Theses supervised by Prof. Dr. Bayram Yılmaz
20 theses · Yeditepe University
Transgenik fare kisspeptin nöronlarında erken dönem sosyal izolasyon ilişkili fonksiyonel ve aksonal plastisitenin araştırılması
Bu çalışmada adolesan dönemde maruz kalınan sosyal izolasyon stresinin, hipotalamik Arkuat Çekirdeğinde (ARC) bulunan Kisspeptin nöronlarının fonksiyonlarının ve aksonal uzantılarının nasıl etkilendiği araştırıldı. Bu amaçla Kiss:Cre transgenik fare hattı kullanıldı. Post-natal 21. günde (P21) fareler 3 haftalık sosyal izolasyona alındı. Bu dönemde fareler opak kafeslerde tek başına yaşarken sosyal gruptaki fareler normal kafeslerde üçerli gruplar halinde yaşatıldı. İzolasyon sonrasında farelerin lokomotor aktiviteleri, beslenme davranışları ve anksiyeteleri gözlendi. Kisspeptin nöronlarını görüntüleyebilmek için farelerin hipotalamik ARC bölgelerine GFP-Adone Associated Virüs (AAV) enjeksiyonu yapıldı. Bu farelerin ARC Kisspeptin nöronlarından elektrofziyoloji kaydı alındı. Projeksiyon analizi yapabilmek için ARC Kisspeptin nöronlarının aksonal uzantıları konfakal mikroskopta görüntülendi. ARC bölgesinde Kisspeptin, NörokininB ve Dinorfin nöropeptid ifadesi immünfloresan tekniği kullanılarak incelendi. Davranış deneylerinden elde edilen sonuçlara bakıldığında gece fazında izole gruptaki dişilerin diğer gruplardan daha aktif olduğu (p<0.005) erkeklerde ise sosyal gruptaki farelerin izole gruptakilerden daha aktif olduğu görüldü. Beslenme davranışlarında gruplar arasında anlamlı bir fark görülmedi. Yükseltilmiş artı labirent testinde izole gruptaki farelerin daha ankisyetik olduğu görüldü (p<0.05). Sosyal gruptaki farelerin ARC Kisspeptin nöronlarının aksiyon potensiyel frekanslarının izole gruptaki farelere göre daha yüksek olduğu (p<0.001), sosyal gruptaki dişi farelerin aksiyon potansiyel frekanslarının izole gruptaki dişi ve erkek farelere göre daha yüksek olduğu (p<0.001) görüldü. İmmünfloresan sonuçlarına bakıldığında izole gruptaki farelerin ARC bölgelerinde Dinorfin miktarının sosyal gruptan daha fazla olduğu (p<0.05) görüldü. Nerokinin B miktarının izole gruptaki farelerde, Kisspeptin miktarının ise sosyal gruptaki farelerde daha fazla olduğu gözlendi ancak istatistiksel anlamlılık görülmedi. Aksonal projeksiyon analizlerine bakıldığında sosyal gruptaki farelerin hipotalamik ARC Kisspeptin nöronlarının Anteroventral Periventricular (AVPV) bölgeye izole gruptakilere göre daha yoğun projeksiyon yaptığı gözlemlendi. Beynin diğer bölgelerine yapılan ARC Kisspeptin aksonal projeksiyonları için gruplar arasında bir fark görülmedi. Elde edilen bu bulgulara göre ergenlik döneminde maruz kalınan sosyal izolasyonun farelerin lokomotor ve anksiyte davranışlarını etkilediği ve Kisspeptin nöronlarının fonksiyonlarını baskıladığı görüldü.
Investigation of epigenetic profiles in exfoliated mammary epithelial cells retrieved from lactating women
Breast cancer is one of the most diagnosed cancer among Turkish women and mortality rate is higher for the younger women. As the breast cancer is a heterogenous disease, identification of the underlying molecular mechanisms of the epidemiologically established risk factors for the breast cancer provide us the possible strategies for prevention and early detection. Hypermethylation of the promoter region of the tumor suppressor genes is a hallmark of the breast cancer and it can be used as a biomarker for the indicator of the breast cancer risk and tool for the diagnosis. Human breast milk is rich in mammary epithelial source and suitable for DNA methylation analysis. Fresh milk samples were collected from lactating women residing in Marmara region, mainly Istanbul. Pyrosequencing analysis was carried on DNA promoter methylation assessment for the six tumor suppressor genes which are PYCARD, CDH1, SFRP1, GSTP1, RBP1 and RASSF1A. The methylation scores were generally found as unmethylated state. DNA methylation analysis results showed that age is significantly positively related with methylation rate of the all CpG sites of RASSF1A, many CpG sites of the PYCARD and sixth and seventh CpG region of the GSTP1 promoter methylation. Statistically positive relationship was found between BMI of the lactating women and fifth CpG of the CDH1 and RASSF1A. Age of the first birth was found as statistically positive with all CpG sites of the RASSF1A and GSTP1 promoter methylation. Statistically higher methylation rate was found in many CpG regions of the PYCARD for women using the intrauterine devices. Significantly higher methylation rate for RBP1 was also seen among participants who had family history of breast and ovarian cancer. Significantly higher methylation percentage for RASSF1A was also detected among women who has family history of ovarian cancer. Dietary habits including dairy, fish, egg and poultry consumption were also significantly linked with methylation rate in several CpG region of PYCARD, CDH1, SFRP1, GSTP1, RBP1 and RASSF1A. In conclusion, breast milk is valuable biological sample for monitoring of early molecular modifications in breast tissue.
