Reticulon Homology Domain Containing Protein Families of the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is the largest membrane bound organelle in a cell and has multiple responsibilities. Execution of the various duties performed by the ER requires it to be shaped in a rather complex and intricate manner. ER’s two major structural motives, namely sheets and tubules, play very complex yet not fully understood role in giving ER its overall structure and function. The ratio of sheet and tubule conformations differ significantly within cell types and during cell cycle. Such a balance is possible only with a well-functioning set of factors that constantly communicate with each other throughout a cell cycle. These factors are specifically responsible for either shaping the ER sheets or tubules in addition to factors that keep the dynamic nature of the ER sound. During mitosis, ER undergoes a major transformation in its structure, where the sheet-tubule ratio shifts more towards tubules. Specific factors keep this process sound by acting actively during the stage of mitosis for proper cell division to occur. Although research on such factors are still on-going, many in-depth details on such factors (e.g. their precise localization) and their mechanism of action plus novel factors for ER shaping still needs to be resolved using techniques involving high end light and electron microscopy. In addition, a constant battle in data analysis for answering key questions also persists. Development of tools to study and analyse data on the lines of image analysis and processing is an unmet need that needs simultaneous attention. The research in this thesis focuses on three family of proteins that we uncover as responsible candidates in shaping the ER. To aid the study, this thesis also discusses the development of a software platform for analysis of microscopic data generated during this study. In this research, Reticulon family of proteins (RTN) were characterised using high-end microscopic techniques. We showed RTN4A and RTN4B to localize to ER tubules and sheet edges using pre-embedding immuno electron microscopy (immuno-EM) and electron tomography. Using qPCR, RTN4A and RTN4B were observed to be the most expressed isoforms in neurons and epithelial cells respectively. FAM134C, a poorly characterised protein was identified as one of the RTN4B interacting proteins. FAM134C localised to the ER where it specifically resided at high curvature ER (sheet edges and tubules) similar to RTN4B. FAM134C, similar to the RTN4B also had the capability to promote ER tubules upon overexpression. In addition, another family of proteins belonging to receptor expression enhancing protein (REEP), namely REEP3 and REEP4 were studied for shaping ER during mitotic stage of cell cycle. REEP3 and REEP4 collectively were observed both in tubulating peripheral ER during mitosis and clearing tubular ER from the chromatin for a normal mitosis to take place. Collectively, this work elaborates on proteins belonging to three classes that shape and position the ER specifically either in interphase or during stages of cell division. Our findings also throws light on the role of different domains in each of these proteins such as the reticulon homology domain (RHD) that was observed to be present in all these proteins under study. The RHD previously known for inserting partially and unsymmetrically in the outer leaflet of the ER gives a strong indication for proteins like RTN4B and FAM134C to localize to ER thus tubulating ER upon overexpression conditions. We uncovered the RHD’s crucial role in ER shaping and positioning in REEP3/4 during mitosis.Endoplasma- eli solulimakalvosto (engl. endoplasmic reticulum, ER) on solun suurin kalvon rajoittama organelli, ja sillä on useita tehtäviä. Pystyäkseen suoriutumaan eri tehtävistään ER:n rakenne on monimuotoinen ja alati muuntautuva. ER:n päärakenteet, laatat ja tubulukset, muodostavat monimutkaisen verkoston, eikä niiden kaikkia toimintoja täysin vielä tunneta. Laattojen ja tubulusten määrällinen suhde on erilainen eri solutyypeissä ja solusyklin vaiheissa. ER:n toimivan tasapainon saavuttamiseksi tarvitaan useita tekijöitä, jotka ovat vuorovaikutuksessa keskenään koko solusyklin ajan. Osa näistä tekijöistä osallistuu ER:n rakenteen muokkaamiseen ja osa on vastuussa ER:n dynaamisesta luonteesta. Solunjakautumisen aikana ER:n rakenne muuttuu, ja tubulaarisia rakenteita muodostuu suhteellisesti enemmän. Eri tekijät toimivat aktiivisesti solunjakautumisen eri vaiheissa mahdollistaen näin solujen jakautumisen. Nämä tekijät ovat edelleen tutkimuksen kohteena, ja yksityiskohtien esim. tarkan paikantumisen ja toimintamekanismin selvittämiseksi sekä vielä tuntemattomien, ER:n rakenteeseen vaikuttavien tekijöiden löytämiseksi, on käytettävä kehittyneitä tekniikoita kuten valo- ja elektronimikroskopiaa. Myös tietoaineiston analysoinnin täytyy edelleen kehittyä pystyäksemme vastaamaan tärkeisiin kysymyksiin, ja sen vuoksi sekä kuvankäsittelyyn että kuvien analysointiin tarvittavien ohjelmien kehittämiseen on kiinnitettävä erityistä huomiota. Tässä väitöskirjatutkimuksessa tutkittiin kolmea proteiiniperhettä, joiden osoitettiin vaikuttavan ER:n rakenteeseen. Tutkimuksen aikana otettujen mikroskooppikuvien analysointi oli tämän tutkimuksen kannalta oleellista ja tästä johtuen työssä käsitellään myös kuvankäsittelyohjelmiston kehittämistä. Tässä väitöskirjatutkimuksessa karakterisoitiin Reticulon-proteiineja (RTN) uusilla, kehittyneillä mikroskooppisilla tekniikoilla. Immuunielektronimikroskopialla ja elektronitomografialla osoitettiin RTN4A:n ja RTN4B:n paikallistuvan ER:n tubuluksiin ja laattojen reunoille. Kvantitatiivisella polymeraasiketjureaktiolla pystyttiin osoittamaan, että RTN4A on eniten ilmennetty muunnos hermosoluissa ja RTN4B vastaavasti pintakudossoluissa. Vähän tutkittu proteiini, FAM134C, tunnistettiin yhdeksi RTN4B:n kanssa vuorovaikutuksessa olevista proteiineista ja se paikallistettiin samankaltaisiin ER:n rakenteisiin kuin RTN4B (laattojen reunat ja tubulukset). FAM134C:n ja RTN4B:n ylituotto aikaansai tubulaaristen rakenteiden muodostamista. Lisäksi, ER:n rakenneproteiiniryhmän REEP (engl. receptor expression enhancing protein) proteiinien REEP3 ja REEP4 vaikutusta ER:n rakenteeseen tutkittiin solunjakautumisvaiheen aikana. Solunjakautumisvaiheessa REEP3 ja REEP4 paikallistettiin tubulaariseen, perifeeraaliseen ER:ään. Nämä proteiinit tarvittiin myös tubulaarisen ER:n irrottamiseksi kromatiinista normaalin solunjakautumisen aikaansaamiseksi. Tämä tutkimus syventää tietoja kolmen ER:ä muokkaavan proteiiniperheen proteiineista ja niiden paikallistumisesta ja vaikutuksista ER:n rakenteeseen niin kasvuvaiheessa kuin solunjakautumisen eri vaiheissa. Tulokset antavat myös lisätietoa eri domeenien rooleista näissä proteiineissa, esim. retikulonihomologia-domeenista (RHD), jonka löydettiin kaikista näistä proteiineista. RHD:n tiedetään olevan osittain kaksoiskalvorakenteen sisällä ja siten aiheuttavan kalvojen kaarevoitumista: tämä havaittiin myös ER:n tubuloitumisena ylituotettaessa RTN4B:tä tai FAM134C:tä. Tutkimuksen mukaan RHD:lla oli ratkaiseva rooli ER:n REEP 3/4 rakenneproteiinien toiminnassa solunjakautumisen aikana

