467 research outputs found
Development of variable and robust brain wiring patterns in the fly visual system
Precise generation of synapse-specific neuronal connections are crucial for establishing a robust and functional brain. Neuronal wiring patterns emerge from proper spatiotemporal regulation of axon branching and synapse formation during development. Several neuropsychiatric and neurodevelopmental disorders exhibit defects in neuronal wiring owing to synapse loss and/or dys-regulated axon branching. Despite decades of research, how the two inter-dependent cellular processes: axon branching and synaptogenesis are coupled locally in the presynaptic arborizations is still unclear.
In my doctoral work, I investigated the possible role of EGF receptor (EGFR) activity in coregulating axon branching and synapse formation in a spatiotemporally restricted fashion, locally in the medulla innervating Dorsal Cluster Neuron (M- DCN)/LC14 axon terminals. In this work I have explored how genetically encoded EGFR randomly recycles in the axon branch terminals, thus creating an asymmetric, non-deterministic distribution pattern. Asymmetric EGFR activity in the branches acts as a permissive signal for axon branch pruning. I observed that the M-DCN branches which stochastically becomes EGFR ‘+’ during development are synaptogenic, which means they can recruit synaptic machineries like Syd1 and Bruchpilot (Brp). My work showed that EGFR activity has a dual role in establishing proper M-DCN wiring; first in regulating primary branch consolidation possibly via actin regulation prior to synaptogenesis. Later in maintaining/protecting the levels of late Active Zone (AZ) protein Brp in the presynaptic branches by suppressing basal autophagy level during synaptogenesis. When M-DCNs lack optimal EGFR activity, the basal autophagy level increases resulting in loss of Brp marked synapses which is causal to increased exploratory branches and post-synaptic target loss. Lack of EGFR activity affects the M-DCN wiring pattern that makes adult flies more active and behave like obsessive compulsive in object fixation assay. In the second part of my doctoral work, I have asked how non-genetic factors like developmental temperature affects adult brain wiring. To test that, I increased or decreased rearing temperature which is known to inversely affect pupal developmental rate. We asked if all the noisy cellular processes of neuronal assembly: filopodial dynamics, axon branching, synapse formation and postsynaptic connections scale up or down accordingly. I observed that indeed all the cellular processes slow down at lower developmental temperature and vice versa, which changes the DCN wiring pattern accordingly. Interestingly, behavior of flies adapts to their developmental temperature, performing best at the temperature they have been raised at. This shows that optimal brain function is an adaptation of robust brain wiring patterns which are specified by noisy developmental processes.
In conclusion, my doctoral work helps us better understand the developmental regulation of axon branching and synapse formation for establishing precise brain wiring pattern. We need all the cell intrinsic developmental processes to be highly regulated in space and time. It is infact a combinatorial effect of such stochastic processes and external factors that contribute to the final outcome, a functional and robust adult brain
Exploring the effects of robotic design on learning and neural control
The ongoing deep learning revolution has allowed computers to outclass humans
in various games and perceive features imperceptible to humans during
classification tasks. Current machine learning techniques have clearly
distinguished themselves in specialized tasks. However, we have yet to see
robots capable of performing multiple tasks at an expert level. Most work in
this field is focused on the development of more sophisticated learning
algorithms for a robot's controller given a largely static and presupposed
robotic design. By focusing on the development of robotic bodies, rather than
neural controllers, I have discovered that robots can be designed such that
they overcome many of the current pitfalls encountered by neural controllers in
multitask settings. Through this discovery, I also present novel metrics to
explicitly measure the learning ability of a robotic design and its resistance
to common problems such as catastrophic interference.
Traditionally, the physical robot design requires human engineers to plan
every aspect of the system, which is expensive and often relies on human
intuition. In contrast, within the field of evolutionary robotics, evolutionary
algorithms are used to automatically create optimized designs, however, such
designs are often still limited in their ability to perform in a multitask
setting. The metrics created and presented here give a novel path to automated
design that allow evolved robots to synergize with their controller to improve
the computational efficiency of their learning while overcoming catastrophic
interference.
