Molecular, Cellular and Mechanical basis of Epithelial Morphogenesis during Tribolium Embryogenesis

Embryonic development entails a series of morphogenetic events which require a precise coordination of molecular mechanisms coupled with cellular dynamics. Phyla such as arthropods show morphological and gene expression similarities during middle embryogenesis (at the phylotypic germband stage), yet early embryogenesis adopts diverse developmental strategies. In an effort towards understanding patterns of conservation and divergence during development, investigations are required beyond the traditional model systems. Therefore, in the past three decades, several insect species representing various insect orders have been established as experimental model systems for comparative developmental studies. Among these, the red flour beetle Tribolium castaneum has emerged as the best studied holometabolous insect model after the fruit fly Drosophila melanogaster. Unlike Drosophila, Tribolium is a short-germ insect that retains many ancestral characters common to most insects. The early embryogenesis of Tribolium shows dynamic epithelial rearrangements with an epibolic expansion of the extraembryonic tissue serosa over the embryo, the folding of the embryo in between the serosa and the second extra embryonic tissue amnion and the folding of the amnion underneath the embryo. These extensive tissues are evolutionarily conserved epithelia that undergo different tissue movements and are present in varying proportions in different insects, providing exceptional material to compare and contrast morphogenesis during early embryogenesis. However, most of the previous work on insects including Tribolium have largely focused on the conservation and divergence of gene expression patterns and on gene regulatory interactions. Consequently, very little studies on dynamic cell behaviour have been done and we lack detailed information about the cellular and tissue dynamics during these early morphogenetic events. During my PhD, I first established a live imaging and data analysis pipeline for studying Tribolium embryogenesis in 4-D. I combined live confocal and lightsheet imaging of transgenic or transiently labelled embryos with mechanical or genetic perturbations using laser ablations and gene knockdowns. Using this pipeline quantifications of cell dynamics and tissue behaviours can be done to compare different regions of the embryo as the development proceeds. In the second and third part of my thesis, I describe the actomyosin dynamics and associated cell behaviours during the stages of serosa epibolic expansion, amniotic fold formation and serosa window closure. I cloned and characterised the cellular dynamics of the Tribolium spaghetti squash gene (Tc-squash) - the non-muscle Myosin II regulatory light chain, which is the main molecular force generator in epithelial cells. Interestingly, the analysis of Tc-squash dynamics indicates a conserved role of Myosin II in controlling similar cell behaviours across short germ and long germ embryos. In the last part of the thesis, I report the dynamics of an actomyosin cable that emerges at the interface of the serosa and amnion. This cable increases in tension during development, concomitant with serosa tissue expansion and increased tensions in the serosa. It behaves as a modified purse string as it’s circumference shrinks due to a decrease in the number of cable forming cells over time. This shrinkage is an individual contractile property of the cells forming the cable. This indicates that a supracellular and contractile actomyosin cable might be functional during serosa window closure in insects with distinct serosa and amnion tissues. Further, the tension in the cable might depend on the relative proportion of the serosa, amnion and embryonic regions. Using these integrated approaches, I have correlated global cellular dynamics during early embryogenesis with actomyosin behaviours, and then performed a high-resolution analysis and perturbations of selected events. The established imaging, image processing and perturbation tools can serve as an important basis for future investigations into the tissue mechanics underlying Tribolium embryogenesis and can also be adapted for comparisons of morphogenesis in other insect embryos. More broadly, correlating the existing genetic, mechanical and biochemical understanding of developmental processes from Drosophila with species such as Tribolium, could help identify deeply conserved design principles that lead to different morphologies through differences in underlying regulation.:Page List of Tables v List of Figures vii 1 Introduction 1 1.1 Evo-Devo of insects 3 1.2 Tribolium castaneum 5 1.3 Fluorescence live imaging and lightsheet microscopy 10 1.4 Morphogenesis 15 1.5 Thesis objective 29 2 4D lightsheet imaging and analysis pipeline of Tribolium embryos 33 2.1 Standardisation of an injection protocol for sample mounting and imaging with the Zeiss LZ1 SPIM 35 2.2 Double labelling of Tribolium embryos 37 2.3 Image processing with Fiji 37 2.4 Long term timelapse imaging of Tribolium embryogenesis with SPIM 44 2.5 2D cartographic projections of 3D data as a method to visualise and analyse SPIM data 47 2.6 Summary 59 3 Cellular dynamics of the non muscle Myosin II regulatory light chain - Tc-Squash 61 3.1 Tc-Squash dynamics during Tribolium embryogenesis 64 3.2 Myosin drives basal cell closure during blastoderm cellularisation 66 3.3 Myosin shows planar polarity in the embryonic tissue 69 3.4 Myosin accumulation and apical constriction of putative germ cells at the posterior pole 71 3.5 Myosin pulses during apical constriction of mesoderm cells 74 3.6 Myosin accumulates at the extraembryonic-embryonic boundary to form a contractile supracellular cable 77 3.7 Summary 77 4 A supracellular actomyosin cable operates during serosa epiboly 79 4.1 Actin and Myosin accumulate at the extraembryonic-embryonic boundary 81 4.2 The actomyosin assembly migrates ventrally till it forms the rim of the serosa window 82 4.3 The actomyosin cable shows dynamic shape changes during serosa window closure 87 4.4 Serosa cells increase in area till circular serosa window stage 89 4.5 Tension in the serosa tissue increases during epibolic expansion 89 4.6 Serosa cells decrease their apical areas after laser ablation 92 4.7 Tension in the actomyosin cable increases during serosa epiboly 93 4.8 Myosin dynamics at the cable changes between early and serosa window stage 96 4.9 Individual cell membrane shrinkage and cell rearrangements decrease the cable circumference 98 4.10 Myosin dynamics at the cable during serosa window closure 101 4.11 Tension in the cable is not relieved after multiple laser cuts 103 4.12 Analysis of the actomyosin cable in Tc-zen 1 knockdown 105 4.13 Summary 109 5 Discussion 111 5.1 Reconstruction of insect embryogenesis using lightsheet microscopy and tissue cartography 111 5.2 Conserved Myosin II behaviours and its implications on morphogenesis across insects 114 5.3 A contractile supracellular actomyosin cable functions serosa window closure in Tribolium 119 6 Materials and Methods 123 6.1 Tribolium stock maintenance 123 6.2 RNA extraction and cDNA synthesis 124 6.3 Cloning of templates for mRNA synthesis and transgenesis 124 6.4 dsRNA synthesis for RNAi experiments 126 6.5 Capped, single stranded RNA synthesis 126 6.6 Fluorescence image acquisition 27 A Appendix 131 Bibliography 14

