The ß-carbolines norharman and harman structurally resemble the synthetic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that is known for its ability to damage neuromelanin-containing dopaminergic neurons of the substantia nigra and thereby induce parkinsonism. MPTP is, however, not normally present in the environment whereas the ß-carbolines are present in cooked food and tobacco smoke. In this thesis it was demonstrated that norharman and harman had affinity to melanin and were retained in neuromelanin-containing neurons of frogs up to 30 days post-injection (the longest survival time examined). It was also demonstrated that norharman induced neurodegeneration, activation of glia cells and motor impairment in mice. Furthermore, this compound induced ER stress and cell death in PC12 cells. An in vitro model of dopamine melanin-loaded PC12 cells was developed in order to study the effect of melanin on norharman-induced toxicity. In this model, melanin seemed to attenuate toxicity induced by low concentrations of norharman. After exposure to the highest concentration of norharman, melanin clusters were disaggregated and there was an increased expression of stress proteins and caspases-3, known to be involved in apoptosis. The polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene was demonstrated to have a CYP1A1-dependent localization in endothelial cells in the choroid plexus, in the veins in the leptomeninges and in the cerebral veins of mice pre-treated with CYP1-inducers. These results demonstrate that the distribution of environmental compounds could be influenced by the presence of neuromelanin and expression of CYP enzymes in the brain and that norharman may induce neurotoxic effects in vivo and in vitro.

Les ciliopathies rétiniennes sont un groupe de maladies rares causés par des mutations de gènes ciliaires. Les défauts des gènes ciliaires peuvent causer des défauts de trafic de protéines et induit l'apoptose des cellules photoréceptrices causés par le stress du réticulum endoplasmique (RE). On a étudié ciliopathies rétiniennes par modèle mourin, amaurose congénitale de Leber, rétinopathie pigmentaire liée à l’X, syndrome de Bardet-Biedl, syndrome d’Alström. Les souris Bbs1-/- , Bbs10-/- et CEP290-/- ont monté une diminution de la fonction rétinienne et sont causée par ER stress. Les souris Rd9/y et Alms1foz/foz présentent une apparition tardive et avec un faible taux de dégénérescence rétinienne et ils pourrait être causée par d'autres mécanismes. Le traitement GV-Ret basé sur le stress du RE pourrait sauver à la fois la fonction de et la morphologie de la rétine dans souris BBS. / Retinal ciliopathies are a group of rare diseases caused by mutations of ciliary genes. Defects in ciliary genes can cause defects in proteins traffics and induces apoptosis of photoreceptor cells caused by stress of the endoplasmic reticulum (ER) .We studied retinal ciliopathies by mice models, Leber congenital amaurosis, Xlinked retinitis pigmentosa, Bardet-Biedl syndrome and Alström Syndrome. The Bbs1-/-, Bbs10-/- and CEP290-/- mice exhibited a decrease in retinal function caused by ER stress. Rd9/y and Alms1foz/foz mice showed a late onset and a low rate of retinal degeneration and they could be caused by other mechanisms. The GV-Ret treatment based on ER stress could save both the function and morphology of the retina in BBS mice .

[ES] Los dobles haploides son una gran herramienta para la mejora genética de híbridos debido a que se puede alcanzar la homocigosis completa en una sola generación. Entre las técnicas usadas para obtener estas plantas, la inducción de la embriogénesis de microsporas, mediante el cultivo de anteras o de microsporas, es la más eficiente y la más usada. La embriogénesis de microsporas es también un ejemplo de totipotencia de las células vegetales gracias a su habilidad de reprogramar su desarrollo gametofítico hacia una ruta esporofítica, donde las células proliferan de forma organizada para crear un nuevo organismo. Como en muchos otros procesos in vitro, las condiciones de cultivo deben ser optimizadas para incrementar la eficiencia. En la presente Tesis Doctoral, hemos usado dos especies como sistemas experimentales para estudiar y optimizar el cultivo de microsporas. Por un lado, usamos berenjena (Solanum melongena) como un ejemplo de cultivo de importancia económica en el que los protocolos todavía tienen mucho margen de mejora. La optimización de la densidad celular y los reguladores de crecimiento han demostrado ser útiles para modificar la eficiencia del cultivo de microsporas de berenjena. Por otra parte, hemos utilizado el cultivo de microsporas de Brassica napus para estudios básicos puesto que ha sido ampliamente usado como modelo para entender procesos celulares que ocurren durante este cambio en el desarrollo. Se detalla un protocolo estandarizado para el cultivo de microsporas de B. napus, el cual ha sido utilizado en todos los cultivos en esta Tesis para explorar una serie de procesos y estructuras celulares potencialmente implicados en el cambio de desarrollo hacia embriogénesis. Estos procesos incluyen estrés del retículo endoplásmico, muerte celular programada, autofagia y estructura y composición de la pared celular. Estudiamos en paralelo el cultivo de microsporas de dos genotipos de B. napus con diferente respuesta androgénica en condiciones estándar y añadiendo Tricostatina A, un modulador epigenético que ha mostrado ser beneficioso para la respuesta androgénica en algunos casos. En conjunto, esta Tesis representa un avance en la optimización del cultivo de microsporas en estas especies y arroja luz sobre el papel de algunos procesos en el contexto de embriogénesis de microsporas. / [CA] Els dobles haploides són una gran eina en millora vegetal