Overall, this dissertation intimates the ability to automatically design
robots that are more general purpose than current robots and that can perform
various tasks while requiring less computation.Comment: arXiv admin note: text overlap with arXiv:2008.0639
New technologies to study helminth development and host-parasite interactions
How parasites develop and survive, and how they stimulate or modulate host immune responses are important in understanding disease pathology and for the design of new control strategies. Microarray analysis and bulk RNA sequencing have provided a wealth of data on gene expression as parasites develop through different life-cycle stages and on host cell responses to infection. These techniques have enabled gene expression in the whole organism or host tissue to be detailed, but do not take account of the heterogeneity between cells of different types or developmental stages, nor the spatial organisation of these cells. Single-cell RNA-seq (scRNA-seq) adds a new dimension to studying parasite biology and host immunity by enabling gene profiling at the individual cell level. Here we review the application of scRNA-seq to establish gene expression cell atlases for multicellular helminths and to explore the expansion and molecular profile of individual host cell types involved in parasite immunity and tissue repair. Studying host-parasite interactions in vivo is challenging and we conclude this review by briefly discussing the applications of organoids (stem-cell derived mini-tissues) to examine host-parasite interactions at the local level, and as a potential system to study parasite development in vitro. Organoid technology and its applications have developed rapidly, and the elegant studies performed to date support the use of organoids as an alternative in vitro system for research on helminth parasites
Kohastumine onkogeense reostusega ja looduslikud vähikaitsemehhanismid veekeskkonnas
Väitekirja elektrooniline versioon ei sisalda publikatsiooneVähki võivad haigestuda kõik hulkraksed organismid. Liikide võime vähki alla suruda on erinev. Kuna suuremaks kasvamine ja kauem elamine suurendavad organismide vähiriski, on tugevaim kaitse suurekasvulistes ja pikaealistel liikidel. Näiteks kui inimeste vähkisuremus on umbes 17%, siis elevantidel on see alla 5%.
Loomade risk haigestuda vähki ei sõltu mitte ainult elueast ja kehasuurusest, vaid ka keskkonnast. Reostus on nii inimeste kui ka teiste loomade vähki haigestumise riski suurendanud. Veekeskkonnas elavad loomad on reostuse vähki tekitava mõju osas eriti haavatavad, kuna reostus levib veekeskkonnas kiiresti ning kuhjub veekogude setetes.
Oma doktoritöös uurisin reostuse ja vähitekke seoseid veeliikidel. Praeguseks on umbes 30 veeliigi puhul seost reostuse ja vähi vahel näidatud, kuid kõige paremini on see seos tõestatud kaladel. Kalad, kes kasvavad suureks ja elavad kaua, peavad suureks kasvamiseks vajalike geenide koopiate suuremat arvu kompenseerima vähikaitsegeenide koopiate arvu suurendamisega. Võrdlev uuring võimaldas ka ennustada, millised kalaliigid on kõige tugevama ja millised kõige nõrgema vähikaitsega.
Merepõhjas elavate kalaliikide, jõelesta ja soomuslesta peal uurisin, miks on esimene neist liikidest vähile haavatavam kui teine. Leidsin, et vähi tekkeks peab jõelestadel olema alla surutud mitmete geenide avaldumise tase. Sama ei ole näha soomuslestade puhul. See viitab võimalusele, et jõelestadel on olemas vähikaitsemehhanismid, mis aitavad neil hoida madalamat vähitaset.
Tänapäevases maailmas on looduslike vähikaitsemehhanismide mõistmine muutunud eriti oluliseks, kuna inimtekkelised keskkonnamuutused on suurendanud vähi esinemissagedust nii inimestel kui ka looduslikel liikidel. Liikide erineva haavatavuse mõistmine vähitekke kontekstis võib tulevikus aidata paremini mõista reostuse mõju looduslikele liikidele ning panustada ka parematesse regulatsioonimehhanismidesse, kaitsmaks looduslikke liike ja populatsioone väljasuremise eest.All multicellular organisms can get cancer, but species differ in their ability to resist this disease. Since large body size and longer lifespan increase the likelyhood of cancer, larger and longer living species have evolved stronger cancer defence mechanisms to protect themselves. For example, when the risk of dying because of cancer is 17% in humans, it is less than 5% in elephants.
The likelihood that an animal develops cancer does not only depend on their lifespan and body size, but also of their environment. Pollution has increased cancer risk in both humans and other animals. Animals that live in aquatic environment are especially vulnerable to oncogenic pollution, since pollution spreads quickly in the water and accumulates for long time in the sediments.
In my PhD thesis, I studied the links between pollution and cancer in aquatic species. This link has been so far demonstrated in 30 aquatic animal species, with most studies on fish. I have also found that fish who live longer and grow larger have to compensate the number of copies of genes allowing them to grow large with additional copies of genes suppressing cancer. My comparative study allowes also to predict which fish species have the strongest and which have the weakest genetic defences against cancer.
By studying two species living in the bottom of the sea, flounder and dab, I asked why one of them is more vulnerable to cancer than the other. I found that for cancer to occur in flounder, many genes need to be suppressed. The same is not seen in dabs. This suggests that flounders have defence mechanisms against cancer that are not present in dab.
