Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic compounds mainly consisting of benzene rings which have become the common threat to our environment as the grievous pollutants. The only cost effective and eco-friendly way to remove these pollutants from the environment is their degradation via microbes. In the present study bacterial cultures were isolated for degradation of Naphthalene (Nap) and Phenanthrene (Phe) representing low molecular weight (LMW) PAHs, Fluoranthene (Fla) representing high molecular weight (HMW) PAH and Iranian light crude oil (ILCO) representing mixture of hydrocarbons. Forty-four bacterial strains (CMG2001-CMG2044) were isolated from water and mud samples enriched on Nap. Two bacterial consortia HP and LP and one bacterial strain (CMGCZ) of LP consortium were isolated from oil contaminated soil samples by enrichment on Fla. Bacterial strains CMG2001-CMG2044 were screened for growth on Nap, Phe and Fla by 96 well microtiter plate assay. Among them eighteen bacterial strains exhibited growth on one or more tested PAHs while two of them (CMG2028 and CMG2042) were selected for further studies on PAHs degradation. CMG2028 and CMG2042 were identified by 16S rRNA gene sequencing as Kocuria flavus and K. rosea, respectively. In minimal medium 36% and 53% Nap (500mg l -1 ) was degraded in ten days of incubation by K. rosea CMG2042 and K. flavus CMG2028, respectively. Addition of yeast extract (YE) in medium as an additional carbon source resulted in enhanced degradation (59%) of Nap in K. rosea CMG2042 and reduced degradation (45%) in K. flavus CMG2028 within ten days. Although both the strains exhibited growth on Phe (10mg l -1 ) and Fla (10mg l -1 ) in YE added and omitted medium but only Phe (9%) was degraded by K. rosea CMG2042 as a sole carbon source. K. flavus CMG2028 and K. rosea CMG2042 exhibited growth on YE added and omitted minimal agar plates coated with ILCO and their colonies accumulated oil but did not grow in liquid medium with 0.5% ILCO. Bacterial strain CMGCZ, isolated from LP consortium, was identified as Rhodococcus erythropolis by 16S rRNA gene sequencing. R. erythropolis CMGCZ formed clear zones on Fla sprayed minimal and LB agar plates. In minimal medium degradation of Nap (500mg l -1 ), Phe (100mg l -1 ) and Fla (100mg l -1 ) by R. erythropolis CMGCZ in one week of incubation was 13.2%, 13.1% and 99.3%, respectively however YE addition in medium resulted in complete inhibition of Nap degradation, slightly enhanced degradation of Phe (14.8%) and a more rapid degradation of Fla (100%). R. erythropolis CMGCZ was capable of growing on xxii R. Z. A. Khan-PhD Thesis OPTIMIZATION OF BIODEGRADATION OF PAHs BY BACTERIA ABSTRACT 1% ILCO in liquid medium and degraded 13.2% and 11% aiphatic fraction of ILCO in YE added and omitted medium, respectively. LP and HP consortia enriched in minimal medium (HPMO/LPMO) and in YE added medium (HPMM/LPMM) were tested for degradation of Fla (100mg l -1 ). LPMM and HPMM consortia degraded 100% and 25.5% Fla, respectively in YE added medium in twenty days. In minimal medium 51.5% Fla was degraded by LPMO consortium but HPMO consortium failed to degrade Fla. Further subculturings of both the consortia in YE added medium resulted in improved Fla degradation by LPMM consortium but Fla degradation by HPMM consortium ceased. LPMM consortium degraded 98.6% and 95.7% Fla in YE added and omitted medium, respectively within a week of incubation. When LPMO consortium was incubated longer (35 days) in minimal medium without further transfer it degraded 97.6% Fla in a week which proved that longer incubation compensated deficiency of additional carbon source. Comparison of aromatic ring dioxygenase expressing bacteria (ARDB) of both the consortia revealed that increase in ARDB was observed only in LPMM and longer (35 days) incubated LPMO consortium. LPMM consortium was proved to be completely bacterial by adding microeukaryotic and different prokaryotic inhibitors in the growth medium. The optimized temperature and pH of the medium for LPMM consortium were found to be 30Ċ and 7.0, respectively. Degradation optimization for Fla concentrations ranging from 100mg l -1 to 1000mg l -1 with the difference of 150mg l -1 Fla resulted in 98.8%, 94.6%, 85.6%, 77.5% and 44.6% Fla degradation by LPMM consortium, respectively. Fla degradation optimization by R. erythropolis CMGCZ for same concentrations of Fla resulted in 100%, 100%, 57.2%, 19.1% and 12.6% degradation of Fla, respectively. PCR amplification of Rieske [Fe 2 -S 2 ] center of PAH dioxygenase genes resulted in 100bp PCR product in HPMM and LPMM consortia, R. erythropolis CMGCZ and only one type of blue colony (CMGBL) in ARDB population of LPMM consortium. Amplified PCR product of R. erythropolis CMGCZ exhibited homologies at nucleotide and deduced amino acid level mainly with Rieske [Fe 2 -S 2 ] domain protein of Mycobacterium species and pahAC gene of uncultured bacterium clones, known for degradation of PAHs. Bacterial strains and LP consortium isolated in the present study are efficient degraders of different PAHs and promising candidates for use in bioremediation operations at hydrocarbons contaminated sites.