Pregnancy in unicornuate uterus without rudimentary horn: a case report

A unicornuate uterus is associated with numerous obstetric and gynaecological complications such as infertility, endometriosis, miscarriage, malpresentations, and intrauterine growth restriction. Around 2.3-13% of Mullerian duct anomalies present as unicornuate uterus. Management of unicornuate uterus is still uncertain and it leads to poorer pregnancy outcome. We present here a case of 26-year-old primigravida who presented to us with 40-weeks pregnancy associated with breech presentation. She was taken for elective caesarean section and intra-operatively she was found to have unicornuate uterus without rudimentary horn. Unicornuate uterus is associated with poor pregnancy outcome but a successful pregnancy is possible. Usual presentation of patients with unicornuate uterus is near their menarche and they have higher than usual gynaecological complications. Pregnancies in unicornuate uterus are prone to intra uterine growth restriction hence serial ultrasound should be done for regular fetal growth monitoring

HARMFUL EFFECTS OF AIR POLLUTION CAUSED DUE TO TRAFFIC IN THE URBAN AREAS

Air pollution has become a major worldwide problem, it has effected millions of life. There are various reasons for this unregulated rise in the pollution levels of the air, one of the major reasons is vehicular pollution which is effecting the life of numerous people who use roads as the major transportation facility which is nearly everyone in the world .The pollutants that are found in vehicle exhausts significantly affect the health of the people living in urban areas. High levels of nitrogen oxide, carbon di oxide are toxic to humans. Sulphur dioxidecausesacid rain. Carbon dioxide contributes to climate change causing global warming. Due this there needs to be change, a change that can help save the environment .There are various methods that can applied to save on our fossil fuels, reduce pollution and also improve our traffic conditions and improve road safety

