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Zebrabalığında hareket bozukluguyla ilişkilendirilen ANO10, WDR81 ve VLDLR genlerinin susturulduğu modelde incelenmesi
Movement Disorders are the neurological symptoms that cause alterations in normal motility, posture and muscle tone. Certain brain areas, such as the cerebellum, mediate correct motor control and functioning. When defects or congenital lesions occur in the cerebellum, neural disruption in motor coordination causes the development of a particular movement disorder known as cerebellar ataxia. The focus of this study was to examine how three genes of interest (ano10, wdr81, vldlr), contributing to multiple varieties of cerebellar ataxias, influence one another and other genes that are associated with this disorder. Mutations in vldlr and wdr81 are associated with Cerebellar Ataxia Mental Retardation Disequilibrium Syndrome type 1 and type 2 (CAMRQ1 and CAMRQ2), respectively, whereas mutations in the ano10 gene is responsible for the development of Autosomal Recessive Cerebellar Ataxia Type 3 (ARCA3). In this work, five key scientific findings were reported. Firstly, in silico analysis predicted a common Ca2+ activated Casein Kinase 2 (CK2) domain in the protein sequences of the genes of interest and also predicted a common interacting UBC protein. These predicted interactions, a common CK2 domain and a UBC interacting protein may explain the observed neurodegenerative phenotype in cerebellar ataxia. Secondly, the transcript level analysis (qPCR and RNASeq) of ano10a, wdr81 and vldlr using zebrafish embryos collected from early embryonic and late larval stages showed that the three genes were expressed relatively higher at 1 hpf, 2 hpf and 5 hpf developmental stages than others and may suggest their importance in developmental processes. Additionally, the comparison of the expression patterns of ano10a, wdr81 and vldlr during early embryogenesis indicated that three targeted genes were co-localized at diencephalon, midbrain (optic tectum) and cerebellum. These spatiotemporal results may restrict the involvement of these three genes selectively in early neurodevelopmental processes. Thirdly, this study also examined the expression level analysis of three targeted genes in 12 different adult tissues in a sexually dimorphic manner. Findings showed that genes of interest were expressed significantly higher at the eyes, brain and gonads (p-values < 0.05). Moreover, the gender specific examination in the 12 adult tissues revealed that ano10a and wdr81 expression differed significantly at eyes, gills, liver and gonads (p-values < 0.05) whereas, vldlr gene expression was significantly different at swim bladder and gonads in male and female individuals (p- values < 0.05). Fourthly, the clustergram analysis indicated that three genes of interest were grouped within close families with each other and 9 additional cerebellar ataxia associated genes and may imply that targeted genes alter functions in the converging pathways. Finally, results from the clustergram analysis helped to design and carry out a study knocking down the expression of ano10a, wdr81 and vldlr separately with MO antisense technology to examine the effect of the silenced mRNA on the expression levels of each other and 9 other highly correlated cerebellar ataxia-related genes. Single MO injections caused the significant upregulation of all investigated genes especially at 72 hours after/post injection (hpi) (p-values < 0.05) when ano10a transcript was silenced suggesting either an activated compensatory mechanism or activated alternative disease specific cascade molecules in response to its absence. Taken together, the outcomes of functional knockdowns can pave the way for the development of novel therapeutic targets using inhibitors or antagonists of activated cellular pathway components or the enhancers of downregulated genes to prevent or at least slow down the progression of not only cerebellar ataxia but also several other neurodegenerative disorders.