Transgenik farelerde hipotalamik arkuat agRP nöronlarının opioid modülasyonu
Hipotalamusun arkuat çekirdeğinde (ARC) lokalize olan Agouti ile ilişkili peptit (AgRP) nöronları iştah, metabolizma ve enerjinin düzenlenmesinde merkezi bir role sahiptir. Bu nöron grubunun manipülasyonlarının, beslenme davranışının akut düzenlenmesinde etkili olduğu kanıtlanmıştır. AgRP nöronları, paraventriküler çekirdeğe (PVH) yoğun aksonal projeksiyonlar gönderir ve PVH'de bulunan diğer nöronlarla sinaptik bağlantılar kurar. ARCAgRPPVH sinaptik bağlantıları enerji dengesi, metabolizma ve daha da önemlisi beslenmenin düzenlenmesinde önemli roller oynadığından, bu bağlantıları modüle eden ilaçların da iştah düzenlemesinde önemli bir rol oynaması olasıdır. Bu çalışmada, µ opioid reseptör antagonizmasının AgRP nöronları üzerindeki olası fonksiyonel değişimlerini ex vivo ve in vivo yöntemlerle karakterize etmeyi amaçladık. Çalışmamızda hem dişi hem de erkek AgRP-Cre fareleri kullanıldı. µ opioid reseptör agonisti DAMGO'nun AgRP nöronları üzerindeki etkileri elektrofizyolojik aktivite deneyleri ile ex vivo olarak incelenirken, µ opioid reseptör inhibisyonunun in vivo etkileri beslenme davranışı, lokomotor aktivite ve fiber fotometri Ca+2 görüntüleme yöntemleri kullanılarak incelenmiştir. Ex vivo deneylerde, DAMGO, AgRP nöronlarının ateşleme frekansını önemli ölçüde azalttı ve bu nöronların dinlenim membran potansiyelini hiperpolarize etti. Ek olarak, DAMGO, bu nöronlara giden uyarıcı post-sinaptik akımların frekansını önemli ölçüde azaltırken, inhibe post-sinaptik akım frekansını değiştirmediği gözlendi. Optogenetik tekniğininden faydalanılarak yapılan ARCAgRPPVH sinaptik bağlantı deneylerinde, ilaç uygulamasından sonra sinaptik tepe genliğinin önemli ölçüde azaldığı gözlendi. DAMGO'nun beş günlük intraperitoneal uygulamasından sonra, farelerde gıda tüketim miktarının artma trendi olduğu gözlendi. DAMGO uygulamasının bir sonucu olarak, hayvanların kat ettikleri mesafe anlamlı derece azalırken hızlarında bir değişiklik olmadı. Sistemik DAMGO uygulamasından sonra AgRP nöronlarının in vivo elektrofizyolojik aktivitesini kaydetmek için fiber fotometri Ca+2 görüntüleme tekniğini kullandık. DAMGO, 16 saat aç bırakılan farelere uygulandığında, in vivo AgRP nöron aktivitesinin azalmaya yönelik bir trend izlediği gözlemlendi. Sonuç olarak, mevcut veriler göz önüne alındığında, µ opioid agonizminin hedonik beslenmeyi güçlü bir şekilde uyardığı ve homeostatik beslenmede AgRP nöronlarının aktivitesinin düzenlenmesinde rol oynadığı önerilebilir.
The effects of hypothalamic paraventricular oxytocin neurons on the hypofrontality in transgenic male mice
Oxytocin is a neuropeptide synthesized in the neurons of hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) and has a modulatory effect on various social behaviors. There is only one oxytocin receptor (OTR) known to date, and oxytocin exerts its neuromodulatory effects through this receptor. Oxytocin receptor is found in many areas of the brain, including the prefrontal cortex (PFC). In addition, several studies have shown that hypothalamic oxytocin neurons project to the PFC. Dopamine has an important role in regulating the cognitive function of the PFC and it is thought that oxytocin and dopamine systems work together and have similarities in the areas of organization and projection. In this study, it was aimed to investigate effects of oxytocin on hypofrontality-induced behavioral disturbances and the interaction of oxytocin and dopamine neurons in PFC. Adult male oxytocin-cre mice were used in this study. Recombinant adeno-associated virus with stimulatory hM3D subunit or inhibitory hM4D subunit were injected to paraventricular area in order to activate and inhibit oxytocin neurons. Then, effects of manipulation of oxytocin neurons on several behaviours were evaluated. Anxiety, locomotor activity, social interaction and short-term memory were examined by using elevated plus maze, open field test, social interaction test and T-maze test, respectively. Fiber photometry Ca2+ imaging was used to investigate interaction of oxytocin and dopamine neurons in the PFC. Oxytocin did not significantly change anxiety and locomotor activity. Inhibition of paraventricular oxytocin neurons significantly decreased the social novelty preference of oxytocin-cre mice and could cause short-term memory impairment. Further, Ca2+ signal recordings from prefrontal dopamine neurons were taken before and during acute activation of paraventricular oxytocin neurons, but no significant effect of acute activation of paraventricular oxytocin neurons on the activity of prefrontal dopamine neurons was found. In this study, the effects of manipulation of oxytocin neurons on various behaviors was revealed. Thus, modulation of prefrontal dopaminergic neuronal activity by chemogenetic activation and inhibition of oxytocin neurons was examined for the first time.
