PhD Thesis

Isolation and characterization of bioactive metabolites from two ethno pharmacologically important Zingiberaceae plants of Bangladesh and their endophytic fungi

2026-08-04T00:00:00Z

Isolation and characterization of bioactive metabolites from two ethno pharmacologically important Zingiberaceae plants of Bangladesh and their endophytic fungi Ferdous, Kazi Jannatul The work explained in this thesis elaborates the isolation and identification of endophytic fungi from two ethnopharmacologically significant Zingiberaceae plants of Bangladesh, namely Curcuma longa L. and Zingiber officinale Rosc. The term “ethnophamacologically” means that these kinds of plants are traditionally used by specific groups of people for medicine purpose from very ancient period. A detail description was given for chemical screening of the crude extracts of the plants as well as their endophytic fungi. Isolation of secondary metabolites was done from the extracts of plants and their endophytic fungi by a combination of repeated column chromatography and preparative thin layer chromatography (PTLC). Structure clarification of pure compounds was made by their spectral data (GC-MS, 1D and 2D NMR). Finally bioassays of different extracts and isolated pure compounds were performed to observe antioxidant, antimicrobial and cytotoxic effect. The antioxidant activity was measured by the DPPH free radical scavenging method and antimicrobial activity was performed by the disc diffusion method. The cytotoxic activity of various extracts was completed by brine shrimp lethality bioassay. But the pure isolated compounds’ cytotoxic activity was analyzed on a variety of cell line cultures, for example, Lung cancer cell line A-549, kidney fibroblast cell line BHK-21, human cervical carcinoma cell line (HeLa). A network pharmacology-based approach in combination with molecular docking analysis was also used to identify a compound’s multiple targets that may have linked to its pharmacological action. A total of seven endophytic fungi were isolated from C. longa and, a total of five endophytic fungi were isolated from Z. officinale. All the endophytes were segregated from diverse parts of the plants (petiole, leaf, root, bark). Through morphological and molecular analysis it was revealed that among twelve endophytic fungi, eight are Fusarium species, three are Clonostacys species, and one is cladosporium species. All crude extracts contain flavonoids, coumarins, anthrocyanins, isocoumarins or their derivatives types of compounds which was revealed through chemical screening by thin layer chromatography (TLC). The crude methanolic extract of C. longa showed significant antioxidant (IC50 value 19.85 μg/ml) and cytotoxic activity (LC50 value1.81 μg/ml) compared to their standards. Its vaccuum liquid chromatography (VLC) fractions also showed significant antioxidant and cytotoxic activity. In case of Z. officinale, the plant methanolic crude extracts showed very significant antioxidant activity (IC50 value 2.08 viii μg/ml) and mild to moderate antibacterial activity (zone of inhibition 7-10 mm). Its fractions obtained through the modified Kupchan partition method also showed good antioxidant and antibacterial activity. The obtained results prove the phytochemical as well as pharmacological significance of these plants. A total of 27 metabolites and 21 metabolites were discovered through GC-MS analysis of crude methanolic extracts of C. longa and Z. officinale respectively. Most of them are bioactive constituents. Two compounds have been isolated from the plant C. longa, which were characterized as 4-hydroxy-3-methoxy cinnamic acid (CL-1) and methyl ferulate (CL-4). Among seven endophytic fungi isolated from this plant, CLRE-3 (Clonostachys rosea) was selected for large-scale cultivation based on chemical and biological investigation. From its ethyl acetate extract four compounds were isolated and characterized as Bassiatin (CLE-7), Nectriafurone (CLE-14), 8-O-methyl nectiafurone (CLE-11) and 8-O-methyl bostrycoidin (CLE-18)). Bassiatin, 8-O-methyl nectiafurone and 8-O-methyl bostrycoidin were obtained from this endophytic fungus for the first time. Among five endophytic fungi isolated from Z. officinale ZOLE-2 (Cladosporium cladosporoides) was selected for large-scale cultivation by observing its chemical and biological analysis. From its ethyl acetate extract two compounds were isolated and characterized as ergosterol (ZOE-1) and 3, 5, dihydroxy-ergosta-7, 22-diene-6-one (ZOE-6). Following biological investigation it was revealed that almost all isolated compounds were bioactive. CLE-18(2) and ZOE-6 indicated mild to moderate antibacterial activity against E. coli, P. aeruginosa and S. aureus bacteria with a zone of inhibition of 10 to 16 mm. CL-4 and CLE-7 showed moderate antibacterial activity against E. coli bacteria with a zone of inhibition of 10 mm. CL-1 showed noteworthy antioxidant activity with IC50 value 4.77 μg/ml in comparison with standards ASA (IC50 value 9.01 μg/ml) and BHA (IC50 value 11.42 μg/ml). It also exhibited cytotoxic activity against A-549 cancer cell line which was done by the trypan blue exclusion method. CLE-14 showed cytotoxic activity against BHK-21 cell line with IC50 value of 663.4μg/ml compared to standard doxorubicin (IC50 value of 327.1 μg/ml). It did not exhibit any activity against the HeLa cell line. CLE-7 also displayed cytotoxic activity against the A-549 cancer cell line. A computational study was done against Bassiatin (CLE-7) and lung cancer. Thus MMP9, PTGS2 and ERBB2 were unveiled as the top three common targets between the disease and compound. CLE-7 targets these proteins with fairly good binding score compared to ix standards based on molecular docking analysis. Thus it can be considered as a potential candidate for lung cancer treatment. The isolation of endophytic fungi and their chemical constituents from these plants have been revealed for the first time in Bangladesh through this investigation. From this study it is clear that these plants and their endophytic fungi would be a good resource of reference in the scientific world because of possessing various types of bioactive compounds. This analysis meets up the overall aims and objectives of the study for isolation and characterization of bioactive metabolites which can be used as a scaffold for innovative medicinal chemistry amendment to build up new drugs. This thesis is submitted for the degree of Doctor of Philosophy