MICRO-STIMULATION OF NEURONS

This project encompasses the basic understanding of neuropharmacology. Neuropharmacology is the study of how drugs affect cellular function in the nervous system, and the neural mechanisms through which they influence behavior. There are two main branches of neuropharmacology: behavioral and molecular. Behavioral neuropharmacology focuses on the study of how drugs affect human behavior (neuropsychopharmacology), including the study of how drug dependence and addiction affect the human brain.Molecular neuropharmacology involves the study of neurons and their neurochemical interactions, with the overall goal of developing drugs that have beneficial effects on neurological function. Both of these fields are closely connected, since both are concerned with the interactions of neurotransmitters, neuropeptides, neurohormones, neuromodulators, enzymes, second messengers, co-transporters, ion channels, and receptor proteins in the central and peripheral nervous systems. Studying these interactions, researchers are developing drugs to treat many different neurological disorders, including pain, neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, psychological disorders, addiction, and many others.Before understanding the effect of drugs we studied firing rates of neurons and neural networks. Initially we started off understanding firing rates and at what frequencies spikes will be recorded on the SpikerBox. We found that at low frequencies we can observe spikes. Later we started stimulating muscles in the cockroach’s leg using music. Upon varying bass, treble, etc we noticed the difference in spikes and recorded the same. After understanding the spikes we injected Nicotine and Mono Sodium Glutamate with control (water) at intervals of 2-4 minutes to observe which drug would have an effect on the neurons. MSG is present in 80% networks however for insects we found Nicotine stimulates and MSG does not

Study of obstetric and fetal outcome of post caesarean pregnancy

Background: With the sky rocketing caesarean section rates an increasing number of women face the issue of mode of delivery in their current pregnancy. There are conflicting reports regarding the safety of a trial for Vaginal Birth After Caesarean delivery (VBAC) in terms of uterine rupture and concern about, maternal and perinatal morbidity. The purpose of this study was to evaluate the obstetric and fetal outcomes of patients presenting at term with a history of previous LSCS.Methods: A one year prospective observational study was conducted where in all patients who had a term pregnancy with a history of prior LSCS were included in the study after obtaining their consent for participation. The obstetric and fetal outcomes of these patients in the present pregnancy were noted and tabulated. A descriptive analysis of these outcomes was carried out.Results: 100 patients at term, with a history of previous LSCS were studied. Of these, trial for a VBAC was attempted by 50 patients of these 46% (23) had a successful VBAC. And remaining 54% went for emergency LSCS. 50% patients underwent an elective repeat caesarean deliver. Scar dehiscence was seen in 2.72% of the patients who opted for a trial for VBAC. Perinatal morbidity was higher in cases of repeat caesarean delivery than in those who had a successful VBAC (12.12% vs. 0 percent). Maternal complications were also higher in patients who had a repeat LSCS compared to those who had a successful VBAC.Conclusions: With an increase in the proportion of patients with a history of previous LSCS, it is essential for health care institutions to have proper antenatal counseling regarding VBAC and a well-defined management protocol in place in an effort to increase the number of VBACs and bring down the overall caesarean rates. Patients with a history of prior vaginal delivery have an increased likelihood for a successful VBAC. A successful VBAC is associated with a lower perinatal and maternal morbidity than repeat caesarean delivery, and this is relevant for counseling women about their choices after a caesarean delivery

Amnion and Chorion Membranes: Potential Stem Cell Reservoir with Wide Applications in Periodontics

The periodontal therapy usually aims at elimination of disease causing bacteria and resolution of inflammation. It involves either resective or regenerative surgery to resolve the inflammation associated defects. Over the years, several methods have been used for achievement of periodontal regeneration. One of the oldest biomaterials used for scaffolds is the fetal membrane. The amniotic membranes of developing embryo, that is, amnion (innermost lining) and chorion (a layer next to it), have the properties with significant potential uses in dentistry. This paper reviews the properties, mechanism of action, and various applications of these placental membranes in general and specifically in Periodontics

Parameterized Complexity of Conflict-Free Matchings and Paths

An input to a conflict-free variant of a classical problem Gamma, called Conflict-Free Gamma, consists of an instance I of Gamma coupled with a graph H, called the conflict graph. A solution to Conflict-Free Gamma in (I,H) is a solution to I in Gamma, which is also an independent set in H. In this paper, we study conflict-free variants of Maximum Matching and Shortest Path, which we call Conflict-Free Matching (CF-Matching) and Conflict-Free Shortest Path (CF-SP), respectively. We show that both CF-Matching and CF-SP are W[1]-hard, when parameterized by the solution size. Moreover, W[1]-hardness for CF-Matching holds even when the input graph where we want to find a matching is itself a matching, and W[1]-hardness for CF-SP holds for conflict graph being a unit-interval graph. Next, we study these problems with restriction on the conflict graphs. We give FPT algorithms for CF-Matching when the conflict graph is chordal. Also, we give FPT algorithms for both CF-Matching and CF-SP, when the conflict graph is d-degenerate. Finally, we design FPT algorithms for variants of CF-Matching and CF-SP, where the conflicting conditions are given by a (representable) matroid

