymphatic and blood vessels over time and in different locations using different tools. This occurred through three main methods 1) the effects on lymphatic vascular cells treated with chemotherapy, 2) in a breast cancer mouse model treated with H-FIRE, 3) in math models of the draining lymphatic organ, called the lymph node and an agent-based math model (ABM) of cancer cell movement due to fluid flow. The work showed that in the lymphatic cells, carboplatin a type of chemotherapeutic used to treat breast cancer, changed lymphatic vasculature through generating stress through oxidation and was reversed through treatment with an anti-oxidant. In the breast cancer mouse model, incomplete ablation with H-FIRE caused time dependent changes to the lymphatic and blood vasculature in the tumor, in the surrounding tissue, and in the lymph node over seven days. This work shows the novel findings of pulsed electric field therapy causing changes to the lymphatic vasculature. The creation of a new method of identifying habitats of the lymph node was used to compare changes to the lymphatic and blood vasculature to treatment. Lastly, the creation of an ABM added measured fluid flow maps from medical imaging methods to build upon existing work, and showed the usefulness in comparing mechanisms of cancer cell invasion due to fluid flow. Altogether, this work presents novel insight into the lymphatic system in cancer within after various treatments are applied and new methods of measuring these changes because of treatment using multiple methods. It is our hope that these findings can be used to further inform the field towards a more comprehensive understanding of treatment effects in breast cancer.

Glioblastoma (GBM) is the most common and deadliest of the malignant primary brain tumors in humans, with a reported 5-year survival rate of only 6.8% despite years of extensive research. Failure to improve local tumor control rates and overall patient outcome is attributed to GBM's inherent therapeutic resistance. Marked heterogeneity, extensive local invasion within the brain parenchyma, and profound immunosuppression within the tumor microenvironment (TME) are some of the unique features that drive GBM therapeutic resistance. Furthermore, tumor cells are sequestered behind the blood-brain barrier (BBB), limiting delivery of effective therapeutics and immune cell infiltration into the local tumor. Electroporation-based therapies, such as irreversible electroporation (IRE) and second generation, high-frequency IRE (H-FIRE) represent attractive alternative approaches to standard GBM therapy given their ability to induce transient BBB disruption (BBBD), achieve non-thermal tumor cell ablation and stimulate local and systemic anti-tumor immune responses without significant morbidity. The following work explores the use of H-FIRE to overcome GBM-induced therapeutic resistance and improve treatment success. Chapter 1 opens with an overview of GBM and known barriers to treatment success. Here, we emphasize the utility of spontaneous canine gliomas as an ideal translational model for investigations into novel treatment approaches. Chapter 2 introduces novel ablation methods (i.e. IRE/H-FIRE) capable of targeting treatment-resistant cancer stem cells. The focus of Chapter 3 is to highlight IRE applications in a variety of spontaneous tumor types. In Chapter 4, we investigate the feasibility and local immunologic response of percutaneous H-FIRE for treatment of primary liver tumors using a spontaneous canine hepatocellular carcinoma (HCC) model. In chapter 5, we characterize the mechanisms of H-FIRE-mediated BBBD in an in vivo healthy rodent model. In Chapter 6, we characterize the local and systemic immune responses to intracranial H-FIRE in rodent and canine glioma models to enhance the translational value of our work. Collectively, our work demonstrates the potential for H-FIRE to overcome therapeutic resistance in GBM, thereby supporting its use as a novel, alternative treatment approach to standard therapy. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadliest form of primary brain cancer in humans, with only 6.8% of people surviving 5-years after their diagnosis. GBM is characterized by a number of unique features that make it resistant to standard treatments, such as surgery, radiation and chemotherapy. Examples include: (1) extensive invasion of tumor cells into the brain, making complete removal via surgery very difficult; (2) tumor cells are protected by a structure called the blood-brain barrier (BBB), which restricts the entry of most drugs (i.e. chemotherapy) and many immune cells, into the brain, thereby preventing them from reaching tumor cells; (3) tumor cells produce substances that block the immune system from being able to detect the tumor itself, which allows it to continue to grow undetected. High-frequency irreversible electroporation (H-FIRE) represents a new approach for the treatment of GBM. H-FIRE uses electric pulses to temporarily or permanently injure cell membranes without the use of heat, which allows for very precise treatment. The following work explores the ways in which H-FIRE can interfere with specific GBM features that drive its resistance to treatment. Here, we demonstrate that H-FIRE is capable of temporarily disrupting the BBB and characterize the mechanisms by which this occurs. This allows for drugs and immune cells within the blood to enter the brain and access the tumor cells, particularly those extending beyond the visible tumor mass and invading the brain. We also illustrate the potential for H-FIRE treatment within the brain to stimulate local and systemic immune responses by causing the release of proteins from injured cells. Similar to a vaccine, these proteins are recognized by the immune system, which becomes primed to help fight off cancer cells within the body. The end result is an anti-tumor immune response. Collectively, this work supports the use of H-FIRE as an alternative treatment approach to standard therapy for GBM given its potential to overcome certain causes of treatment resistance.