Genotypic and Biochemical Investigations in Bangladeshi Patients with Papillary Thyroid Carcinoma

2026-08-03T00:00:00Z

Genotypic and Biochemical Investigations in Bangladeshi Patients with Papillary Thyroid Carcinoma Islam, MD. Ariful Background Papillary thyroid cancer (PTC) is the most frequent type of thyroid carcinoma, and exhibits significant molecular and biochemical heterogeneity both within and between populations. It is indolent in nature, but both its diagnosis and prognosis are difficult to predict given its multiple oncogenic pathways and hormonal derangements. This study explores novel somatic variants and their biochemical correlates in a Bangladeshi PTC cohort, aiming to improve risk assessment and promote personalized cancer treatment in underrepresented populations. Methods A cross-sectional comparative study was conducted involving 110 patients with PTC, 100 individuals with benign thyroid conditions, and 100 healthy controls. Targeted next-generation sequencing (NGS) and Sanger validation were employed to identify hotspot and novel intronic variants in NRAS, RB1, HRAS, and RET genes. Concurrently, biochemical profiling included serum thyroid-stimulating hormone (TSH), parathyroid hormone (PTH), calcium, vitamin D, and thyroglobulin (Tg), with correlations analyzed against genotypic status and clinical features. Results Three novel variants -NRAS g.7775T>A, NRAS g.7797G>A, and RB1 c.2039T>A (p.Ile680Asn)—were identified, showing significant enrichment among mutation-positive PTC patients (p < 0.0001), particularly in non-smoking, non-familial cases, suggesting a sporadic mutational origin. The NRAS g.7797G>A variant was associated with early-stage tumors (T1). In contrast, the RB1 c.2039T>A variant was observed in both early and advanced stages, supporting their involvement in early tumorigenesis. Biochemically, PTC patients exhibited markedly elevated TSH levels and reduced serum calcium (p = 0.0305) compared to benign and healthy controls. Individuals carrying the mutation demonstrated more pronounced hypocalcemia and a significant positive correlation between calcium and vitamin D (r = 0.3955, p = 0.0455), a pattern not observed in wild-type carriers. No xxv significant associations were found for Tg or PTH, possibly reflecting heterogeneous expression or unmeasured confounders, such as anti-Tg antibodies. These findings suggest genotype-specific disruptions in calcium–vitamin D–TSH homeostasis and highlight a potential endocrine–genetic crosstalk in PTC pathophysiology. Conclusion The current study provides the first integrative molecular–biochemical profile of Bangladeshi PTC patients, identifying novel NRAS and RB1 variants with potential as early detection and intermediate-risk biomarkers. The observed endocrine dysregulation in mutation carriers underscores the interplay between genetic alterations and systemic metabolism. These findings highlight the clinical utility of combining genotypic screening with metabolic markers for cost-effective, precision-guided management of PTC, especially in resource-limited settings. Future studies should focus on functional validation of these variants and the development of region-specific diagnostic and prognostic frameworks. This thesis is submitted for the degree of Doctor of Philosophy.