Islam where considers the superiority of law, provision of justice and equity, building and purification of civilization and emphasis on the welfare of society, there ensures the first priority to humanity, peace and prosperity in the external relations. Islamic state keeps relations on the basis of equality with the world and non-Muslim citizens living within the state. On this belief and ideology Islam invites the world to set together. Islam on these principles of Islamic ideology and belief sets the foundation of collectiveness. On this principle the whole philosophy of life and living system are embraced, and the same Islamic law is the foundation of nations, on this behalf the Islamic state organized the relation with other states. In this way Islamic state on these principles keep relations with other states and within the state relations between Muslim and non-Muslim citizens on the basis of brotherhoods, equality, mercy and the principles of dignity of human being. Along with peace Islam set the principles of war which comprise ethical and prison limitations, duties and ethics amongst warrior, difference between the rights of fighters and non-fighters, treatment with pact holders and prisoners, and specified the way of better treatment with the defeated nations. He thought the manners of war to bloody man who consider everything right during the war. Islam lays great stress on equality, social justice, brotherhood and peace not only in state but across the boarders too. In this article a deep study is done to explain the relations of an Islamic state with other states. Islamic foreign policy emphasizes on the principles of equality among all the human beings and all the races and nations. Islam builds international relation on humanitarian basis.
Numerous genetic conditions have been described clinically but the molecular etiology for most of them is still unknown. With the advancement in the field of molecular biology powerful techniques have been developed to localize these conditions in the human genome and subsequent identification of causative genes. Functional analysis of causative genes leads to the discovery and understanding of novel genetic processes and pathways underlying disease conditions including normal developmental pathways. Linkage analysis studies in Mendelian disorders to identify the causative genes and mutations are possible using large pedigrees with multiple affected individuals. Analysis of alleles using microsatellite markers and genome wide SNPs lead to the discovery of novel genes and loci for specific disorders. The main aim of this thesis was to analyze families with autosomal recessive primary microcephaly and families with inherited limb disorders particularly polydactyl, syndactyly and brachydactyly to identify the causative mutations or chromosomal loci. Autosomal recessive primary microcephaly (MCPH) is a neurogenic disorder characterized by reduced head circumference (≤4 SD) and variable degree of mental retardation without any other neurological manifestations. The normal brain architecture is preserved despite the fact that brain size is reduced to three folds. In the first part of this study, genetic analysis of eleven primary microcephaly families was carried out. Linkage analysis using highly polymorphic microsatellite markers confirmed linkage in six families to ASPM (MCPH5), two CENPJ (MCPH6), one MCPH2 locus and haplotype analysis in two families demonstrated compound heterozygosity for ASPM. Sequencing of ASPM in six potentially linked families (MCP3, MCP6, MCP7, MCP9, MCP11 and MCP17) revealed six homozygous mutations in the affected subjects (A1160fs1181X, Y2245fs2258X, R3233X, Y3164X, S3186X, and R3244X respectively) and two possible compound heterozygous families (MCP35 and MCP18) demonstrated compound heterozygous mutations (W1326X/R3107X and R1019X/Q2632X, respectively). Compound heterozygous patients (W1326X & R3107X) also have additional clinical symptoms of seizures. Two families linked to MCPH6 locus (MCP21, MCP22) demonstrate 1bp deletion mutation c.17_18delC (T6fsX3) in exon 2 of CENPJ leading to premature termination of protein. This mutation was previously reported in two Northern Pakistani families. XVIFamily MCP15 established linkage to MCPH2 locus on chromosome 19q (19q13.1-q13.2). MCPH2 locus was defined by markers D19S416 and D19S420 which was about 7.6 cM in two consanguineous families from Northern Pakistan. However, the region is significantly reduced in MCP15 which is defined by markers D19S416 and D19S47. This substantially decreases the minimum critical interval from 7.6 cM to about 4.4 cM containing 162 genes. Family MCP36 has only single affected child. Molecular analysis using microsatellite markers revealed that affected individual is homozygous for the MCPH1 locus. By sequencing I have identified a novel nonsense mutation in exon 4 of MCPH1/microcephalin. The mutant protein lacks both of the C-terminal BRCT domains required for the normal functioning of protein during cell cycle progression and DNA repair mechanism. The second part of thesis comprised of genetic analysis of inherited limb disorders. Inherited limb malformations are genetically heterogeneous group of conditions with wide range of