Development of Single and Multimodality Imaging Probes for PET, SPECT and Fluorescence Imaging

This dissertation is in an integrated format discussing three major projects centered around probe development. In the first project, novel metal-chelated and fluorinated GLP-1 derivatives were prepared all containing D-Ala-8, a modification known to improve resistance towards degradation by dipeptidyl-peptidase IV. The effect of increased distance between DOTA and the peptide chain was investigated using a spacer, in order to reduce steric effects imposed by DOTA. Placement of linker and DOTA moieties were varied within the GLP-1 sequence to test for optimal metal-complex location. Binding affinity of peptide derivatives was determined in vitro with CHO/GLP-1R cell line and shown to be in the nM range. In vivo imaging was carried out using C57BL/6 mice. Modifications made to the peptide backbone, based on charge distribution, were shown to improve pharmacokinetics. Our results suggest developed GLP-1 tracers to be potential candidates for the non-invasive imaging of pancreatic islets in vivo. In the second project, Gallium-chelated protoporphyrin IX (PPIX) derivatives containing the tripeptide RGD αvβ3-targeting moiety were prepared and evaluated. Reaction conditions for both naturally occurring 69/71Ga-labeling as well as 68Ga-radiolabeling were optimized using a microwave reactor. Optical properties were evaluated in order to study the effects of ligand conjugation and gallium chelation on absorption/emission properties of PPIX. Quantum yields pre- and post gallium chelation were comparable, indicating that the presence of gallium does not quench the inherent fluorescence of PPIX. The targeted compound was preferentially taken up by αvβ3 integrin-expressing cancer cells versus the non-targeted form, as assessed by fluorescence microscopy. Our results suggest that gallium-PPIX conjugates could be used as dual modality positron emission tomography/fluorescence microscopy probes and can assist in bridging imaging agent discovery from in vitro microscopy through to in vivo imaging. In the third and final project, a novel high-throughput cell-based technique was developed for screening one-bead-one-compound (OBOC) libraries. In order to validate this technique, a small library consisting of heptameric peptides, and the αvβ3 integrins targeting RGD sequence, was synthesized on Tentagel beads via a photo-labile linker. Compounds were screened against the αvβ3-expressing MD 435 cell line. After fluorescent-based sorting, peptides were photolytically cleaved off resin and analyzed using MALDI-TOF/TOF. Deconvolution of all peptide sequences was carried out successfully indicating this screening process to be a facile and efficient technique for discovery of novel targeting entities

Molecular Genetics of Bartter Syndrome

Bartter syndrome (BS) is a heterogeneous disorder, caused by mutations in several genes which mostly encode proteins involved in ions transportation across renal cells in the thick ascending limb of the nephron. It is characterized by deficient renal reabsorption of sodium and chloride, which results in a group of certain symptoms. Different types of BS can be distinguished from different clinical manifestations, and most importantly, via analyzing possible affected gene(s) for its confirmation. A close associated syndrome which was primarily considered as a mild variant of BS, Gitelman syndrome (GS), is characterized by hypokalemic metabolic alkalosis with hypocalciuria, and hypomagnesemia. In this review, we discuss different features of BS and also GS, including clinical and genetic alterations which correspond to each type.  Keywords: Bartter Syndrome; Molecular Genetics; Child

Genetic Study of Nephrotic Syndrome in Iranian Children- Systematic Review

Idiopathic nephrotic syndrome is a heterogeneous disease with a spectrum of age at presentation, phenotype, renal pathology, and response to treatment. Many mutations are recognized to be implicated in sporadic or hereditary forms. The aim of this review was to summarize the results of the genetic studies which have already been carried out in Iran considering their limitations.A literature search was conducted from March 1970 to September 2015 through MEDLINE, EMBASE, Google Scholar, Google, Iran Medex, Magiran, and SID. Eleven studies were relevant. Three articles were excluded due to insufficient data, duplicated case, and a syndromic nephrotic case without genetic studies. Our results showed that in the southwest of Iran, 80% of the patients had mutations in NPHS1 while in Fars Province, one third showed mutations in NPHS2 when all exons were assessed. In two different studies conducted in one center in Tehran, no mutation was detected in exon 5 but when all exons were studied, more than 65% had hot spot mutation in exon 8 of NPHS2. Interestingly, none of adolescents with FSGS showed mutation in p.R229Q (NPHS2, exon 5). This review revealed that both NPHS1 and NPHS2 were prevalent in Iranian children with SRNS. No mutation of p.R229Q was reported in Iranian adolescent with SRNS.Keywords: Nephrin; NPHS2 protein; Nephrotic Syndrome; Glomerulosclerosis; Focal Segmental