Identification and characterization of genes from wild halophytic rice (Porteresia coarctata), for using in development of salt tolerant rice

2026-04-19T00:00:00Z

Identification and characterization of genes from wild halophytic rice (Porteresia coarctata), for using in development of salt tolerant rice Habiba, Most Umme Soil salinity is a major abiotic constraint affecting rice cultivation in coastal and estuarine regions, where rising sea levels and irrigation-induced salinization increasingly threaten global food security. The halophytic wild rice species Oryza coarctata—the only naturally salt-tolerant species in the genus Oryza, which can set rice-like grains—offers a promising genetic reservoir for improving salt stress tolerance in cultivated rice (Oryza sativa). This study presents a comprehensive functional and molecular characterization of O. coarctata, along with its application in wide hybridization and genetic engineering strategies aimed at enhancing salt tolerance in rice. Oryza coarctata exhibits exceptional salt tolerance supported by unique physiological and anatomical features. Its leaf structure includes deep adaxial invaginations, multiple vascular bundles per ridge, and salt-secreting hairs, while its roots possess a thickened exodermis, large xylem vessel, and well-developed aerenchyma—traits that aid in ionic regulation and survival in saline, waterlogged conditions. Notably, O. coarctata reduced electrical conductivity (EC) of saline hydroponic media by 2.77–8.51 dS/m across 100–300 mM NaCl, demonstrating a desalination ability absent in salt-sensitive rice varieties. Co-cultivation of O. coarctata with BRRI Dhan28 improved the latter’s growth and yield under 100 mM salt stress, increasing yields from 34% to 67%. Gas exchange measurements in O. coarctata showed only modest declines in photosynthesis at 100–300 mM NaCl (14–26% reduction in CO₂ assimilation), with strong light response (r = 0.932) and moderate Jₘₐₓ reduction (19–32%, p < 0.05). Unlike O. sativa, which fails at 80 mM salt, O. coarctata maintains high photosynthetic efficiency and survival under extreme salinity. These traits underscore its potential for use in salt-affected rice ecosystems through ecological facilitation and genetic improvement strategies. To explore gene transfer from the tetraploid O. coarctata (4n=2x=48) into rice, a wide hybridization approach was adopted using a tetraploid O. sativa (var. Latisail 4n) as the maternal parent. This approach is referred to as the bulbosum technique, where chromosomal loss occurs resulting in half of the original chromosomes (2x). Despite high genomic divergence, two partial 18 hybrids were recovered out of 1,191 pollinated spikelets, indicating successful albeit low-frequency gene introgression. These partial hybrids exhibited intermediate phenotypes, including fibrous and tap root systems, variable seed morphology, and in some cases, the absence of leaf midribs—a signature trait of O. coarctata. Molecular analysis confirmed that the hybrids carried specific chromosomal segments from O. coarctata, particularly on chromosomes 3 and 12. Partial hybrid lines A2-06-01, A2-10-01, and B2-03-01 exhibited significantly higher chlorophyll content than diploid and tetraploid O. sativa following 100 mM salt stress. Lines B-02-01 and B-03-01 showed significantly greater plant height compared to O. sativa (2n), while A2-06-01 and A2-10-01 also demonstrated a significantly higher tiller number than O. sativa (2n). A significant advancement of this work was the development of O. coarctata-specific SSR markers to facilitate molecular screening of hybrids lines. Initially, known SSR markers from the Gramene database showed limited polymorphism between O. sativa and O. coarctata. Subsequently, 90 markers from the O. officinalis genome (CC genome), developed under the OMAP project, were tested. Of these, 19 markers were optimized to uniquely detect O. coarctata alleles across most chromosomes (except 5 and 8). In addition, seven new SSR markers were developed from O. coarctata genomic sequences. These 26 markers together form a robust toolkit for identifying and validating introgressions in future breeding programs. To directly assess the functional contribution of O. coarctata genes, three candidate genes—OcAsr1 (abscisic acid stress ripening protein), OcPVA1 (vacuolar H⁺-ATPase subunit c), and OcMT3 (metallothionein type 3)—were cloned and overexpressed in O. sativa using in planta Agrobacterium-mediated transformation method. This non-tissue culture-based transformation system enabled the successful generation of transgenic lines in the high-yielding indica background BRRI Dhan75 (for OcAsr1and OcPVA1) and BRRI Dhan67 (for OcMT3). Gene expression profiling revealed distinct stress-inducible expression patterns of the genes in O. coarctata: OcAsr1 peaked at 24 hours under 200 mM NaCl, OcPVA1 showed strong and consistent expression across both 100 and 200 mM NaCl, and OcMT3 exhibited a late response, peaking at 48 hours. Transgenic lines overexpressing OcAsr1 (notably P_73_2, P_70_1, and P_76_2) displayed enhanced root and shoot biomass, chlorophyll retention, and a 30–50% reduction in root and shoot Na⁺/K⁺ ratios. These lines maintained 40–50% higher grain yield under 100 mM NaCl 19 and exhibited minimal yield penalty under normal conditions. OcPVA1-expressing lines (MUH_113, MUH_117, MUH_99) demonstrated strong early responses, enhanced membrane stability, improved ionic balance, and significant grain yield retention under salinity. These results support its role in ion transport and vacuolar compartmentalization of sodium ions. Transgenic lines expressing OcMT3 (P-14-1, P-13-2) showed reduced oxidative damage (as measured by electrolyte leakage and histochemical staining), improved Na⁺/K⁺ balance, and stable yield under stress, highlighting OcMT3's role in ROS detoxification and metal ion sequestration. Importantly, all three gene constructs did not compromise yield under non-saline conditions, underscoring their suitability for future breeding or biotechnological applications. This study highlights the remarkable salt tolerance of Oryza coarctata and its potential to enhance salinity resilience in cultivated rice through wide hybridization and genetic engineering. Functional traits, molecular markers, and transgenic lines expressing O. coarctata genes demonstrated improved growth, yield, and stress tolerance under saline conditions, offering promising avenues for climate-resilient rice breeding. This thesis is submitted for the degree of Doctor of Philosophy.