Currently, understanding natural cancer defence mechanisms has become crucial, as human induced environmental change has increased cancer prevalence in both humans and wild species. Understanding the differences between species in cancer defences can help to predict the effect of pollution on wild species, and contribute to the development of better regulatory mechanisms for protecting wild species.https://www.ester.ee/record=b555737
Use of omic tools for environmental risk assessment of emerging contaminants in marine species of commercial interest
La presente tesis doctoral busca evaluar los posibles efectos toxicológicos que pueden
tener los contaminantes ‘emergentes’ (CE) en los organismos marinos expuestos a
ellos. En concreto, se han evaluado dos filtros solares, un repelente de insectos y un
biocida, todos ellos presentes en productos de cuidado personal (PCP). Estas
sustancias llegan a los sistemas marinos a través de los vertidos de aguas residuales,
así como las entradas directas procedentes de actividades recreativas, tales como la
natación y el baño. La exposición a estos contaminantes se hace a muy bajas
concentraciones y de forma crónica, por tanto, evaluar sus posibles efectos a nivel
molecular resulta un paso clave para determinar su toxicidad. Las tecnologías “ómicas”
permiten el estudio global de los diferentes niveles biológicos (transcriptoma, proteoma,
metaboloma…) desde un punto de vista holístico e integrado. La integración de datos
ómicos, junto con la fusión de datos de bioconcentración y toxicocinética, es un método
extremadamente útil para dilucidar el modo de acción (MoA) de los contaminantes. De
este modo, en la presente tesis doctoral se ha implementado el uso y la integración de
herramientas ómicas (metabolómica y transcriptómica) para evaluar el efecto
toxicológico de dos filtros solares (4-metilbencilideno alcanfor (4-MBC) y sulisobenzona
(BP-4)), un repelente de insectos (N,N-Dietil-meta-toluamida (DEET)) y un biocida
(triclosán (TCS)) en dos especies marinas de interés comercial, la dorada (Sparus
aurata) y la almeja japonesa (Ruditapes philippinarum). Adicionalmente se realizaron
estudios de bioconcentración y toxicocinética.
Primero, se realizaron siete experimentos de exposición con los contaminantes y
organismos anteriormente mencionados usando un flujo continuo para reproducir
escenarios de exposición ambiental bajo condiciones controladas de laboratorio. Tras la
aplicación de técnicas analíticas y de alto rendimiento, se observó que la
bioacumulación de los contaminantes es superior en la almeja que en los peces. Esto
evidencia la importancia de tener en cuenta organismos de distintos niveles tróficos para
evaluar el potencial de bioacumulación de los contaminantes ambientales. Además, se observó que el filtro solar 4-MBC presentó el factor de bioacumulación (BCF) más alto
(368 565 L Kg -1) y la tasa de eliminación más baja (61.65%) que, junto con la
persistencia de este compuesto en el medio, son probablemente indicativos de un riesgo
potencial para el medio acuático marino que puede llegar a magnificarse a través de la
red alimentaria. También se observó que este compuesto sufría varias
biotransformaciones (reducción, oxidación, hidroxilación…) con el fin de ser excretado,
afectando al metabolismo de las drogas y xenobióticos y al del glutatión, llegando a
producir estrés oxidativo. Por otro lado, se observó que, aunque el TCS y BP-4 tenían
altos BCF en la almeja (1309 y 850 L Kg -1 respectivamente), sus tasas de eliminación
también eran altas (97.12 y 99.99%). Sin embargo, la exposición a estos contaminantes
produjo, entre otros, alteraciones en el metabolismo de los lípidos lo que puede deberse
a su capacidad como disruptores endocrinos. El DEET presentó el BCF más bajo en la
almeja (9.9 L Kg -1) y una alta tasa de eliminación (98.85%). El principal impacto de la
exposición de este compuesto a nivel transcriptómico se observó en el metabolismo de
biodegradación de xenobióticos y en el de los carbohidratos, lo que sugiere que se
estaba realizando un gran consumo energético para poder llevar a cabo la excreción del
compuesto.