Arkuat çekirdekte bulunan AGRP nöronlarının, kainik asit ile indüklenmiş epilepsi üzerine etkileri
Agouti ile ilişkili peptit (AgRP) nöronlarının hipotalamusun kavisli çekirdeğindeki gama aminobütirik asit (GABA) ile modülatör etkisi, enerji metabolizması için önemlidir. Epilepside enerji metabolizmasının bozulduğu bilinmektedir. AgRP nöronları ile ilişkili GABA ve nöropeptid Y'nin antikonvülsan etkileri nedeniyle, bu nöronların epilepside rol oynayabileceği varsayılmıştır. AgRP nöronlarının akut ve kronik uyarılmasının, davranışsal ve elektrofizyolojik olarak kainik asit (KA) kaynaklı deneysel epilepsi modelini etkileyebileceğini varsaydık. Otuz dişi ve erkek transgenik AgRP-cre faresi kullanıldı. Epileptik aktivite, 100 nl KA (serum fizyolojik içinde 15mM) ile indüklendi. AgRP nöronlarının akut ve kronik uyarılmasının epileptik aktivite üzerindeki etkilerini gözlemlemek için optogenetik ve kemogenetik yöntemler kullanıldı. Sonuç olarak, kontrol ve epilepsi grupları elektroensefalografi (EEG), dinlenim zar potansiyeli kaydı, beslenme davranışı, açık alan, obje yerleşim ve yeni nesne tanıma testlerine göre karşılaştırıldı. İstatistiksel analiz için One-Way-ANOVA ve Student-t-test uygulandı. Nöbetler racine cetveline göre skorlandı. Üç ve üzeri nöbet olarak kabul edildi ve kontrol grubu ile karşılaştırıldı (p<0.05). EEG'de epilepsi ve kontrol grupları aynı düzende devam etse de, epilepsi grubu amplitüdde daha yüksek bir sayısal eğilim izledi. Açık alan testi sırasında AgRP nöronları kronik olarak uyarılmış epilepsi grubunun kontrol grubuna göre iç bölgede daha az zaman geçirdiği görüldü (p<0.05). AgRP nöronlarının akut ve kronik uyarımı epilepsi grubunda anlamlı bir fark yaratmasa da sonuçların kontrol grubuna benzer bir seyir izlediği görüldü. Bulgularımız, AgRP nöronlarının epilepside modülatör bir etkiye sahip olabileceğini düşündürmektedir.
Effects of methylphenidate on the onset of puberty and reproductive system in male and female rat models
Methylphenidate (MPH) is a central nervous system stimulant, which blocks the reuptake of dopamine and noradrenaline. It is widely used to treat attention deficient hyperactivity disorder (ADHD) in children and adolescents. This study aimed to investigate the effect of chronic administration of MPH on reproductive functions in both male and female rats and the reversibility of those effects. Sprague Dawley rats from both sexes were administered with 5 mg/kg of MPH or physiological saline via oral gavage from postnatal day (PND) 21 to PND 60 and from PND 21 to PND 90. In addition, recovery groups from both sexes in which stopped receiving MPH from PND 60 to PND 90 were included. In the end of the study, the results showed slightly but significantly early puberty in male models, slight decrease in testosterone levels in treated groups to increase again in the recovery group. The histological findings showed seminiferous tubule degeneration in the testes in MPH-treated groups. Leydig and Sertoli cells were affected in the MPH-treated groups, and all the findings were improved in the recovery group. In female animals, a significant delay in puberty onset was observed. Estradiol levels were decreased in MPH-treated group up to PND 90 compared to control group and a surge in luteinizing hormone levels was detected in recovery group. Histological findings showed morphological deterioration in the basement membrane of the ovaries of MPH-treated groups, more atretic follicles were detected and these findings were improved in the ovaries of recovery group. This study demonstrates that MPH could affect the reproductive functions in both male and female rats and those effects are transient. Key words: Methylphenidate, Ovary, Puberty, Sex hormones, Testes
Investigation of obesogenic effects of hexachlorobenzene, DDT and P,P-DDDE in male rat model
Obesity continues to be a global public health problem. Recent studies reported that some environmental pollutants may have obesogenic effects. Peroxisome proliferator activating receptor gamma (Pparγ) and uncoupling protein-1 (UCP1) genes play roles in adipogenesis. Their expression in white adipose tissue (WAT) and brown adipose tissue (BAT) is of importance. In the present study, we aimed to investigate obesogenic effects of dichlorodiphenyltrichloroethane (DDT), hexachlorobenzene (HCB) and dichlorodiphenyldichloroethylene (DDE). Secondly, we aimed to investigate the serum sex hormones (luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone), as well as testicular histology. Thirty-two adult male Sprague-Dawley rats were randomly divided into four groups: control, HCB, DDT and DDE. Organochlorine pesticides (OCPs; 5mg/kg) dissolved in corn oil were administered to the animals by oral gavage every other day for five weeks. Controls received vehicle alone. The body weights of the animals were monitored throughout the experiments. At the end, the animals were decapitated, BAT and WAT samples were collected to analyse Pparγ and UCP1 levels. Testes were dissected out for histological examination. Serum samples were collected to measure levels of LH, FSH, testosterone and serum cholesterol and triglyceride levels. Body weights of the animals did not significantly change among the groups. Serum cholesterol and triglyceride were similar. Serum LH and FSH levels were not significantly altered, but testosterone was significantly reduced in all groups (p<0.05). HCB administration significantly increased Pparγ expression only in WAT (p<0.05). DDT significantly decreased Pparγ and UCP1 expressions in both WAT and BAT (p<0.01). DDE significantly decreased Pparγ expression only in WAT (p<0.001). Testicular histology was found to deteriorated by OCPs. In conclusion, HCB-induced expression of Pparγ in WAT obesogenic activity of HCB. In addition, DDT and DDE-induced decreases in expression of Pparγ and UCP1 in WAT suggest that these two pesticides may reduce adipogenesis.