phenotypic manifestations. Inherited limb disorders occur as an isolated entity or syndromic form and are of clinical significance due to their severity and overall frequency. Limb development is a cascade of complex pathways involving patterning, growth and differentiation. Molecular characterization of inherited limb disorders may lead to the identification of novel genes and signalling pathways important for normal limb development during organogenesis. Family PD1 with preaxial polydactyly and triphalangeal thumb revealed autosomal dominant inheritance. Linkage analysis using microsatellite markers D7S550, D7S559 and D7S2423 was performed and maximum multipoint LOD score of 1.93 at recombination frequency θ= 0.1 was obtained. This region spans SHH and its cis-acting regulatory element (ZRS), which is well conserved among various species lying in intron 5 of LMBR1. Direct Sequencing of ZRS identified a novel point mutation (T>G) in ZRS element at base position 4976 in intron 5 of LMBR1. Many point mutations have been identified in ZRS leading to disruption of SHH expression during limb development leading to preaxial duplication in upper limbs. Electrophoretical mobility shift assay (EMSA) demonstrated a marked difference between wild and the mutant probe which uniquely bound a specific subset of nuclear transcription factors extracted from Caco-2 cells. It is suggested that altered transcription factor affinity may be important for our understanding of how single nucleotide substitutions in long distance regulatory elements changes cis-regulation of its target gene. XVIIGreig cephalopolysyndactyly syndrome (GCPS) is an autosomal dominant disorder which affects limb and craniofacial development. GCPS was mapped to chromosome 7p13. Mutations in GLI3 had been described in GCPS patients. In the present study four novel GLI3 mutations in four distinct families have been identified. In family PD2, a single nucleotide substitution mutation [c.1702A>T (p.R568X)] leading to immediate stop codon in exon 13 is identified. Two base pairs deletion mutation [c.1853_1854delAC (p.Y618fs)] leading to frameshift and premature terminated protein product of 673 amino acids is identified in family PD316 in a family from Denmark. Both the mutations R568X and Y618fs lie in zinc finger domain in the first third of GLI3 producing truncated protein product which may affect the DNA binding property of zinc finger domain leading to possibly haploinsufficiency of GLI3. In family PD7, a novel C to T substitution at coding nucleotide 4574 (p.P1525L) in exon 15 of GLI3 is identified. The third mutation which is a missense (c.4574C>T (p.P1525L) lies in the last third of GLI3. Missense mutation P1525L lies in the C terminal region of GLI3 protein in the transactivation domain. In family PD16, at coding nucleotide position 3557, C to T substitution leading to missense incorporation of amino acid (p.P1186L) is identified. The variability in phenotype with respect to mutation in the affected family members may help to understand the phenotypic spectrum of GLI3 mutation. Brachydactyly is a rare and genetically heterogeneous disorder. In the present study a novel locus in a large consanguineous family with recessive form of brachydactyly type E is localized on chromosome 6p22.3 by homozygosity mapping using 10K SNP analysis. The physical linkage interval lies between 15,837,143 to 16,579,402 bp which is about 742 Kb. Maximum two point LOD score (Zmax) of 5.00 at recombination fraction (θ=0.0) was calculated at marker locus D6S18xAG. This region spans only seven genes including four pseudogenes. Sequencing of protein coding genes which include MYLIP, GMPR and ATXN1 did not reveal any mutation. Analysis using SNP6 array also did not identify any homozygous deletion or duplication in the region. However, smaller deletions or duplication (≤30 kb) cannot be excluded. Family PD14A shows cutaneous syndactyly of 3 rd and 4 th digit in hands. After exclusion using microsatellite markers on chromosome 2q34-q36 (syndactyly type I), 3p21.31 (zygodactyly), 2q31 (SPD1 locus, HOXD13), 6q22.31 (GJA1, syndactyly type III), 22q13.31 (SPD2), 14q11.2- q12 (SPD3) and 17p13.3 (syndactyly type IX) genome wide 10K SNP analysis was performed. XVIIIAfter fine mapping using microsatellite markers a single homozygous region on chromosome 9 flanked by markers, SNP_A-1518820 and D9S21AT (marker not available in Marshfield genetic map) was identified. The physical positions of flanking markers are 12018387 bp to 15340449 bp on chromosome 9 with maximum LOD score (Zmax) of 2.35 for given locus (θ=0.00). The region spans only 22 genes. A novel single nucleotide G to A substitution at coding nucleotide position 1289 (c.1289G>A) in exon eight leading to missense incorporation of glutamine instead of arginine at amino acid position 430 of Frem1 (p.R430Q) is identified. Arginine at 430 amino acid position of Frem1 is not only conserved among different vertebrate species but also conserved among Frem family of genes. However, possibility of missense mutation in Frem1 producing a defect in digit separation requires more families to study in addition to the functional studies in experimental models to prove the pathogenic nature of this mutation." xml:lang="en_US