per a la producció d'híbrids, a causa de la seua total homozigosi, que es pot aconseguir en només una generació in vitro. Entre les diverses tècniques que s'utilitzen per tal d'obtenir aquestes plantes, la inducció de l'embriogènesi de microspores, mitjançant cultiu d'anteres o microspores, és la més comuna i eficient. L'embriogènesi de microspores també és un exemple de la totipotència de les cèl·lules vegetals, capaços de reprogramar-se d'una via gametofítica a una via esporofítica, on proliferen de manera organitzada per crear un nou organisme. Com en moltes altres tecniques in vitro, s'han d'optimitzar les condicions del cultiu per tal d'augmentar l'eficiència. En la present Tesi Doctoral, hem utilitzat dues espècies de plantes com a sistemes experimentals per estudiar i optimitzar el cultiu de microspores. Per una banda, hem utilitzat l'albergínia (Solanum melongena) com a exemple de cultiu d'importància econòmica on els protocols encara tenen marge per a l'optimització. La optimització de la densitat de cèl·lules en cultiu i la concentració de reguladors de creixement van demostrar ser útils per modificar l'eficiència de la resposta dels cultius de microspores d'albergínia. D'altra banda, hem utilitzat cultius de microspores de Brassica napus principalment per a estudis bàsics, ja que s'utilitza àmpliament com a model per entendre els processos cel·lulars que es produeixen durant aquest canvi de desenvolupament. Es detalla un protocol estandarditzat per al cultiu de microspores de B. napus, que s'ha utilitzat en tots els cultius inclosos en aquesta Tesi per explorar una sèrie de processos i estructures cel·lulars potencialment implicades en el canvi de desenvolupament cap a l'embriogènesi. Aquests inclouen l'estrès del reticle endoplasmàtic, la mort cel·lular programada, l'autofàgia i l'estructura i composició de la paret cel·lular. Vam estudiar en paral·lel cultius de microspores de dos genotips de B. napus amb diferent resposta androgènica, cultivats en condicions estàndard i afegint-hi Tricostatina A, un modulador epigenètic que s¿ha demostrat beneficiós per a la resposta androgènica en alguns casos. En conjunt, aquesta Tesi representa un avanç en l'optimització dels cultius de microsporas en aquestes espècies i aporta llum sobre el paper d'alguns processos en el context de l'embriogènesi de microspores. / [EN] Doubled haploids are a great tool for hybrid breeding due to their complete homozygosity achievable in only one in vitro generation. Among the several techniques used to obtain these plants, induction of microspore embryogenesis, via anther or microspore culture, is the most common and efficient approach. Microspore embryogenesis is also an example of totipotency of plant cells due to their ability to reprogram themselves from a gametophytic to a sporophytic pathway, where cells proliferate in an organized way to create a new organism. As in many other in vitro procedures, culture conditions must be optimized in order to increase efficiency. In the present Doctoral Thesis, we used two plant species as experimental systems to study and optimize microspore culture. On one hand, we used eggplant (Solanum melongena) as an example of economically important crop where protocols have still room for optimization. Optimization of cell density and growth regulators demonstrated to be useful to modify the efficiency of eggplant microspore cultures. On the other hand, we used B. napus microspore cultures principally for basic studies since it is widely used as a model to understand cellular processes occurring during this developmental switch. A standardized protocol for Brassica napus microspore culture is detailed, which was used in all the cultures included in this Thesis to explore a series of processes and cellular structures potentially involved in the developmental switch towards embryogenesis. These included endoplasmic reticulum stress, programmed cell death, autophagy, and cell wall structure and composition. We studied in parallel microspore cultures from two B. napus genotypes with different androgenic response cultured in standard conditions and adding Trichostatin A, a epigenetic modulator shown to be beneficial for the androgenic response in some cases. Together, this Thesis represents an advance in the optimization of microspore cultures in these species, and sheds light on the role of some processes within the context of microspore embryogenesis. / Thanks are due to the Electron Microscopy Service of Universitat Politècnica de València, Marisol Gascón (IBMCP Microscopy Service). This work was supported by grant AGL2017-88135-R to JMSS from MICINN jointly funded by FEDER and by a Marie Skłodowska-Curie Individual Fellowship (656579) to PC-M This work was supported by grant AGL2017-88135-R to JMSS from MINECO jointly funded by FEDER. / Camacho Fernández, C. (2021). Microspore embryogenesis: cell wall dynamics and reprogramming of cell fate [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/163698 / TESIS