COMPARISON OF SERUM VITAMIN A LEVELS BETWEEN NEONATES WITH CONGENITAL HEART DISEASE AND CONTROLS

Objective: Prevention of congenital heart disease (CHD) has been hampered by a lack of information about the known modifiable risk factors for abnormalities in cardiac development. Vitamin A plays an important role in the periods of rapid cellular growth and differentiation, especially during pregnancy. Assuming a link between Vitamin A levels and congenital malformations, hypothetical different levels of Vitamin A were evaluated in neonates with and without CHD, in this study.Methods: In a case–control study that was conducted in 2015 in Mashhad/Iran, serum levels of Vitamin A in 30 neonates with CHD were compared to 30 healthy controls. The cases were diagnosed by echocardiography and recruited by convenience sampling. Demographic and laboratory data including age, sex, and serum Vitamin A level in each group were collected. Data analysis was done in SPSS V 20 software, and descriptive statistics, t-test, and analysis of covariance were used.Results: The mean age in cases was 11±3.4 days and in controls was 12.5±4.8 days. A total of 18 patients (60%) were male. In CHD patients, 10 cases (33.3%) had cyanotic heart disease, and 20 cases (66.7%) had non-cyanotic heart disease. The mean serum Vitamin A values in subjects (11.54±9.56 μg/dL) and controls (21.84±14.3 μg/dL) were significantly different, (p<0.05) and in case group was lower than the normal range.Conclusion: There was a significant difference in serum Vitamin A values in subjects and controls. Therefore, awareness of people about the importance of this vitamin in preventing CHD in children seems necessary

Report of a novel mutation in Rb1 gene from an Iranian retinoblastoma patient and its effect on splicing pattern of mRNA

زمینه و هدف: رتینوبلاستوما شایع ترین تومور جامد درون چشمی در کودکان زیر شش سال است. جهش در هر دو نسخه ژن Retinoblastoma1 (RB1) مسئول شکل گیری این بیماری می باشد. طیف وسیعی از جهش ها تاکنون در سرتاسر ژن RB1 گزارش شده است. بسیاری از جهش های نقطه ای گزارش شده در ژن های انسان، علی رغم نوع آنها، وضعیت پیرایش را دستخوش تغییر می کنند. در این مطالعه جهش جدید در ژن RB1 در یک بیمار مبتلا به رتینوبلاستوما معرفی و تاثیر آن روی پیرایش mRNA بیان می شود. گزارش مورد: در مطالعه حاضر، آنالیز جهش ژن RB1 بر روی یک بیمار ایرانی مبتلا به فرم تک گیر و یک طرفه رتینوبلاستوما (دختر بچه 4 ساله) با استفاده از تعیین توالی نواحی کد کننده و همچنین Multiplex Ligation dependent of Probe Amplification (MLPA) انجام گرفته است. در ادامه با استفاده از روش RT-PCR وضعیت پیرایش mRNA ژن RB1 نیز مورد بررسی قرار گرفت. در نتیجه این بررسی ها یک جهش هم معنی (g.70320C>T) در نزدیکی انتهای اگزون 12 شناسایی شد. این تغییر نوکلئوتیدی، توالی مورد توافق یک عنصر افزاینده پیرایش، که محل اتصال پروتئین SC-35 می باشد را از بین می برد. بررسی ساختاری cDNA این بیمار نشان دهنده اختلال در فرایند پیرایش و حذف اگزون 12 از رونوشت بالغ ژن RB1 است. نتیجه گیری: بر اساس یافته های این مطالعه می توان تغییر هم معنی فوق را تحت عنوان یک جهش پاتوژن جدید در نظر گرفت، همچنین برای اولین بار وجود یک توالی مورد توافق افزاینده پیرایش، در اگزون 12 ژن RB1 گزارش می شود

A rare case of Sjogren-Larsson syndrome with recurrent pneumonia and asthma

Sjogren-Larsson syndrome (SLS) is a rare autosomal recessive neurocutaneous disorder with worldwide incidence of 0.4 per 100,000 people. It is characterized by the triad of congenital ichthyosis, spastic diplegia or quadriplegia, and mental retardation. Herein we report a 2-year-old male child with SLS, asthma, and recurrent pneumonia. SLS was confirmed by a molecular genetics study that revealed a deletion mutation in the ALDH3A2 gene. An ALDH3A2 gene mutation results in dysfunction of the microsomal enzyme fatty aldehyde dehydrogenase and impaired metabolism and accumulation of leukotriene B4, which is a key molecule and a pro-inflammatory mediator in developing allergic diseases, especially asthma. An increased level of leukotriene B4 has been reported in SLS patients. As far as we are aware, this is the first report of SLS associated with asthma and recurrent pneumonia. In conclusion, pediatricians should be aware of and evaluate patients with SLS for possible associated asthma and allergic disorders

