The Drosophila Neuroblasts: A Model System For Human Ribosomopathies

This dissertation describes the use of Drosophila neuroblasts (NBs) to model human ribosomopathies; the overall goal is to understand why specific stem cell and progenitor cell populations are the primary targets in nucleolar stress as seen in the ribosomopathies. Chapter 1 provides an overview of relevant literature. Chapter 2 describes nucleolar stress in Drosophila neuroblasts as a model for human ribosomopathies. For this, we induce nucleolar stress by using the UAS-GAL4 system to express RNAi that depletes Nopp140 transcripts, and we also employ homozygous, CRISPR-Cas9-generated Nopp140 gene disruptions with a systemic null phenotype (Nopp140-/-). Embryonic lethality was observed under RNAi depletion of Nopp140 as well as homozygous and heterozygous Nopp140 disruption. Larval lethality occurred at the second instar stage in Nopp140-/- line, similar to the previously generated complete Nopp140 deletion line, KO121. Larval brain development was severely impaired in Nopp140-/- larvae and in larvae that expressed neuron-specific RNAi that depletes Nopp140. The hypoplastic brain phenotype was due to reduction in NB populations as well as the proliferative capacity of the dividing NBs. While the majority of NB lineages in wild-type brains are at S-phase and proliferative at day 3 after larval hatching as indicated by EdU labeling assay, only the Mushroom Body (MB) NBs are at S-phase and proliferative in the Nopp140-/- larval brain. Furthermore, these MB NBs retained fibrillarin within their nucleoli, while fibrillarin redistributed to the nucleoplasm in the surrounding cells. Hence, we conclude that MB NBs are more resilient to nucleolar stress induced by the loss of Nopp140 compared to other neuroblast lineages. This finding strengthens the use of Drosophila neuroblasts as a model for the human ribosomopathies, and we hypothesize that different neuroblast lineages respond variably to nucleolar stress. Chapter 3 describes repeat polymorphisms in the Nopp140 gene that result in two Nopp140 alleles, Nopp140-Long and Nopp140-Short, that differ by exactly 96 bps within the central domain. We provide evidence showing preferential amplification of the Nopp140-Long allele compared to that of the Nopp140-Short allele, which we determined to be a PCR artefact, for reasons that remain unknown. Chapter 4 closes with conclusions and future studies

The Nopp140 gene in Drosophila melanogaster displays length polymorphisms in its large repetitive second exon

Nopp140, often called the nucleolar and Cajal body phosphoprotein (NOLC1), is an evolutionarily conserved chaperone for the transcription and processing of rRNA during ribosome subunit assembly. Metazoan Nopp140 contains an amino terminal LisH dimerization domain and a highly conserved carboxyl domain. A large central domain consists of alternating basic and acidic motifs of low sequence complexity. Orthologous versions of Nopp140 contain variable numbers of repeating basic-acidic units. While vertebrate Nopp140 genes use multiple exons to encode the central domain, the Nopp140 gene in Drosophila uses exclusively exon 2 to encode the central domain. Here, we define three overlapping repeat sequence patterns (P, P\u27, and P \u27\u27) within the central domain of D. melanogaster Nopp140. These repeat patterns are poorly conserved in other Drosophila species. We also describe a length polymorphism in exon 2 that pertains specifically to the P\u27 pattern in D. melanogaster. The pattern displays either two or three 96 base pair repeats, respectively, referred to as Nopp140-Short and Nopp140-Long. Fly lines homozygous for one or the other allele, or heterozygous for both alleles, show no discernible phenotypes. PCR characterization of the long and short alleles shows a poorly defined, artifactual bias toward amplifying the long allele over the short allele. The significance of this polymorphism will be in discerning the largely unknown properties of Nopp140\u27s large central domain in rDNA transcription and ribosome biogenesis

Synthesis and Characterization of pH Responsive D-Glucosamine Based Molecular Gelators