En el músculo de la dorada se observó que tanto el DEET como la BP-4 presentaban
bajos BCF (2.6 y 0.7 L Kg -1 respectivamente) mientras que el TCS presentaba un BCF
de 113 L Kg -1. Sin embargo, los análisis transcriptómicos en el hígado de las doradas
revelaron que tras la exposición a DEET y BP-4, se expresaron 250 y 371 genes
diferencialmente, mientras que no se encontraron genes diferencialmente expresados
en la dorada tras la exposición a TCS. Tras la integración con los datos metabolómicos
realizados en las mismas muestras, se determinó que el DEET causaba en la dorada
agotamiento energético a través de la alteración de los metabolismos de carbohidratos
y aminoácidos, estrés oxidativo que daba lugar a daños en el ADN, peroxidación lipídica
y daños en la membrana celular y apoptosis. También se observó la activación del
metabolismo de los xenobióticos, así como una reacción inmune-inflamatoria. Finalmente, la integración de datos multiómicos reveló que la BP-4 causaba en la dorada
impacto en el metabolismo energético y oxidación lipídica, así como, impacto en la
biosíntesis de las hormonas esteroideas y tiroideas y en el metabolismo de los
nucleótidos. En conclusión, esta tesis muestra la utilidad de integrar datos a distintos
niveles biológicos y demuestra que el enfoque multiómico desarrollado y aplicado
supone una gran ventaja para dilucidar modos de acción de los compuestos estudiados
que, con enfoques diferentes, como el uso de una sola herramienta ómica, podrían
haberse pasado por alto
Visualization and manipulation of repair and regeneration in biological systems using light
Tissue repair after an injury is a fundamental process in biomedicine. It can involve regeneration, which uses new growth to restore tissue function. The interest in repair and regeneration is motivated by the desire to treat injuries and diseases and has attracted researchers for centuries. In the last decades, it evolved in the field of regenerative medicine, which has the ultimate goal of providing strategies for regenerating human cells, tissues, or even organs, for instance, via engineering principles.
Already since the first experiments on regeneration by Abraham Trembley, novel findings in biomedicine, repair, and regeneration have been enabled or accompanied by research in optics, for example, on the development of novel microscopy techniques. Nowadays, novel optical techniques are advancing, which allow to understand the role of single cells in tissue repair processes. Moreover, repair processes within cells can be visualized and manipulated. Ultimately, optics can provide enabling techniques for regenerative therapies. This habilitation thesis aims to present several of these advances.
On a single cell level, femtosecond laser nanosurgery was used to target specific intracellular structures during concurrent imaging in vitro. The relation of femtosecond laser nanosurgery to the cell state and cellular staining was investigated. Manipulation of single Z-discs in cardiomyocytes using a femtosecond oscillator laser system was accomplished, which allows to better elucidate the role of a single Z-disc in cardiomyocyte function. In particular, measurements on cell survival, (calcium-) homeostasis, and morphology yielded only minor deviations from control cells after single Z-disc ablation. A reduction in force generation was elucidated via traction force microscopy and gene expression level changes, for instance, an upregulation of -actinin were examined.
Additionally, light-based systems to influence single cells in their alignment or to trigger single cells, for example, to activate other cells via optogenetics were applied. On the tissue scale, imaging via confocal microscopy or multiphoton microscopy has been applied for various contexts of regenerative approaches. Furthermore, a fiber-based imaging approach, which could later be used for longitudinal imaging in vivo and builds upon a fluorescence microscope system and an imaging fiber bundle in combination with reconstruction via a neural network, was developed. As another imaging strategy, an abdominal imaging window served to image the mouse liver in vivo via multiphoton microscopy in successive imaging sessions. Manipulation in tissue was applied in colonoids, which resemble the structure of the colon on an in vitro scale, and revealed different cell dynamics dependent on the location of the damage. In particular, activation of the Wnt signaling pathway after crypt damage was observed. Cell ablation via a femtosecond laser amplifier system during concurrent two-photon microscopy was also established during in vivo liver imaging to study micro-regenerative processes.