Investigation of organochlorinated pollutant levels in human breast milk and their genotoxic effects on mammary epithelial cells
Persistent organic pollutants (POPs) pose a threat for the environment and human health. In this study, we investigated concentrations of organochlorinated pesticides (OCPs) and polychlorinated biphenyls (PCBs) accumulated in the fat of the human breast milk. Lactating mothers were asked to fill the questionnaire form including established risk factors associated with breast cancer development. In addition, potential DNA damage in exfoliated epithelial cells was analyzed by using Comet Assay. A total of 62 healthy lactating mothers living in Istanbul volunteered to participate in this study. Yeditepe University Clinical Research Ethics Committee approved the study protocol, and written consent was obtained from the volunteers. The genotoxicity level of the DNA isolated from human breast milk was determined by Comet Assay. The amount of OCPs and PCB congeners accumulated in human breast milk was analyzed by gas chromatography. These indicator PCB congeners were analyzed: PCB 28, PCB 52, PCB 101, PCB 118, PCB 138, PCB 153, PCB 180 and PCB 202. The OCPs investigated in the present study were α-benzenehexachloride, β-benzenehexachloride, δ-benzenehexachloride, hexachlorobenzene (HCB), 2,4′-dichlorodiphenyldichloroethylene (o,p-DDE), 4,4′-dichlorodiphenyldichloroethylene (p,p-DDE), 2,4′-dichlorodiphenyltrichloroethane (o,p-DDT), 4,4′-dichlorodiphenyltrichloroethane (p,p-DDT), 2,4′-dichlorodiphenyldichloroethane (o,p-DDD), and 4,4′- dichlorodiphenyldichloroethane (p,p-DDD). Demographic, dietary and reproductive health information was obtained by filling a questionnaire from each lactating mother. The analysis showed that highest levels of PCB congeners were PCB 153 (1983±2050 ng/g lipid), PCB 101 (274.4±292.7 ng/g lipid) and PCB 180 (96.21±156.2 ng/g lipid) detected in human breast milk. The lowest PCB congener was determined as PCB 202 (7.51±7.78 2 ng/g lipid) in the milk samples. The highest OCPs detected were p,p-DDT (139.8±271.1 ng/g lipid) and remained metabolites of DDTs had nearly the same levels. The lowest OCP was determined as HCB (1.44±0.69 ng/g lipid) in fat fraction of the human breast milk. The highest level of HCH was detected as Gamma-HCH (91.62±207.3 ng/g lipid) which is also known as lindane. Our findings showed that there was a significantly positive association between age and PCB 180 (p<0.01) and p,p-DDD (p<0.05) levels. However, significantly negative correlation was also detected between age and o,p-DDE (p<0.05). A significantly positive relationship was observed between number of nursed children and delta-HCH (p<0.05) and DDTs (p<0.05). Significantly positive association between age of first birth was seen in p,p-DDD (p<0.001), whereas significantly negative correlation was seen in o,p-DDE (p<0.01). Significantly higher amount of p,p-DDE (p<0.02) and o,p-DDE (p<0.01) were detected in breastfeeding mothers who had lived in İstanbul less than five years. PCB 52 (p<0.05) and p,p-DDT (p<0.01) had significantly positively correlated with weekly dairy consumption. There was a significantly positive association between tail moment (TM) and PCB 202 (p=0.042), whereas the significantly negative correlation was observed for gamma-HCH (p<0.001). Our findings show that traces of OCPs and PCBs are still detectable in human breast milk of lactating women living in Istanbul. Detection of these pollutants showed an age-dependent pattern in the breast milk samples and declined in multipara women compared to primipara mothers. We also confirm that breast milk is a useful and important biological tool for monitoring the burden of POPs in human.
Investigation of neuroendocrine effects of methylphenidate on gonadotropin releasing hormone and kisspeptin in cell culture models
Methylphenidate (MPH) is widely used among children for attention deficit hyperactivity disorder. Though there are discussions about the growth-related effects of its use, the neuroendocrine mechanisms of MPH around puberty have not been studied. This study aims to investigate the role of MPH on gonadotropin-releasing hormone (GnRH) and Kisspeptin (Kiss1) expression and secretion in vitro using cell culture techniques. GnRH and Kiss1 secreting cells, GT1-7 and rHypoE-8 cell lines respectively, were maintained in DMEM containing 10% FBS and 1% PSN at 37°C under 5% CO2 humidified atmosphere. The effect of MPH on proliferation and migration at different doses (1 nM-200 μM) for 24 hours was assessed by trypan blue dye exclusion and wound healing (scratch) assays, respectively. To investigate GnRH expression and secretion, cells were cultured in maintenance medium without FBS with or without MPH (200 μM) and samples at different were obtained to investigate GnRH expression by real-time qRT-PCR and GnRH release to the medium by ELISA. Normal distribution of the data was analyzed by Shapiro-Wilk normality test. Data from dose-response experiments were either analyzed one-way ANOVA or Kruskal-Wallis followed by Dunnet's multiple comparison or Dunn's multiple comparison tests, respectively. Real-time qPCR and ELISA data were analyzed by Student's t-test and 2-ΔΔCT method. There were no significant differences between untreated cells and MPH-treated cells in terms of proliferation and migration. As the proliferation and migration of the cells were not affected by the different concentrations of MPH, the highest concentration of 200 μM was selected to assess gene expression and hormone secretion. GnRH expression has remained comparable to the control arm with no statistical difference at any time point. GnRH release was significantly higher under MPH compared to the control group at 2 hours after treatment (p<0.05), while no differences were found between groups at other time points. Kiss1 expression was assessed for time points of 30 minutes, 2 hours, and 24 hours. Kiss1 expression was significantly increased in the first 30 minutes of MPH treatment. As, MPH affected GnRH and Kiss1 expression at multiple time points, to further investigate the response of the cells, the recovery in the expression after the cessation of MPH treatment was analyzed. It was observed that after the cessation of MPH treatment there is no statistical significance between MPH treatment and control arms in GnRH expression. Though the cellular evidence suggests that MPH affects the expression and release of GnRH and Kiss1 to some extent, it is far from reflecting the accumulating clinical data. Lack of statistical difference should not be interpreted as no differences between the MPH use over neuronal cell lines. When results are modeled for larger data sets (n= 30-60) statistically the majority of the timepoints become significant in comparison to the control arm. Our findings suggest puberty-delaying effects of MPH may be partly through GnRH and Kiss1 expression and release. Further investigations are needed to understand a medication that possibly have direct effects on 40 million people worldwide.
