Adult stem cells are vital for tissue maintenance. Stem cell over proliferation results in tumour formation, whilst loss of stem cells causes tissue degeneration and a variety of diseases. Stem cell maintenance and proliferation is regulated through somatic structures called niches. The germline stem cell niche in Drosophila ovary has been well defined and it is useful to better understand the interactions between niche and stem cells. Notch signalling is needed for germline stem cell niche creation and maintenance. The aim of this thesis is to better understand both the regulation of Notch signalling during development and its requirement in the adult niche. The first paper, "Reversible regulation of stem cell niche size through dietary control of Notch signalling", revolves around the dynamicity of the niche. The niche is found to respond to diet stimuli and has the ability to be restored. Notch was previously found to be involved in the maintenance of the niche. We found that Notch signalling is altered by diet, and we dissect its different maintenance and recovery roles in the ovary. In the second paper, "ZO-1 controls stem cell niche assembly by acting as an upstream regulator of Deltex-dependent Notch signalling", we show how Notch signalling is finely regulated during niche formation through interplay with the proteins Polychaetoid and Deltex. This paper leads to a better understanding of how the niche is assembled and how Notch signalling is regulated in a context-dependent way. The obtained results from both papers will help understand the dynamics of the model germline stem cell niche, and how Notch signalling is found at the convergence between internal and external stimuli regulating the ovary's response to a changing environment.

Stem cell niches are specialized signalling microenvironments that allow maintenance of the stem cells. According to the traditional model of the stem cell niche, the niche signalling input is integrated by a cell towards a binary decision between stemness and differentiation. I have studied the regulation of somatic cyst stem cell (CyCS) proliferation in the testicular stem cell niche of Drosophila melanogaster by performing the DamID screen for targets of the transcriptional regulator Zfh1, a shared target of Jak/STAT and Hedgehog niche signalling. I have found that Zfh1 binds to the regulatory regions of kibra and salvador, tumour suppressors of the Hippo/Yorkie pathway, and downregulates them, restricting Yorkie activity to the Zfh1 positive CySCs. Clonal inactivation of the Hippo pathway is sufficient for CySC proliferation, but does not affect their differentiation ability. I therefore proposed a different stem cell niche model, whereby the niche signalling directly “micromanage” stem cell behavior, not involving the cell fate decision making.