Small molecular gelators are a class of compounds with potential applications for soft biomaterials. Low molecular weight hydrogelators are especially useful for exploring biomedical applications. Previously, we found that 4,6-O-benzylidene acetal protected D-glucose and D-glucosamine are well-suited as building blocks for the construction of low molecular weight gelators. To better understand the scope of D-glucosamine derivatives as gelators, we synthesized and screened a novel class of N-acetylglucosamine derivatives with a p-methoxybenzylidene acetal protective group. This modification did not exert a negative influence on the gelation. On the contrary, it actually enhanced the gelation tendency for many derivatives. The introduction of the additional methoxy group on the phenyl ring led to low molecular weight gelators with a higher pH responsiveness. The resulting gels were stable at neutral pH values but degraded in an acidic environment. The release profiles of naproxen from the pH responsive gels were also analyzed under acidic and neutral conditions. Our findings are useful for the design of novel triggered release self-assembling systems and can provide an insight into the influence of the the structure on gelation

UV Spectrophotometric Determination of Luliconazole Semisolid Dosage Form

Luliconazole mostly prescribed drug for the management of superficial problems, the very simple, Fast, accurate and economical methods have been proposed for the determination of Luliconazole cream. Luliconazole  was measured by using Uv spectroscopy method with the solution of mrthanol and purified water the linearity was found to be 0.9987 and the accuracy showed mean % RSD of 0.921776 and with total meam % RSD 1.10130 in intermediate precision, robustness %RSD 0.543539 all the paranmeters values were within standard limit thus Analytical method was validated according to ICH guideline for the determination of Luliconazole cream. The method was found to be precise and validated as per ICH guidelines

“Estimation and Validation of Methylcobalamin in Tablet Dosage form using UV-Visible Spectrophotometric Method”

An ultraviolet (UV) spectrophotometric method was developed and validated for quantitavie determination of Methylcobalamin (Mecobalamin) in tablet dosage form. Methylcobalamin is a cobalamin, a form of vitamin B12 and used to prevent or treat pathology arising from a lack of vitamin B12 , such as pernicious anemia and is also used in the treatment of peripheral neuropathy, diabetic neuropathy ans as a preliminary treatment for amyotrophic latera sclerosis. The very simple, Fast, accurate and economical methods have been proposed for the determination of Mecobalamin. Mecobalamin was measured by using Uv spectroscopy method with the solution of methanol, the linearity was found to be 0.9981 and the accuracy showed mean % RSD of 0.791694 and with total meam % RSD 0.97923 in intermediate precision, range %RSD 0.652005 all the parameters values were within standard limit thus Analytical method was validated according to ICH guideline for the determination of Methylcobalamin. The method was found to be precise and validated as per ICH guidelines

A study of the prevalence and risk factors leading to HIV infection among a sample of street children and youth of Kathmandu

Background: The true prevalence of HIV and other sexually transmitted diseases among street children in Nepal is virtually unknown while information on related behavioural risk factors in this population is non-existent. The risk of HIV infection among street children and adolescents may be especially high due to their marginalized social and economic conditions. This study was conducted to determine the prevalence of HIV infection among a sample of street children and youth of Kathmandu and to identify risk factors associated with HIV infection in this group. A sample of street children and youth was recruited based on the purposive sampling of ten streets in Kathmandu, Nepal, known to have a high density of street children and youth. A total of 251 street children (aged 11–16 years) and youth (aged 17–24 years) were enrolled, with informed consent, from November, 2008 through June, 2009. Most of the participants (95%) were male. Case status was determined by serological assessment of HIV status; data on risk factors were obtained using structured survey interviews. HIV prevalence and rates of a number of behavioural risk factors suspected to play a role in HIV transmission among street children and youth were determined, including unprotected sex, intravenous drug use, and other risky sex and substance use behaviours. Results: Among the 251 children and youth, we found an overall HIV prevalence of 7.6%. As the sample size of females was small (n = 13) and the behavioural risk factors are likely to be quite different for boys and girls, we conducted separate analyses by gender. As our small sample of females is unlikely to be representative and lacks power for statistical testing, our report focuses on the results for the males surveyed.The strongest behavioural risk factor to emerge from this study was intravenous drug use; 30% of the male subjects were injecting drug users and 20% of those were HIV positive. Furthermore, frequency of drug injection was a highly significant predictor with a dose–response relationship; males reporting occasional injection drug use were nearly 9 times more likely to be HIV positive than never users, while weekly drug injectors had over 46 times the risk of non-users, controlling for exposure to group sex, the only other significant risk factor in the multivariate model. Conclusions: This sample of street children and youth of Kathmandu has a nearly 20-fold higher prevalence of HIV infection than the general population of Nepal (0.39%). The children and youth engage in number of high risk behaviours, including intravenous drug use, putting them at significant risk of contracting HIV and other sexually transmitted infections