<p>The goal of this thesis is to algorithmically identify candidate modifiers for <i>retinitis pigmentosa</i> (RP) to help improve therapy and predictions for this genetic disorder that may lead to a complete loss of vision. A current research by (Chow et al., 2016) focused on the genetic contributors to RP by trying to recognize a correlation between genetic modifiers and phenotypic variation in female <i>Drosophila melanogaster</i>, or fruit flies. In comparison to the genome-wide association analysis carried out in Chow et al.’s research, this study proposes using a K-Means clustering algorithm on RNA expression data to better understand which genes best exhibit characteristics of the RP degenerative model. Validating this algorithm’s effectiveness in identifying suspected genes takes priority over their classification.</p><p>This study investigates the linear relationship between <i>Drosophila </i>eye size and genetic expression to gather statistically significant, strongly correlated genes from the clusters with abnormally high or low eye sizes. The clustering algorithm is implemented in the R scripting language, and supplemental information details the steps of this computational process. Running the mean eye size and genetic expression data of 18,140 female <i>Drosophila</i> genes and 171 strains through the proposed algorithm in its four variations helped identify 140 suspected candidate modifiers for retinal degeneration. Although none of the top candidate genes found in this study matched Chow’s candidates, they were all statistically significant and strongly correlated, with several showing links to RP. These results may continue to improve as more of the 140 suspected genes are annotated using identical or comparative approaches.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / The islet beta cell is unique in its ability to synthesize and secrete insulin for use in the body. A number of factors including proinflammatory cytokines, free fatty acids, and islet amyloid are known to cause beta cell stress. These factors lead to lipotoxic, inflammatory, and ER stress in the beta cell, contributing to beta cell dysfunction and death, and diabetes. While transcriptional responses to beta cell stress are well appreciated, relatively little is known regarding translational responses in the stressed beta cell. To study translation, I established conditions in vitro with MIN6 cells and mouse islets that mimicked UPR conditions seen in diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Chronic exposure of beta cells to proinflammatory cytokines (IL-1 beta, TNF-alpha, IFN-gamma), or to the saturated free fatty acid palmitate, led to changes in global beta cell translation consistent with attenuation of translation initiation, which is a hallmark of ER stress. In addition to changes in global translation, I observed transcript specific regulation of ribosomal occupancy in beta cells. Similar to other privileged mRNAs (Atf4, Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes during the UPR, whereas the mRNA encoding a proinsulin processing enzyme (Cpe) partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5’ untranslated region of mouse Pdx1 (between bp –105 to –280) contained elements that promoted translation under both normal and UPR conditions. In contrast to regulation of translation initiation, deoxyhypusine synthase (DHS) and eukaryotic translation initiation factor 5A (eIF5A) are required for efficient translation elongation of specific stress relevant messages in the beta cell including Nos2. Further, p38 signaling appears to promote translational elongation via DHS in the islet beta cell. Together, these data represent new insights into stress induced translational regulation in the beta cell. Mechanisms of differential mRNA translation in response to beta cell stress may play a key role in maintenance of islet beta cell function in the setting of diabetes.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes manifests from peripheral insulin resistance and a loss of functional beta cell mass due to decreased beta cell function, survival, and/or proliferation. Beta cell stressors impair each of these factors by activating stress response mechanisms, including endoplasmic reticulum (ER) stress. The glucolipotoxic environment of the diabetic milieu also activates a stress response in beta cells, resulting in death and decreased survival. Whereas the cell cycle machinery (comprised of cyclins, kinases, and inhibitors) regulates proliferation, its involvement during beta cell stress in the development of diabetes is not well understood. Interestingly, in a screen of multiple cell cycle inhibitors, p21 was dramatically upregulated in INS-1-derived 832/13 cells and rodent islets by two independent pharmacologic inducers of beta cell stress - dexamethasone and thapsigargin. In addition, glucolipotoxic stress mimicking the diabetic milieu also induced p21. To further investigate p21’s role in the beta cell, p21 was adenovirally overexpressed in 832/13 cells and rat islets. As expected given p21’s role as a cell cycle inhibitor, p21 overexpression decreased [3H]-thymidine incorporation and blocked the G1/S and G2/M transitions as quantified by flow cytometry. Interestingly, p21 overexpression activated apoptosis, demonstrated by increased annexin- and propidium iodide-double-positive cells and cleaved caspase-3 protein. p21-mediated caspase-3 cleavage was inhibited by either overexpression of the anti-apoptotic mitochondrial protein Bcl-2 or siRNA-mediated suppression of the pro-apoptotic proteins Bax and Bak. Therefore, the intrinsic apoptotic pathway is central for p21-mediated cell death. Like glucolipotoxicity, p21 overexpression inhibited the insulin cell survival signaling pathway while also impairing glucose-stimulated insulin secretion, an index of beta cell function. Under both conditions, phosphorylation of insulin receptor substrate-1, Akt, and Forkhead box protein-O1 was reduced. p21 overexpression increased Bim and c-Jun N-terminal Kinase, however, siRNA-mediated reduction or inhibition of either protein, respectively, did not alter p21-mediated cell death. Importantly, islets of p21-knockout mice treated with the ER stress inducer thapsigargin displayed a blunted apoptotic response. In summary, our findings indicate that p21 decreases proliferation, activates apoptosis, and impairs beta cell function, thus being a potential target to inhibit for the protection of functional beta cell mass.

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