Em busca de novas estratégias para o reparo de tecidos pulpares com células-tronco faz-se necessário compreender melhor o nicho em que elas se encontram. Uma forma é observar o mecanismo de ativação sobre estas células durante o reparo de tecidos pulpares após sofrer uma injúria. Assim, foi proposto estudar a localização e composição do nicho das célulastronco da polpa dental através da exposição pulpar induzida in vitro em dentes humanos e in vivo em dentes de ratos, através da incorporação de Bromodeoxiuridina (BrdU) e Iododeoxiuridina (IdU) e co-expressão com proteínas de células-tronco. Para o ensaio in vitro, 33 terceiros molares humanos com rizogênese incompleta foram divididos em 3 grupos: dentes com exposição pulpar induzida e mantidos em cultivo (GCE, grupo cultura exposição) (n=15); dentes sem injúria e mantidos em cultivo (GCC, grupo cultura controle) (n=15); dentes hígidos apenas (GH, grupo hígido) (n=3). Os grupos GCE e GCC, foram cultivados com BrdU (10 l/ml) por 24h e posteriormente analisados após 2, 5 e 14 dias. Para o ensaio in vivo, 12 ratos Wistar receberam IdU (1mg/ml) diluído na água por 30 dias. Após 45 dias foram divididos em 2 grupos: grupo in vivo com exposição pulpar (GIVE) (n=17 dentes) e grupo in vivo controle (GIVC) (n=16 dentes). No GIVE foi realizada a exposição pulpar no primeiro molar e em seguida restaurado com Coltosol. Após 2, 4 e 8 dias pós-operatórios as amostras foram coletadas e processadas para análise histológica e imuno-histoquímica. As proteínas CD90, CD146, PDGFr e BrdU foram analisadas em duas áreas: lesão e centro em molares humanos enquanto, as proteínas CD90, NG2 e IdU foram analisadas em uma área única, em molares de rato. As análises estatísticas foram efetuadas pelos testes de Mann- Whitney e Kruskal-Wallis, p 5%. Em dentes humanos, na área de lesão do grupo GCE, células BrdU positivas foram encontradas nos três períodos, sendo presentes em maior número células PDGFr positivas nos dias 2 e 5 e células CD90 positivas no 14º dia; no centro pulpar, no 2º dia pós injúria houve um número semelhante de células CD90, CD146 e PDGFr positivas, sendo encontradas em maior porcentagem no 5º dia células PDGFr e CD90 positivas e 14º dia células CD90 positivas. No grupo GCC na área equivalente à lesão, células CD90 (2 e 5 dias) e PDGFr (14 dias) positivas foram encontradas em maior percentual; no centro pulpar, o número de células positivas para CD90, CD146 e PDGFr foi semelhante em todos os períodos, sendo encontradas células BrdU positivas no 2º dia. Em molares de ratos, no GIVE houveram mais células NG2 positivas no 2º dia, IdU no 4º dia e CD90 no 8º dia. No GIVC, o CD90 foi altamente expresso em todos os períodos analisados, a proteína NG2 também foi expressa em odontoblastos e CD90 na camada subodontoblástica em molares de rato. Todas as proteínas mencionadas coexistem no tecido pulpar humano e animal formando populações celulares heterogêneas perivasculares e perineurais encontradas em maior concentração em regiões próximas à área de injúria, sugerindo que nichos perivasculares e perineurais participam em conjunto na homeostase pulpar em resposta à injúria. / In pursuit of new strategies for pulp tissue repair with stem cells, it is necessary to better understand the niche in which they are found. One way is to observe the mechanism of these cells activation during the repair of pulp tissues after suffering an injury. Thus, it was proposed to study the location and composition of the dental pulp stem cell niche through induced dental pulp exposure in vitro in human teeth and in vivo in rat teeth by incorporating Bromodeoxyuridine (BrdU) and Iododeoxyuridine (IdU) and co-expression with stem cell proteins. For the in vitro assay, 33 human third molars with incomplete rhizogenesis were divided into 3 groups: teeth with induced pulp exposure and maintained in culture (GCE, group cultured with pulp exposure) (n = 15); healthy teeth maintained in culture (GCC, control cultured group) (n = 15); healthy teeth only (GH, healthy group) (n = 3). GCE and GCC groups were cultured with BrdU (10 l / ml) for 24 h and subsequently analyzed after 2, 5 and 14 days. For the in vivo assay, 12 Wistar rats received IdU (1mg / ml) diluted in water for 30 days. After 45 days were divided into 2 groups: in vivo group with pulp exposure (GIVE) (n = 17 teeth) and in vivo control group (GIVC) (n = 16 teeth). In GIVE, the pulp exposure was performed on the first molar and then restored with Coltosol. After 2, 4 and 8 postoperative days the samples were collected and processed for histological and immunohistochemical analysis. CD90, CD146, PDGFr and BrdU proteins were analyzed in two areas: injury and center in human molars whereas, CD90, NG2 and IdU were analysed in a single area in rat molars. Statistical analyzes were performed by the Mann-Whitney and Kruskal-Wallis tests, p 5%. In human teeth, in the lesion area of the GCE group, BrdU positive cells were found in the three periods, while a greater number of PDGFr positive cells on days 2 and 5 and CD90 positive cells on the 14th day was found; in the pulp center, on the second day after injury there were a similar number of CD90, CD146 and PDGFr positive cells, and found in greater percentage in the 5th day PDGFr and CD90 positive cells and in 14th day CD90 positive cells. In the GCC group, in the area equivalent to injury site, CD90 (2 and 5 days) and PDGFr (14 days) positive cells were found in a higher percentage; in the pulp center, the number of cells positive for CD90, CD146 and PDGFr was similar in all periods, and BrdU positive cells were found on day 2. In rat molars, GIVE presented more positive cells for NG2 on day 2, for IdU on day 4 and CD90 on day 8. In the GIVC, CD90 was highly expressed in all periods analysed, the NG2 protein was also expressed in odontoblasts and CD90 in the subodontoblastic layer in rat molars. All mentioned proteins coexist in the human and animal pulp tissue forming heterogeneous perivascular and perineural cellular populations found in greater concentration in regions near the area of injury, suggesting that perivascular and perineural niches participate together in pulpal homeostasis in response to injury.