High Density Lipoprotein in Bangladeshi Adults: Characterization and the Basis of its Variation

2026-03-02T00:00:00Z

High Density Lipoprotein in Bangladeshi Adults: Characterization and the Basis of its Variation Saiedullah, Muhammad Background and objectives: High-density lipoprotein cholesterol (HDL-c) constitutes a vital cardioprotective factor, yet low levels are prevalent among the Bangladeshi population, and its biochemical and genetic determinants remain poorly characterized. This study aimed to determine the prevalence of low HDL-c and associated components of dyslipidemia, along with their demographic and biochemical factors, among healthy Bangladeshi adults. The main focus of the study was to determine the effect of twelve single nucleotide polymorphisms (SNPs) in genes involved in lipid dynamics, i.e., ApoA1 (–75 G/A and +83 C/T), ApoB (7673C/T [rs693], 10108A/G [rs1801701], 12669G/A [rs1042031]), ABCA1 (–565C/T, 1051G/A, 2868G/A), PON1 (163T/A [rs854560, L55M] and 575A/G [rs662, Q192R]), and CETP (–629C/A, 277C/T [Taq1B]) was explored. Methodology: This cross-sectional study recruited 409 healthy adults from different areas of the Dhaka division. Participants were free from diabetes, hypertension, kidney, liver, or other chronic diseases. After obtaining informed consent, demographic measurements and clinical histories were recorded. Fasting blood samples (5 mL) were collected following aseptic procedures and processed for biochemical analyses using automated spectrophotometric instruments. Measurements of serum lipids, specifically total cholesterol (TC), triglycerides (TG), and HDL-c, were conducted by spectrophotometric end-point methods. Low-density lipoprotein cholesterol (LDL-c) was subsequently derived via the Friedewald formula. Additionally, Apolipoprotein A1 (ApoA1) and Apolipoprotein B (ApoB) concentrations in serum were quantified using immunoturbidimetry on an automated platform. A column-based genomic DNA extraction kit was used to extract DNA from homogenized whole blood leukocytes, and genotyped SNPs in the ApoA1, ApoB, ABCA1, PON1, and CETP genes using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) followed by agarose-gel electrophoresis. We categorized the participants based on median HDL-c levels or according to reference values into lower HDL-c and higher HDL-c groups following NCEPATPIII guidelines for statistical analysis. Logistic or multiple linear regressions and Fisher’s exact tests were employed to assess associations between genotypes, allele frequencies, lipid profiles, and demographic variables. Results: The median value of serum HDL-c of the total participants was 34.0 (95%CI: 33.0– 35.0) mg/dL. Females exhibited significantly higher levels of HDL-c than males [37.1 (35.5– xvii 38.5) mg/dL vs. 31.6 (30.7–32.6) mg/dL, p < 0.001]. Lower levels of HDL-c (< 40 mg/dL for males and < 50 mg/dL for females) were prevalent in 91.9% of the participants with similar prevalence across genders (90.4% vs 93.7%, p = 0.229). Among the participants, elevated levels of triglycerides (>150 mg/dL) were found in 34.0%, total cholesterol (>200 mg/dL) in 21.0%, and LDL-c (>130 mg/dL) in 8.8%. In contrast to HDL-c, gender-based differences