Formulation & Evaluation of Fluconazole Gel for Topical Drug Delivery System

Fluconazole is a recent triazole antifungal drug that is used in the treatment of superficial and systemic fungal infection. The oral use of fluconazole is not much recommended as it has many side effects. Thus this formulation is made for better patient compliance and to reduce the dose of the drug and to avoid the side effects like liver damage and kidney damage. This research was designed to formulate & evaluate different formulation of a topical gel containing fluconazole by using a polymer with different concentration as Carbopol 940 & NaCMC. Methanol was used as a penetration enhancer. The evaluation of formulated fluconazole topical gel was carried out for a physical appearance, pH-value, spreadability, homogeneity, drug content. The formulated gel showed good physical characteristics. The formulation F3 (101.18%) & F6 (105.4%) show good drug content as the polymer concentration in them was higher. The percentage yield of F4 (98.26%) was the highest. The spreadability of gel decreases with an increase in polymer concentration. The pH of the formulation was in the range of 5-8 which is considered acceptable to avoid the risk of irritation upon application to the skin

Quantification of run-of-river hydropower potential in the Upper Indus basin under climate change

IntroductionDespite ambitious plans to quadruple hydropower generation in the Indus basin, a quantitative assessment of the impact of climate change on hydropower availability in the basin is missing. To address this gap, we combine downscaled CMIP6 projections with the Hydropower Potential Exploration (HyPE) model to quantify future hydropower potential available in the upper Indus basin.MethodsHyPE uses a spatial cost-minimization framework to evaluate four classes of hydropower potential, namely theoretical, technical, financial and sustainable, considering various constraints on the siting and sizing of two run-of-river hydropower plant configurations.ResultsUnder future discharge projections, all classes of potential increase while subbasin changes align with the spatial patterns projected in hydro-climatology. Theoretical potential changes by 3.9–56 %, technical potential by −2.3–46.8 %, financial potential by −8.8–50.4 % and sustainable potential by −6.1–49.7 %. A small decline is observed in the northwestern subbasins where increase in potential is lower than in the southeast. In contrast, with increasing variability in the Indian Summer Monsoon in the future, the southeastern subbasins have the strongest increase in sustainable potential accompanied by higher increase in plant size, decrease in costs and higher variability. The southeastern Satluj subbasin is the hotspot where sustainable potential has the highest increase of up to 145 %. The northwestern Kabul subbasin has the highest decrease of up to −27 %. The Swat subbasin has the lowest variability in sustainable potential while the Jhelum and Indus main subbasins remain the subbasins with the cheapest potential into the future. The performance of future sustainable portfolios differ from the performance of historical portfolios by −11.1–39.9 %.DiscussionHence, considering future climate in the present-day planning of hydropower will lead to improved performance under a majority of scenarios. The sufficiency of hydropower potential to fulfill energy security depends on future population growth. Energy availability is projected to decline in the northwest as population increases faster than hydropower potential. The per capita sustainable potential In the Kabul subbasin reduces to a third of the historical value. A socio-hydrological approach is necessary to address the complexity of achieving sustainable and equitable hydropower development in the Indus basin under such spatial mismatch between hydropower availability and energy demand in a resource-limited world

Diagnostic host gene signature for distinguishing enteric fever from other febrile diseases.

Misdiagnosis of enteric fever is a major global health problem, resulting in patient mismanagement, antimicrobial misuse and inaccurate disease burden estimates. Applying a machine learning algorithm to host gene expression profiles, we identified a diagnostic signature, which could distinguish culture-confirmed enteric fever cases from other febrile illnesses (area under receiver operating characteristic curve > 95%). Applying this signature to a culture-negative suspected enteric fever cohort in Nepal identified a further 12.6% as likely true cases. Our analysis highlights the power of data-driven approaches to identify host response patterns for the diagnosis of febrile illnesses. Expression signatures were validated using qPCR, highlighting their utility as PCR-based diagnostics for use in endemic settings