Adult neural precursor cells (NPCs) are a heterogeneous population of mitotically active, self-renewing multipotent cells of both adult and developing CNS. They can be expanded in vitro in the presence of mitogens. The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks) and significant PC loss (24 weeks). A stereological count demonstrates that mice with significant cell loss exhibit highest survival of grafted NPCs and migration to the vicinity of PCs as compared to wt and younger grafted animals. These animals showed improved motor skills as compared to sham animals. Confocal analysis and profiling shows that many of implanted cells present in the cerebellar cortex have formed gap junctions with host PCs and express connexin43. Grafted cells did not adopt characteristics of PCs, but stereological and morphometric analysis of the cerebellar cortex revealed that grafted animals had more surviving PCs and a better preserved morphology of these cells than the control groups. Perforated patch clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. In this study, evidence for a neuroprotective effect came, in addition to motor behavior improvement, from stereological and electrophysiological analyses and suggest that timing of stem cell delivery is important to determine its therapeutic effect. In a brain stem cell niche, NSCs reside in a complex cellular and extracellular microenvironment comprising their own progeny, ependymal cells, numerous blood vessels and various extracellular matrix molecules. Recently, it was reported that blood vessel ECs-NSCs crosstalk plays an important role in tissue homeostasis. Bloodstream offers a natural delivery vehicle especially in case of diffuse neurodegenerative diseases which require widespread distribution of exogenous cells. As NSCs are confronted with blood-brain barrier endothelial cells (BBB-ECs) before they can enter into brain parenchyma, we investigated their interaction using primary cultures in an in vitro BBB model. We isolated human fetal neural precursor cells (hfNPCs) from aborted fetal brain tissues and expanded in vitro. We showed that in an in vitro model, human BBB endothelium induces the rapid differentiation of hfNPCs and allows them to cross the endothelial monolayer, with the differentiated progeny remaining in close contact with endothelial cells. These results are not reproduced when using a non-BBB endothelium and are partly dependent on the cytokine MCP1. Our data suggest that, in the presence of attractive signals released by a damaged brain, intravascularly administered NPCs can move across an intact BBB endothelium and differentiate in its vicinity. Overall, our findings have implications for the development of cellular therapies for cerebellar degenerative diseases and understanding of the brain stem cell niche.

The discovery of neural stem cells within the adult CNS has indicated an enormous potential in facilitating neuronal regeneration after injury. Studies from our laboratory have suggested that manipulation of the Rb/E2f pathway can directly impact embryonic and adult neurogenesis, thereby having tremendous potential for neuronal regeneration therapies. Recently, two new members of the Rb/E2f pathway have been discovered, the atypical E2fs: E2f7 and E2f8. Initial studies have suggested that atypical E2fs regulate diverse processes such as cell proliferation, DNA-stress response, apoptosis, however, their importance in the brain are unknown. To analyze their function during brain development, we crossed Nestin-Cre mice with mice bearing a conditional E2f7/E2f8 allele to delete both E2f7 and E2f8 in neural precursor cells. Whereas cortical development was largely unaffected by E2f7/E2f8 deficiency, we observed an enlargement of the lateral ventricles occurring postnatally, a brain condition named ventriculomegaly. We then looked at the ependymal cells, which are the cells lining the wall of the lateral ventricles, to determine if these cells were affected by the absence of atypical E2fs. We found progressive denaturation of the ependymal cell layer, distortion of the ependymal motile cilia and reactive astrocytes within the layer. We identified Gli3, a component of the Sonic hedgehog pathway (Shh), as a target for E2fs, including atypical E2fs. We unravelled a novel mechanism by which atypical E2fs regulate the expression of Gli3, leading to up-regulation of Numb/NumbL, which in consequence destabilizes cadherins organization within the ependymal cell layer. In conclusion, our work suggests that E2f7 and E2f8 transcription factors play an essential role in maintaining the ependymal cell barrier.

Stem cell niches are specialized signalling microenvironments that allow maintenance of the stem cells. According to the traditional model of the stem cell niche, the niche signalling input is integrated by a cell towards a binary decision between stemness and differentiation. I have studied the regulation of somatic cyst stem cell (CyCS) proliferation in the testicular stem cell niche of Drosophila melanogaster by performing the DamID screen for targets of the transcriptional regulator Zfh1, a shared target of Jak/STAT and Hedgehog niche signalling. I have found that Zfh1 binds to the regulatory regions of kibra and salvador, tumour suppressors of the Hippo/Yorkie pathway, and downregulates them, restricting Yorkie activity to the Zfh1 positive CySCs. Clonal inactivation of the Hippo pathway is sufficient for CySC proliferation, but does not affect their differentiation ability. I therefore proposed a different stem cell niche model, whereby the niche signalling directly “micromanage” stem cell behavior, not involving the cell fate decision making.