Furthermore, laser-based delivery processes with novel materials or in the context of genome editing using CRISPR/Cas9 technology were investigated as enabling technologies for regenerative medicine. In conclusion, this thesis addresses the question of how optics can help to illuminate future directions in research on tissue repair and regeneration, as well as, regenerative therapies by addressing (longitudinal) imaging in a complex environment, sophisticated cell-manipulation strategies, and the application of novel materials for laser-based delivery
Quinazolin-4-one/3-cyanopyridin-2-one Hybrids as Dual Inhibitors of EGFR and BRAF: Design, Synthesis, and Antiproliferative Activity
A novel series of hybrid compounds comprising quinazolin-4-one and 3-cyanopyridin-2-one structures has been developed, with dual inhibitory actions on both EGFR and BRAFV600E. These hybrid compounds were tested in vitro against four different cancer cell lines. Compounds 8, 9, 18, and 19 inhibited cell proliferation significantly in the four cancer cells, with GI50 values ranging from 1.20 to 1.80 µM when compared to Doxorubicin (GI50 = 1.10 µM). Within this group of hybrids, compounds 18 and 19 exhibited substantial inhibition of EGFR and BRAFV600E. Molecular docking investigations provided confirmation that compounds 18 and 19 possess the capability to inhibit EGFR and BRAFV600E. Moreover, computational ADMET prediction indicated that most of the newly synthesized hybrids have low toxicity and minimal side effects
Snow crab (Chionoecetes opilio) hemocytes and hepatopancreas transcriptomes: identification, validation, and application of immune-relevant biomarkers of exposure to noise
The snow crab (Chionoecetes opilio) fishery off the east coast of Newfoundland and Labrador, Canada, has been impacted by annual seismic oil and gas surveying in recent decades, and commercial crab harvesters are concerned that it could potentially affect this resource. A laboratory study was performed to investigate the effects of chronic noise exposure on the transcriptomes of snow crab hemocytes and hepatopancreas, which are important in immunity, metabolism and response to environmental stress. Snow crab were held in replicate control or experimental tanks; on alternating days, the experimental tanks were exposed to noise from an underwater speaker which played a recording of a seismic airgun firing every 10 sec in a continuous loop for ~ 22 weeks (chronic noise). RNA sequencing was used to identify candidate noise-responsive molecular biomarkers in both hemocytes and hepatopancreas. The quantified transcripts of individuals (i.e. n=10 from each of the four groups) were compared using DESeq2, identifying over 300 noise-responsive transcripts in each cell/tissue type. Real-time quantitative polymerase chain reaction (qPCR) assays were designed and run for 61 of these transcripts with significant BLASTx hits. One transcript (rgs2) was significantly (p < 0.05) higher expressed in both hemocytes and hepatopancreas in response to noise; five and seven other transcripts were either significant or showed a non-significant trend of differential expression (0.05 < p ≤ 0.1) in hemocytes (e.g. cfb upregulated, sacs and myof downregulated) and hepatopancreas (e.g. gpx3 and atf3 upregulated, sult1c4 downregulated), respectively. To further investigate the performance and utility of these candidate biomarkers in the field, expression levels of 25 selected transcripts were then examined in the hepatopancreas of snow crab that had been subjected to 2D and 3D seismic surveying using both qPCR and multivariate statistical analyses. Overall, few of these biomarkers showed consistent results in field-collected snow crab, highlighting the need to combine lab and field-based studies to fully evaluate the utility of biomarkers. Nonetheless, our study still identified noise-responsive biomarkers in both settings (including stress and immune relevant genes), providing valuable information for understanding the impact of ocean noise on snow crab physiology and health
Form, function, mind: what doesn't compute (and what might)
The applicability of computational and dynamical systems models to organisms
is scrutinized, using examples from developmental biology and cognition.
Developmental morphogenesis is dependent on the inherent material properties of
developing tissues, a non-computational modality, but cell differentiation,
which utilizes chromatin-based revisable memory banks and program-like
function-calling, via the developmental gene co-expression system unique to
metazoans, has a quasi-computational basis. Multi-attractor dynamical models
are argued to be misapplied to global properties of development, and it is
suggested that along with computationalism, dynamicism is similarly unsuitable
to accounting for cognitive phenomena. Proposals are made for treating brains
and other nervous tissues as novel forms of excitable matter with inherent
properties which enable the intensification of cell-based basal cognition
capabilities present throughout the tree of life
Developmental scRNAseq Trajectories in Gene- and Cell-State Space—The Flatworm Example
Single-cell RNA sequencing has become a standard technique to characterize tissue development. Hereby, cross-sectional snapshots of the diversity of cell transcriptomes were transformed into (pseudo-) longitudinal trajectories of cell differentiation using computational methods, which are based on similarity measures distinguishing cell phenotypes. Cell development is driven by alterations of transcriptional programs e.g., by differentiation from stem cells into various tissues or by adapting to micro-environmental requirements. We here complement developmental trajectories in cell-state space by trajectories in gene-state space to more clearly address this latter aspect. Such trajectories can be generated using self-organizing maps machine learning. The method transforms multidimensional gene expression patterns into two dimensional data landscapes, which resemble the metaphoric Waddington epigenetic landscape. Trajectories in this landscape visualize transcriptional programs passed by cells along their developmental paths from stem cells to differentiated tissues. In addition, we generated developmental “vector fields” using RNA-velocities to forecast changes of RNA abundance in the expression landscapes. We applied the method to tissue development of planarian as an illustrative example. Gene-state space trajectories complement our data portrayal approach by (pseudo-)temporal information about changing transcriptional programs of the cells. Future applications can be seen in the fields of tissue and cell differentiation, ageing and tumor progression and also, using other data types such as genome, methylome, and also clinical and epidemiological phenotype data
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