Diyabet modeli oluşturulmuş AGRP transgenik farelerde sistemik apelin uygulamasının iştah düzenlenmesi üzerine etkilerinin araştırılması
Diyabet, son yıllarda giderek yaygınlaşan bir halk sağlığı problemidir. Tip 2 diyabetin sebepleri arasında ağırlıklı olarak sedenter yaşama ve aşırı yemeye bağlı gelişen aşırı kilo ve obezite sonucu gelişen insülin direnci ve daha az sıklıkla yaşlanma ve genetik faktörler gelirken, tip 1 diyabet asıl olarak, insülin üreten beta hücrelerinin otoimmün yıkımından kaynaklanmaktadır. Besin alımı, hipotalamik arkuat çekirdekte (ARC) bulunan oreksijenik agouti-ilişkili peptit/nöropeptit Y (AgRP/NPY), anoreksijenik proopiomelanokortin/kokain-amfetamin ile düzenlenen transkript (POMC/CART) nöronları ve periferik sinyallerle sağlanmaktadır. Apelin bir adipokin olup kan glukozu düzenlenmesi gibi fizyolojik rolleri bulunmaktadır. Çalışmamızda, streptozotosin (STZ) ile indüklenmiş diyabet modeli oluşturulan transgenik farelerde sistemik apelin uygulamasının besin alımına, hiperglisemiye ve AgRP nöronlarıın aktivitesine olası etkilerini incelemeyi amaçladık. Bu amaçla, genç erişkin AgRP-Ires-Cre hayvanları kullandık. ARC hedeflenerek stereotaktik olarak AAV-CAG-Flex-GFP enjeksiyonunu takiben STZ ve/veya apelin uygulanarak hayvanların besin alımı, vücut ağırlığı ve kan şekerleri takip edildi. Dekaptiasyon gününde ELISA ölçümleri için serum toplandı ve beyin kesitleri alınarak AgRP nöronlarının elektrofizyolojik aktiviteleri incelendi. STZ uygulaması süresince besin alımı anlamlı şekilde azalırken STZ sonrası dönemde artmaya başladı. 50 mg/kg apelin uygulaması hayvanlarda besin alımını ve vücut ağırlığını STZ'den bağımsız olarak düşürdü. 100 mg/kg apelin uygulaması ve uzun dönem apelin sonrası takipte STZ'nin kesilmesinden sonra besin alımı anlamlı şekilde yükselmeye devam etti. Apelinin STZ kaynaklı kan şekeri yükselişine ve serum insülin düşüşüne bir etkisi gözlemlenmedi. STZ sonrası yükselen serum trigliserit seviyeleri 100 mg/kg apelin sonrasında anlamlı derecede düşmüştür. Apelin AgRP nöron aktivitesini değiştirmemekte olup STZ AgRP nöron aktivitesi anlamlı şekilde arttırmıştır. Apelin ise STZ kaynaklı AgRP nöronal aktivitesindeki artışı düşürme eğilimindedir. Bu sonuçlara göre, apelinin diyabet sebepli AgRP nöron aktivitesindeki artışa etki etmediği düşünülebilir.