The intracellular bacteria Wolbachia infect up to 40% of all insect species, including the vectors of prevalent infectious diseases such as Dengue and malaria. Even though Wolbachia infections are the largest pandemic on this planet, the cellular and molecular mechanisms for bacterial spreading in nature are still unknown. Wolbachia are mainly vertically transmitted through the egg cytoplasm, however there is also evidence of extensive horizontal transmission. We have found that Wolbachia target the stem cell niches in the Drosophila ovary to enhance germline colonization and subsequent vertical transmission. This tropism is pervasive across the Drosophila genus, with the pattern of targeting being evolutionarily conserved. Phylogenetic analyses, confirmed by hybrid introgression and transinfection experiments, demonstrate that bacterial factors are the major determinants of differential patterns of niche tropism. Furthermore, bacterial load is increased in germline cells passing through infected niches, supporting previous findings suggesting a contribution of Wolbachia from stem cell niches towards vertical transmission. If niche tropism is important for Wolbachia transmission through the germline, evolutionary theory predicts that there should be no selective pressure to maintain niche tropism in males. Indeed, we have found that tropism to the stem cell niche in the testis, known as the hub, is not evolutionarily conserved. Towards identifying the cellular and molecular mechanisms of stem cell niche tropism, we investigated hub targeting of closely related Wolbachia strains (wMel-like strains: wMel, wMel2, and wMel3; wMelCS-like strains: wMelCS, wMelCS2, and wMelPop). wMel-like and wMelCS-like Wolbachia strains differ in their frequencies and densities of hub infection. The targeting differences of these strains of Wolbachia indicate that this phenotype is rapidly evolving, as they shared a common ancestor only 8,000 years ago. With the plethora of tools available in D. melanogaster, a candidate gene approach was used to target host proteins enriched in the stem cell niche in the testis for RNAi mediated gene knockdown in the hub. We have identified Drosophila stem cell related signaling pathways that promote Wolbachia accumulation. Unraveling the cellular and molecular bases of tissue tropism is fundamental to understanding Wolbachia-host interactions. / 2017-01-01T00:00:00Z

Wolbachia are widespread obligate intracellular bacteria that are maternally transmitted and modulate reproduction of their invertebrate host. Mosquitoes transinfected with Wolbachia have reduced capacity for transmitting vector borne diseases and can replace native populations in the field because of a reproductive advantage. The cellular mechanisms of how reproduction is altered by Wolbachia are poorly understood. In this work Wolbachia-induced reproductive changes in the model organism Drosophila were used to pinpoint underlying cellular processes affected by the bacteria. Specifically, egg production (or fecundity) of Wolbachia-infected Drosophila mauritiana was compared to non-infected flies that had been generated by antibiotic treatment of infected flies. Immediately before the fecundity experiment backcrossing of both fly lines ensured an equivalent nuclear genetic background. Initially egg production in Wolbachia-infected flies was increased by 4-fold but in less than 30 generations this changed to a 0.84 fold decrease with a slight advantage for the non-infected line. Additional backcrossing experiments determined that selection on the host nuclear genome is one of the factors underlying this reversion of fecundity gains. Other non-Mendelian factors, such as the microbiota, may also play a role in this rapid change. Wolbachia alterations in egg production were always linked to Wolbachia induced changes in programmed cell death (PCD) in the germarium during oogenesis and germline stem cell (GSC) division. Germline stem cells are maintained and regulated through their interaction with the germline stem cell niche (GSCN). Interestingly, these cells are both frequently infected with Wolbachia and possess a high bacterial titer. A developmental time course revealed the mechanism of how Wolbachia accumulate in the niche cells. The data suggest that the bacteria actually coordinate their replication with the differentiation of the niche cells. Future work on understanding the cellular and molecular basis of Wolbachia - host interaction will not only give insight into novel mechanisms of host manipulation by a pathogen, but will also expand our current understanding of stem cell niche morphogenesis and modulation of stem cell proliferation. / 2018-06-05T00:00:00Z

The potential of physiologically relevant in vitro cell culture models for studying physiological and pathophysiological phenomena has been widely recognized as replacements for animal and conventional in vitro models. To create models that accurately replicate the structure and function of tissues and organs, it is essential to comprehend the biophysical and mechanical features of the extracellular matrix (ECM) and incorporate them into the in vitro cell culture models. Therefore, we first aimed to investigate how nanotopography can modulate cell behaviors by studying cell behaviors on nanostructures of various aspect ratios on a cobalt-chromium-molybdenum (CoCrMo) alloy surface. We also explored the impact of nanofibrous membranes on the formation of alveolar epithelium, which is critical for lung alveolar interstitium chips. In addition, we investigated the effect of mechanical stretch on cell behaviors and focused on how the dimensionality of the stretch affects cell behaviors. To create physiologically relevant in vitro models based on our findings, we engineered a stem cell niche using a combination of nanofibrous membranes, mechanical stretch, and a soft substrate, and evaluated its impact on stem cell behaviors. Finally, we created a biomimetic human lung interstitium chip for application in physiological and pathophysiological in vitro studies.

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