were observed in the prevalence of other lipid abnormalities. Elevated TG levels were significantly more common among males (40.2%) than females (26.8%, p = 0.005). Conversely, females exhibited a higher prevalence of elevated LDL-c (12.6% vs 5.5%, p = 0.013) and increased ApoA1 concentrations (62.6% vs 50.2%, p = 0.012). Their concentrations were also differed between genders, males with HDL-c below the median (< 31.6 mg/dL) showed higher TG (156 vs 114 mg/dL, p < 0.001) and BMI (24.65 vs 23.05 kg/m², p = 0.002) with lower ApoA1 (108 vs 132 mg/dL, p < 0.001), whereas females with lower levels of serum HDL-c (< 37.1 mg/dL) had lower levels of serum TC (159 vs 178 mg/dL, p < 0.001), LDL-c (102 vs 114 mg/dL, p < 0.001), and ApoA1 (123 vs 148 mg/dL, p < 0.001) along with higher TG (128 vs 103 mg/dL, p < 0.001). Logistic regression statistics identified increased TG and decreased ApoA1 as significant predictors of lower HDL-c across both genders. Across the gene loci studied, ApoA1 (–75 G/A and +83 C/T), ApoB (7673C/T, 10108A/G, 12669G/A), ABCA1 (–565C/T, 1051G/A, 2868G/A), PON1 (163T/A and 575A/G), and CETP (–629C/A and 277C/T), majority of the subjects exhibited wild-type homozygous genotypes. Heterozygous genotypes were less frequent than wild homozygous, and mutant homozygous genotypes were rare. The genotype distributions for most loci conformed to Hardy–Weinberg equilibrium (HWE). Exceptions were noted for ABCA1 2868G/A (p = 0.034), where the distribution deviated from HWE. We found no significant differences in HDL-c and ApoA1 or other lipid variables across the ApoA1 –75G/A and +83C/T genotypes among the overall subjects or males and females. In multivariable linear regression for HDL-c (n = 392), after adjusting for age, BMI, TG and LDL-c neither the –75GG (β = 0.806, p = 0.277) nor the +83CC (β = –1.212, p = 0.330) genotype showed any significant association with HDL-c in the overall sample. Within the male subgroup (n = 210), associations for –75GG (β = –0.665, p = 0.462) and +83CC (β = 1.535, p = 0.325) were non-significant. In contrast, in females the –75GG genotype was linked to a 2.78 mg/dL xviii increase (β = 2.783, p = 0.023) and the +83CC genotype to a 4.28 mg/dL decrease (β = –4.281, p = 0.031) in HDL-c. For serum ApoA1, the overall and male subgroup models showed no significant associations with either –75GG (overall: β = 0.629, p = 0.796; males: β = –2.401, p = 0.459, females: β = 4.606, p = 0.220 ) or +83CC (overall: β = –1.151, p = 0.778; males: β = 4.375, p = 0.434, females: β = –7.796, p = 0.201). Thus, ApoA1 –75 and +83 variants do not independently affect serum ApoA1 levels, they modulate HDL-c in a gender-specific manner, with significant associations observed only in females. Both the ApoB 12669G/A genotype distribution (p = 0.019) and allelic frequencies (p = 0.016) differed between normal and elevated LDL-c groups. Additionally, the ApoB 12669 A allele frequency was higher in subjects with elevated ApoB levels (p = 0.042). Logistic regression revealed the carriers of ApoB 10108 GA+AA had higher risk of elevated TG (OR = 3.36, 95% CI: 1.20–9.45, p = 0.021). Conversely, the ApoB 12669 GA+AA genotype was protective for elevated LDL-c (OR = 0.33, 