Evaluation of hepatotoxic and nephrotoxic effects of hexachlorobenzene, DDT and p,p-DDE in rats
Persistent Organic Pollutants (POPs) pose a threat to the human health because of their long-lasting presence in the environment and bio-accumulative properties in organisms. There have been numerous reports on neurotoxic, carcinogenic, immunotoxin, hepatotoxic, nephrotoxic, cytotoxic, and endocrine disruptor effects of several persistent compounds. In the present study, three OCPs have been selected to be examined for their hepatotoxic and nephrotoxic effects in male rats. While the current literature reports on some indicators, no empirical findings were found for the effects of DDT and DDE on liver enzymes: ALT, AST and ALP. In the experiment, four groups of 8 adult male Sprague-Dawley rats were researched: Control, HCB (5 mg/ kg), p,p-DDE (5mg/ kg), and DDT (5 mg/ kg). Each of the experimental group rats received daily dosages for 18 days. Concentrations of liver and kidney indicators in serum were analyzed. The findings of the experiment shows that the majority of liver indicators: bilirubin (TBIL), cholesterol (CHOL), lactate dehydrogenase (LDH), triglycerides (TRIG), glucose (GLU), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were not significantly affected by the administration of OCP dosages among the DDE, DDT, and HCB groups based on the one-way ANOVA analysis at the 0.05 level. However, results from alkaline phosphatase (ALKP) indicated that its levels were significantly lower for the DDE and DDT group in comparison with the control group at the same significance level. The kidney indicators in the experiment: creatinine (CREA) and urea, were not significantly affected by the administration of OCP dosages among the DDE, DDT, and HCB groups based on the one-way ANOVA analysis at the 0.05 level. It is concluded that OCP dosage and length of exposure play major roles in toxicity levels. Keywords: Persistent Organic Pollutants (POPs); Liver, Kidney
Hipotalamik arkuat çekirdekte FAM163A proteininin ve mitokondriyal aktivitenin beslenme davranışı üzerine etkilerinin farelerde araştırılması
Mitokondriyal fonksiyonun nöronal aktivitede önemli rollere sahip olup hipotalamusun arkuat çekirdeğinde (ARC) bulunan ve beslenme davranışını düzenleyen aguti-ilişkili peptit (AgRP) ve proopiomelanokortin (POMC) nöronlarının aktivitelerinin düzenlenmesinde rol oynadığı bildirilmiştir. FAM163A yakın dönemde keşfedilen bir protein olmakla birlikte fonksiyonu ve hücre içindeki lokalizasyonu net değildir. Çalışmamızın amacı ARC'de Fam163a sessizleştirilmesinin ve mitokondriyal disfonksiyonun AgRP nöron aktivitesi ve mitokondriyal aktiviteye olan etkisini incelemektir. Fam163a sessizleştirilmesi, kısa dönemde kümülatif besin tüketimini arttırırken uzun dönemde kümülatif besin tüketimine etkisi olmamıştır. ARC'de Fam163a'nın sessizleştirilmesi lokomotor aktivite ve anksiyete benzeri davranışta anlamlı bir değişikliğe sebep olmazken, rotenon uygulaması açık alan testinde merkezde geçirilen süreyi anlamlı şekilde arttırmıştır (p<0,05). ARC'de Fam163a sessizleştirilmesi ve rotenon uygulaması AgRP aktivitesini anlamlı şekilde düşürmüştür (p<0,05). Kan glukoz ve serum insülin seviyeleri rotenon uygulaması ve Fam163a sessizleştirilmesinden etkilenmemiştir. Fam163a sessizleştirilmesi mitofaji, mitokondriyal sağlık ve mitokondriyal füzyon ilişkili genlerin ifadelerinde anlamlı değişiklikler gözlenmiştir (p<0,05). Benzer şekilde rotenon uygulaması da ARC'de mitokondri ilişkili transkriptlerin ifadesinde değişime sebep olmuştur. Çalışmamızın sonuçları FAM163A'nın hipotalamik mitokondriyal aktivitede ve mitokondriyal genlerin düzenlenmesinde rolleri olduğunu göstermektedir.
Kronik yüksek kalorili diyetin mediobazal hipotalamik tokluk sinir ağlarının işleyişine etkileri
Son 30 yıldır prevalansı giderek artan obesite kronik olarak yüksek kalorili beslenme ve buna bağlı olarak yağ dokusunun artışı ile ilişkilidir. Hipotalamik arkuat çekirdekte bulunan (ARC) proopiomelanokortin (POMC) nöronları, tokluk hissinin oluşmasında hayati bir rol oynar. Yüksek yağlı gıdaların kronik tüketiminin, leptin gibi hormonlara karşı nöronal duyarlılığı azalttığı, dolayısıyla obezitenin gelişmesine ve kalıcılığına katkıda bulunduğu bilinmektedir. Ancak kronik yüksek yağlı diyetlere (YYD) maruz kalan POMC nöronlarının morfolojik yapısı, elektrofizyolojik özellikleri ve enerji metabolizmasındaki rolü üzerindeki adaptasyonlar ve etkilerin kalıcı olup olmadığı anlaşılamamıştır. Bu tezde, kronik YYD'ye maruz kalan fare modellerinde ARC-POMC nöronlarının adaptasyonlarını araştırmak amaçlanmıştır. 6 aylık YYD'ye maruz kalan farelerde POMC nöronal projeksiyonları değişmezken, POMC nöronları leptine karşı elektrofizyolojik olarak geri döndürülemez direnç geliştirdi. Dahası, POMC aktivasyonu YYD tüketimini artırdı. Standart diyete geçişte gözlemlenen tek adaptasyon, homeostatik yeme davranışında tokluğu ortaya çıkarma rolünün yeniden sağlanmasıydı.