Meme ve karaciğer kanserlerinde özgün hedefler olarak mineralokortikoid ve glukokortikoid reseptörlerinin incelenmesi
Cell signaling is a complex phenomenon and is maintained through intertwined signal transmissions within and in-between the cells. Anti-cancer therapies are often challenged by this fact due to crosstalk-associated activation of alternative survival routes. Hence, development of new treatment strategies and identification of novel prognostic markers depends on in-depth knowledge on cell signaling routes altered in cancer and possible crosstalk paths. Herein, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) signaling, two closely related members of steroid receptor hormone family, and their possible crosstalk were studied across breast and liver cancer cell lines. In breast cancer cell lines, estrogen responsive and MR expressing T47D was used in order to study possible crosstalk among Estrogen receptor (ER) and MR. MR-GR ligand aldosterone (ALDO) and ER ligand estrogen (E2) administered to breast cancer cells alone and in combination and, MR, ER and GR and their downstream signaling members were studied employing qRT-PCR and Western blot assays. Furthermore, ALDO, E2, ALDO-E2 hormone administrations were also used for cell viability assessments. Our results implied possible interactions of ALDO-E2 signaling at the level of cell viability, and at mRNA levels of progesterone receptor. In liver cancer cell lines, MR and GR was investigated as targets of a novel treatment. Liver cancer subtype hepatocellular carcinoma (HCC) has high mortality rate with limited treatment options. Multi-kinase inhibitor Sorafenib (SFB) with mild effectivity is most known systemic therapy against HCC. To potentiate the effectiveness of SFB and overcome to the crosstalk associated limitations, combinatorial drug treatment approach targeting multiple signaling modalities has been adopted in literature. Previously in our lab, SFB was combined with repurposed anti-psychotic drug TFP as a novel combinatorial treatment against hepatocellular carcinoma (HCC) and liver cancer cell lines. Cellular viability was synergistically reduced by SFB-TFP in HCC cell line Hep3B, while antagonistic effects on viability in SkHep1 was apparent. Herein, two liver cancer cell lines Hep3B and SkHep1, were used in comparison to unravel mechanism of action of SFB-TFP combination at the protein level. Apoptosis, cell cycle, PI3K/AKT/mTOR and MAPK pathways were investigated in addition to MR and GR. Our results revealed several markers indicating success of drug combinations and targeted pathways at protein level which needs to be pursued further.
Kokteyl partisi sırasında beyindeki doğal konuşma temsilleri
Humans are remarkably adept in selectively listening to a desired speaker in a crowded environment, while filtering out non-target speakers in the background. Attention is key to solving this difficult cocktail-party task, yet a detailed characterization of attentional effects on speech representations is lacking. It remains unclear across what levels of speech features and how much attentional modulation occurs in each brain area during the cocktail-party task. Besides, it should be clarified whether unattended speech is represented in cortex during selective listening and if so, at what feature levels its representations are maintained. To address these questions, we recorded whole-brain blood-oxygen-level-dependent (BOLD) responses while subjects either passively listened to single-speaker stories, or selectively attended to a male or a female speaker in temporally-overlaid stories in separate experiments. Spectral, articulatory, and semantic models of the natural stories were constructed to enable comprehensive assessments on the hierarchy of speech features. Intrinsic selectivity profiles were identified via voxelwise models fit to passive listening responses. Attentional modulations were then quantified based on model predictions for attended and unattended stories in the cocktail-party task. We find that acoustic representations are confined to the early auditory cortex whereas linguistic representations are broadly distributed across cortex, that attention causes broad modulations at multiple levels of speech representations (articulatory and semantic) while growing stronger towards later stages of processing, and that unattended speech is represented up to the semantic level in parabelt auditory cortex. These results provide insights on speech perception and attentional mechanisms that underlie the ability to selectively listen to a desired speaker in noisy multi-speaker environments.
Diyet ve diyet-mimetiği manipülasyonların gen anlatımı açısından yaşlanan beyne etkileri
Aside from many genetic and environmental influences on the brain, aging itself is a significant risk factor for accelerated cognitive decline, making aging research crucial due to the increasing population age in our era. We aimed to discover gene expression differences in the aging zebrafish brain using three age groups in the first aim. We identified gjc2 (CX47) and alcamb (ALCAM) cell adhesion genes showing consistent downregulation with age across all experiments. ALCAM is also known to be associated with neuroinflammation, which has been implicated to be lowered using anti-aging, non-genetic nutrient interventions. In the second aim, we applied 12 weeks of two opposing nutrient interventions, caloric restriction (CR) and overfeeding (OF) in aging zebrafish, in order to be able to propose a reliable therapeutic approach for reversing age-related neurobiological changes. We measured protein and expression level differences of selected genes related to proliferation to inflammation with these diets. The results showed that sox2 gene expression was significantly upregulated following OF treatment than CR diet, and myca and tp53 mRNA levels were significantly downregulated with advanced age. Alcamb and tfdp1 expression levels were also marginally significantly lowered with CR compared to other groups. Meanwhile, we also conducted another transcriptomic approach using microarray to assess gene expression differences with CR compared to Ad-libitum (AL) feeding. Thus, lastly, in the third part, we found that CR causes changes in cell cycle regulation among several other functional regulatory pathways in zebrafish brains. We identified the tfdp1 gene, which showed downregulation with CR, as a possible CR regulator. Then, to create a CR mimic, we performed morpholino oligo (MO) injections to zebrafish embryos and adult brains to knock down tfdp1 gene expression levels. The injections were not successful in altering Tfdp1 protein levels in neither embryos and adults. However, 8ng tfdp1-MO injections in embryos significantly increased myca and tp53 expression levels, which are among the downstream targets of tfdp1. Our examinations shed light on healthy brain aging and possibly propose new drug targets.