KINETICS MODELING AND ISOTHERMS FOR ADSORPTION OF NITRATE FROM AQUEOUS SOLUTION BY WHEAT STRAW

Nitrate is a colorless, odorless chemical substance with a chemical formulation of NO3- and average mass of 62.0049 gr/ Mol. According to an announcement of the world health organization (WHO), the standard amount of Nitrate in potable water is at most 50 ml/ lit (based on nitrate). Nitrate enters into the body and is transformed to nitrite by digestive system’s bacteria, then enters to the circulatory system and oxides the exiting iron in Hemoglobin of blood which converts the iron capacity from 2 to 3. As a result of this process Hemoglobin is converted to Methemoglobin which has far more less capacity in oxygen delivery. Therefore, the tissues cannot receive sufficient oxygen and it causes a disease called “Methemoglobinemia”. The objective of this study was to investigate the nitrate removal using wheat straw and determining the adsorption isotherms and kinetics. In this study, nitrate solutions were prepared from potassium nitrate salt. The pH values of the solutions were adjusted by NaOH and HCl at a concentration of 0.1 molar. The pH of the solution was adjusted to different values (4 to 13). Kinetics models of Ho et al and Lagergren were used to describe the data. Isotherm models of Langmuir and Freundlich were used to describe the data. The results showed that the maximum capacity of wheat straw in nitrate adsorption occurred at pH=6 and contact time 140 minutes. Equilibrium models (Langmuir and Freundlich) and non-equilibrium (Ho et al and Lagergren) were used to investigate the adsorption process. Comparing the determination coefficients between measured data and obtained value from Ho’s model (R2= 0.97) and Lagergren model (R2= 0.91) showed that the Ho’s model describes experimental data better. Also, comparing the Langmuir and Freundlich isotherm for nitrate adsorption by wheat straw showed that Freundlich isotherm (R2= 0.98) was more proper than Langmuir isotherm (R2= 0.83) in describing adsorption process

The effect of intrathecal delivery of bone marrow stromal cells on hippocampal neurons in rat model of Alzheimer's disease.

OBJECTIVES: Intracerebral injection of bone marrow stromal cells (BMSCs) is being investigated as a therapeutic tool to prevent Alzheimer's disease (AD). Our aim was to investigate the effects of BMSCs by intrathecal injection in AD rat model. MATERIALS AND METHODS: BMSCs were obtained from the bone marrow of Wistar rat and transplanted into AD rat model via intrathecal injection. The rat model had received an injection of β amyloid into the hippocampus for histological and immunohistochemical studies. RESULTS: Histological examination of the brains in transplanted rats compared to controls demonstrated the migration of BrdU-labeled BMSCs from the site of delivery, confirmed the differentiation of BMSCs transplanted cells into the cholinergic neurons, and increased number of healthy and decreased number of dark neurons. CONCLUSION: Our results showed that BMSCs intratechal administration could be a promising method for treatment of Alzheimer's disease in rat model

Retinitis Pigmentosa GTPase Regulator (RPGR) protein isoforms in mammalian retina:insights into X-linked Retinitis Pigmentosa and associated ciliopathies

AbstractMutations in the cilia-centrosomal protein Retinitis Pigmentosa GTPase Regulator (RPGR) are a frequent cause of retinal degeneration. The RPGR gene undergoes complex alternative splicing and encodes multiple protein isoforms. To elucidate the function of major RPGR isoforms (RPGR1–19 and RPGRORF15), we have generated isoform-specific antibodies and examined their expression and localization in the retina. Using sucrose-gradient centrifugation, immunofluorescence and co-immunoprecipitation methods, we show that RPGR isoforms localize to distinct sub-cellular compartments in mammalian photoreceptors and associate with a number of cilia-centrosomal proteins. The RCC1-like domain of RPGR, which is present in all major RPGR isoforms, is sufficient to target it to the cilia and centrosomes in cultured cells. Our findings indicate that multiple isotypes of RPGR may perform overlapping yet somewhat distinct transport-related functions in photoreceptors