Investigation of effects of repeated hyperthermia on skeletal muscle and motor function in mice
This study explored the impact of repeated hyperthermia on mitochondrial function, proteostasis in mice, and consequent effects on motor and anxiety-like behaviors. Forty Swiss male mice were divided into five groups: Control Exposure (con-E), 39°C/15 minutes/7 days (39E), 41°C/15 minutes/7 days (41E), Control Exposure and Recovery (con-ER), 39°C/15 minutes/7 days with 7-days recovery (39ER), and 41°C/15 minutes/7 days with 7-days recovery (41ER). Post-exposure, the 41E group showed muscle weakness (p<0.05), and both 39E and 41E exhibited anxiety-like behavior on the EPM test (p<0.05). Gene analysis from gastrocnemius tissues demonstrated altered mRNA expression. Both 39E and 41E had reduced Pgc-1α levels (p<0.05). Pgc-1β expression notably differed, between 39E and 41ER (p<0.01), 39ER and 41ER (p<0.01), and 41ER (p<0.01). There was a significant difference in TFAM expression between 39E and 41ER (p<0.01). The 39ER group had increased NRF1 (p<0.05). PINK1, affected by temperature, differed between 39E and 41E (p<0.05). PARKIN showcased recovery effects between 41E and 41ER (p<0.01). Ucp2 levels contrasted between 39ER and 41ER (p<0.05). In summary, mild temperatures (39°C) suppressed autophagy, maintaining strength, and initiating the Nrf1-Tfam-Ucp2 pathway post-recovery whereas muscle strength decreased. Severe temperatures (41°C) caused mitochondrial dysfunction and muscle strength decline. Recovery induced increase in Pgc-1β modulated mitochondrial biogenesis and in Parkin indicates mitophagy. Moderate hyperthermia induced apparent anxiety-like behaviors in the acute phase, which subsided after the recovery period. Key words: Repeated Hyperthermia, Mitochondria, Skeletal Muscle, Motor Function
HSD3B2 gen mutasyonunun fonksiyonel analizi
HSD3B3 deficiency is rare form of the congenital adrenal hyperplasia arised from the inactivating mutation of HSD3B2 gene (3β-hydroxysteroid dehydrogenase). Even though it is not frequently seen in the world, the prevalence of the HSD3B2 deficiency is higher specifically in the Turkey. This disease is defined as defects in adrenal steroid synthesis in adrenal gland and gonads. Therefore, understanding and characterisation of the mutational area is very crucial. Also, the possible influence of the mutation can be changed depending on the position of the mutation in HSD3B2 gene. The aim of this study to determine in vitro characterisation of the HSD3B2 enzyme with p.L304P mutant and try to understand the relationship the molecular structure and clinical symptoms. Also, influence of the p.L304P mutation on HSD3B2 enzymatic activity and rate of 17 OH pregnenolone conversion was investigated. In the experiment, pCMV3-HSD3B2 vector was used and target p.L304P mutation was created with site directed mutagenesis. All plasmids were transformed into the DH5 alpha. The presence of the p.L304P mutation and wild type HSD3B2 gene were controlled by sanger sequencing. Later, the plasmids with wild type and mutant were transfected into the HEK293T cell line. After the transfection, the cells were treated with 17 hydroxypregnenolone that is dissolved in the DMEM. The treatment was done at different time interval and different substrate concentrations. At last step, the DMEM was removed and the amount of 17 hydroxyprogesterone was measured with LCMS/MS. In conclusion, our experimental finding showed that the mutation dramatically cause decline in 17 hydroxyprogesterone. Also, the amount of the 17 hydroxypregnenolone was detected higher in wildtype as compared with mutant cell lines.
Investigation of genotoxic changes in mammary epithelial cells and bioactivity of dioxins at reporter gene level in breast milk
Organochlorinated chemicals including dioxins and dioxin-like compounds threat environment and human health. They can easily contaminate living fields and bioaccumulate in adipose tissue in living organisms because of their chemical structures. Because these chemicals have toxic effect on several systems in the organism we considered that bioactivity of these substances should detect at reporter gene level using body fluid. Dioxins and dioxin-like compound can accumalated in breast milk which contain fat. So, human breast milk is one of the critical samples for both mother and baby in order to observe for dioxins. In this study, 50 milk samples were collected from lactating mothers living in İstanbul. Mothers were asked to fill a questionnaire form including personal, medical and nutritional information. Exfoliated epithelial cells were isolated from collected breast milk. Comet assay was used to determine DNA damage in epithelial cells. DR-CALUX assay was used to determine dioxin level in the breast milk. Dioxin level of 27 of samples was above the detection limit. It was observed that DNA damage were high at samples which include high dioxins. These results provide evidence that dioxins can cause genotoxicity in mammary epithelial cells. Furthermore, it was shown that CALUX assay can be used for human biomonitoring and also the human breast milk is very suitable biological sample for this purpose.
The investigation of relationship between oro-gustatory perception of dietary lipids and spontaneous fat preference with maternal nutrition status in offspring Sprague Dawley rats
Recent studies have shown that lingual fatty acid receptors CD36 and GPR120 play an important role in the perception of fat taste and the preference for fatty food consumption. However, the relationship between these receptors and maternal nutrition is not known. The aim of this study was to investigate the change in the expression levels of lingual CD36 and GPR120 proteins in rats exposed to different concentrations of fat containing diets during gestation-lactation and maturation periods and to determine the possible effect of exposure of different fat-containing diets to the regulation of these receptors. In our study, rats were fed with low-fat, standard-fat and high-fat diets during the first stage of the study (gestation and lactation) and in the second stage of study (maturation). Daily caloric intake and weekly weight measurements of the rats were followed during the study. Afterwards, the fat perceptions and oily taste preferences of the rats were measured by two-bottle preference tests, blood and tongue tissues were taken and the experiment was terminated. In order to demonstrate the effect of nutritional status on the appetite regulation, the plasma insulin and leptin levels were measured by ELISA and blood glucose and triglyceride levels were measured by a metabolic meter. Expression levels of CD36 and GPR120 receptors on the tongue were determined by immunofluorescence staining and western blot methods. mRNA levels of these proteins were determined by RT-PCR. Our study shows that maternal high fat diet exposure increases the higher fatty solution preference and decreases lingual CD36 and GPR120 expression levels. These findings allow us to identify the dysregulation of the oro-sensorial signalling pathways through the diet in the maternal process and will contribute to the development of new anti-obesity strategies, especially for childhood obesity. The thesis student was supported with 2228-B program within the scope of Tubitak-Bideb 2211 Domestic PhD Scholarship Program. In addition, this project was supported by TUBITAK (117S384).