95% CI: 0.12–0.87, p = 0.023) and elevated ApoB levels (OR = 0.51, 95% CI: 0.27–0.99, p = 0.045). None of the ABCA1 –565C/T, 1051G/A and 2868G/A SNPs differed in genotype distribution and allele frequency between the two groups of HDL-c (P>0.05). The ABCA1 –565TT, 1051AA, and 2868 GA+AA genotypes were not associated with low HDL-c. This was evident from the adjusted OR statistics: 0.80 (95% CI: 0.49 - 1.31, p = 0.186) for –565TT; 1.46 (95% CI: 0.74 - 2.89, p = 0.403) for 1051AA; and 1.12 (95% CI: 0.49 - 1.63, p = 0.809) for 2868 GA+AA. The median (95%CI) of the PON1 arylesterase (PON1-ARE) was 2.50 (2.41 – 2.56) kU/L in the total subjects and higher in males compared to females 2.56 (2.49 – 2.67) vs 2.41 (2.28 – 2.53, p = 0.028). PON1-ARE was highest in 163TT and 575GG genotypes, followed by heterozygous 163TA and 575AG, homozygous 163AA and 575AA. The 163TT, TA and TT genotypes and T, A alleles were almost similar in the two groups of HDL-c (p > 0.05). Similarly, the 575 AA, AG and GG genotypes and A, G alleles showed no difference between the two HDL-c groups (p >0.05). Logistic regression statistics revealed no association of 163T/A and 575A/G with HDL-c [OR(95%CI): 1.06 (0.68 – 1.65), p = 0.804; 0.90 (0.59 – 1.38), p = 0.630]. No significant differences in genotype distribution or allele frequencies of CETP –629CA and 277CT SNPs were observed between HDL-c groups (p > 0.05). For the 277C/T polymorphism, the combined 277(CC+CT) genotypes differed significantly from the TT genotype between HDL-c groups (p = 0.011, OR = 0.37, 95% xix CI = 0.18–0.78). Higher HDL-c was observed in –629AA (p = 0.023) and CA+AA (p = 0.043) carriers compared to CC carriers. Similarly, higher HDL-c was observed in 277TT (p = 0.002) and CT+TT (p = 0.019) carriers compared to CC genotype. Finally, multiple linear regression statistics revealed negative effects of -629CC (β = –1.106, p = 0.038) and 277(CC+CT) (β = – 0.963, p = 0.016) on serum HDL-c levels. Conclusion: Low levels of HDL-c are exceedingly prevalent among the Bangladeshi population and are associated with male gender, elevated TG and decreased ApoA1 levels, potentially contributing to a higher risk of atherosclerotic cardiovascular disease (ASCVD) in this group. The ApoA1 –75GG showed positive and +83CC showed a negative significant impact on HDL-c only in females. The ApoB 10108G/A polymorphism was associated with elevated TG, and the 12669G/A variant was linked to elevated LDL-c and ApoB levels, although ApoB gene polymorphisms did not affect serum HDL-c levels. No association was found between circulating HDL-c levels and the common ABCA1 genotypes –656CT (heterozygous), 1051GA (heterozygous), and 2868GG (wild homozygous). PON1 163T/A and 575A/G polymorphisms contributed to circulating PON1 esterase activity but were not associated with serum HDL-c. Notably, the CETP –629CC, 277CC, and 277CT genotypes were associated with low levels of HDL-c in the Bangladeshi population, suggesting that screening for CETP gene variants may serve as a valuable biomarker for diagnosing low HDL-c levels and potentially guiding interventions to reduce ASCVD risk in this population. This thesis is submitted for the degree of Doctor of Philosophy.