Optimization of recipe for development of Aonla squash- A Response surface methodology approach

Central Composite Design (CCD) was employed for optimizing the recipe for development of Aonla squash with juice content (25 to 40%), acidity (1.00 to 1.10%) as independent variables and adjusting 50% total soluble solids. Thirteen different experimental combinations given by RSM design were used to investigate the effect of independent process variables on four product responses developed squash. RSM evaluated responses of the 13 squash recipes by second order quadratic equations and found that pH ranged from 2.69-3.00, ascorbic acid from 102-212 mg/100ml, browning from 0.428- 0.579 and overall acceptability scores from 6.25-7.75. ANOVA analysis showed that the product responses of beverages were significantly affected (at 5%) by changes in juice and acidity of the squash recipes. Overall acceptability scores of the beverage increased with the increase in proportion of juice content from 25 to 40%. The coefficient of determination i.e., R2 for all responses was higher than 0.95 and nonsignificant lack of fit was observed for all models; which was desirable. Through numerical optimization, standardized recipe selected by RSM for aonla squash was with 40% juice and 1% acidity getting highest desirability of 0.991. The pH, ascorbic acid, browning and overall acceptability for the optimum recipe predicted by the design were 2.96, 197.8 mg/100 ml, 0.557 and 7.7, respectively. Thus, present study was first in its kind to optimize aonla squash recipe by using RSM as a tool which gave advantage of not only selecting best recipe but also provided impression of the influence of ingredients used in squash’s preparation on its physico-chemical profile

Ultrasonographic measurement of placental thickness and its correlation with estimated fetal weight

Background: The human placenta develops with the principal function of providing nutrients and oxygen to the fetus. Objective of present study was to assess the relationship between placental thickness with estimated fetal weight.Methods: The present study was a prospective observational study and includes 152 pregnant women with known last menstrual period, history of regular menstruation, singleton pregnancy and aged between 20 and 35 years. After Institutional Ethics Committee approval all recruited women were observed for baseline demographic and obstetric data including age, parity and past medical events at first antenatal visit. All women provided an informed written consent and underwent ultrasound evaluation of placental thickness at 18 to 40 weeks of gestation.Results: In the present study the mean placental thickness between the ranges of 18-40mm was 31.63±4.79mm and the mean estimated fetal birth weight was 2145.86±121.24grams. The pearson’s correlation coefficient between the two was 0.982. Thus, proving the significant positive correlation between placental thickness and estimated fetal birth weight (p-value <0.001).Conclusions: Estimated fetal weight is a very important component of antenatal care in which ultrasonography plays an important role. Placental thickness measured at the level of umblical cord insertion can be used as an accurate sonographic indicator in the assessment of fetal weight because of its linear correlation. Therefore, it can be used as an additional sonographic tool in assessing fetal weight

Potential drug-drug interactions among elderly in-patients with cardiac illness at a tertiary care centre

Background: Drug interactions are major cause of concern in hospitalized patients with cardiac illness especially in elderly population. Therefore, the study was conducted to determine the prevalence and pattern of potential drug-drug interactions (pDDI) and risk factors, if any.Methods: It was a prospective observational study involving 75 elderly in-patients with cardiac diseases. IHEC approval was taken before commencement of study and written informed consent was taken from all the study participants. Data was collected using structured data collection tool. pDDI were analyzed using MEDSCAPE databse. Data was analyzed using SPSS 20.0 in terms of descriptive statistics. Pearson correlation coefficient was used to find the association between the risk factors and potential DDIs. P value of ≤0.05 was considered statistically significant.Results: The prevalence of pDDI was found to be 100%. Total 593 pDDI and 33 interacting drug pairs were observed in the study. The common drug interacting pairs were aspirin and furosemide 140 (23.61%), followed by aspirin+ enalapril 98 (16.53%) and heparin and clopidogrel 56 (9.44%). Majority of pDDI 480 (81%) were found to be of moderate severity. A significant association was documented between length of hospital stay (p=0.041) and occurrence of pDDI. A statistically significant correlation (r =0.621; p<0.01) was noted between number of drugs prescribed and total number of pDDIs.Conclusions: A high prevalence of pDDI was observed. The prevalence rate is directly related to number of drugs prescribed and length of hospital stay. Therefore, close monitoring of hospitalized patients is recommended