Investigation of the relationship cognitive functions, sensory and motor behavior, and extracellular matrix proteins in mice on the high-fat diet
Obesity is a worldwide health problem, and many people suffer from obesity and related diseases. Eating habits play an important role in developing obesity, and previous studies demonstrate that low-quality diets have adverse effects on the brain and behavior such as memory loss or impairment in the appetite and satiety mechanism. The effects of different diets have been investigated widely in the cellular mechanisms of the brain, and diets contained a high amount of fat damage to the cellular homeostasis and synaptic plasticity in the brain. However, how the extracellular matrix is modulated by a high-fat diet has not been evaluated enough. In this study, we assessed how lard based high-fat diet affects extracellular matrix proteins and the behavior of mice. For this purpose, male mice fed by the high-fat diet for 8 weeks, and then the behaviors of mice were assessed by the elevated plus-maze, light-dark box test, open field test, novel object recognition test, and rotarod. Then in different regions of the brain, the protein level of Reelin, tenascin C, and neuroligin 2 was evaluated with western blot, and the number of perineuronal nets positive somas was assessed by immunofluorescence staining. Our results reveal that 8 weeks cause the tendency of decrease in memory, and difficulties in learning of mice, and decrease performance in involuntary motor function. Also, changes in the protein amounts and perineuronal nets showed regional differences, and these differences are mostly related to synaptic plasticity and long-term potentiation. In conclusion, the high-fat diet may alter the behavior of mice by affecting the extracellular matrix structure that provides the proper function of the synaptic plasticity.
The role of chat in arcuate nucleus on anxiety behaviour
Anxiety and stress are extremely crucial for the consistency of survival under the conditions of wild nature. For human beings, the level of stress becomes deeper and could be observed in every aspect of our daily lives. Impairments of the mechanism which controls this system lead to anxiety disorder and depression that affect the lives of more than 300 million people worldwide. The stress suppressive effect of nicotine by causing an addiction commonly in everyday life became a focus of interest for researchers. The pathways that nicotine stimulates are physiologically stimulated by the neurotransmitter acetylcholine. The mechanism of anxiety, insomnia, muscle tension, careful reduction, and forgetfulness, which are came along with anxiety disorder and depression, are associated with acetylcholine. The development of effective treatment for these conditions is possible with a correct understanding of the connected systems. Chemogenetic activation technique allows us to target cholinergic neurons of ChAT-IRES-Cre transgenic mice. The aim of this study is chemogenetic activation of cholinergic neurons in the arcuate nucleus in order to understand the role of it in the anxiety behavior. Elevated plus test, open field test, and light-dark box tests were performed. The determined stress factor for those test are; the height, the open and unprotected area, and the over-lightened area. Spent time at the areas related with stress factors was associated with anxiolytic effects and it was shown that the cholinergic neuron group in the arcuate nucleus has an effect of tolerance enhancement of the stress; however, does not show any effect on locomotor activity.
Investigation of the effect of hexagonal boron nitrides (hBNs) on mouse hippocampal cell line
It is a challenge to treat a neurological disease and brain cancer not only due to poor understanding of their molecular basis but also delivery of therapeutic drugs. In this thesis, it is aimed to understand the potential of hBNs as a therapetic agent to relieve cellular stress. First, the cytotoxicity of hBNs and their possible degradation product, boric acid (BA), on Embryonic Mouse Hippocampal Cell Line (mHippoE-14) was assessed in the concentration range of 10-1000 μg/mL for both materials for 24 h and 72 h exposure. It was found that both hBNs and BA showed concentration and time dependent cytotoxicity while hBNs were showing less cytotoxic behavior. Further; cell death mechanism, reactive oxygen species (ROS) generation, cell cycle, and apoptotic body formation were investigated using hBNs and BA concentrations with 4.4, 22, and 44 μg/mL boron content. hBNs drove the cells to apoptosis with the lowest concentration at 24 h exposure and with the increased exposure time the higher concentrations caused apoptosis as well. For BA, an increase in necrosis was observed at lowest two concentrations with 72 h of exposure. BA drove cells to apoptosis starting from the lowest concentration with both exposure times. Both hBNs and BA caused increased ROS production with the increased concentration and exposure times but the increase was more significant with BA exposure. 24 h exposure of hBNs and BA had no significant effect on cell cycle while 72 h exposure caused significant changes on cell cycle, especially on G0-G1 phase. Apoptotic bodies were not observed with any hBNs and BA concentrations and exposure times. The study was further expanded by exposing the cells to doxorubicin (DOX) to cause a stress on cells, and then were treated with hBNs and BA concentrations containing 4.4, 22 and 44 μg/mL boron and were exposed for 24 h and 72 h. It was found that both hBNs and BA increased cell viability when exposed for 72 h. This study suggests that hBNs and BA have very low toxicity and could be used as therapeutic agents at low concentrations to decrease oxidative stress caused by various drugs used for treatment of neurological diseases and brain cancer. However, since hBNs slowly degrade in biological medium, they can be used as a controlled releasing agent as compared to ionic BA in addition to their nanocarrier feature.