Protection of nitrogenase levels in dark-incubated cultures of Anabaena sp. strain CA by various carbon sources, and restoration of nitrogenase activity by oxygen

Photoautotrophically grown, nitrogen-fixing cultures of Anabaena sp. strain CA lost nitrogenase activity completely after 4 h of incubation in the dark. The original level of nitrogenase activity was restored within 3 h of re-illumination and was apparently dependent on de novo protein synthesis. Several organic carbon sources protected nitrogenase activity. The heterocysts isolated from photoautotrophically grown cultures incubated in the dark (35 min) showed negligible nitrogenase activity. When these heterocysts were exposed to oxygen, glucose or fructose during isolation, normal activity was observed only with O2. Oxygen also enhanced the rate of initial H2 evolution from isolated heterocysts

Effects of some inhibitors and carbon sources on acetylene reduction and hydrogen production of isolated heterocysts of Anabaena sp. (strain CA)

Metabolically active heterocysts isolated from wild-type Anabaena sp. strain CA showed high rates of light dependent acetylene reduction and H2 evolution. Fructose and erythrose significantly stimulated nitrogenase activity but not H2 evolution. DCMU and cyanide were not effective. DBMIB significantly inhibited both nitrogenase and nitrogenase-catalysed H2 evolution. This inhibition was overcome by a catalytic amount of TMPD. These data suggest that in the isolated heterocysts all electrons, irrespective of source, must pass through the plastoquinone pool before reducing ferredoxin, which in turn can reduce dinitrogen to ammonia

Achieving Millennium Development Goals and India Vision 2020: Evidences from slums of Varanasi City

The objective of this paper is to examine the achievements of Millennium Development Goals (MDGs) and India Vision 2020 in the context of the slums of Varanasi city. India has been facing the problem of growing urban poverty due to rapid urbanisation. According to the Census of India 2011, about 65 million people live in urban slums, which are overcrowded, polluted, unhygienic, and deprived of basic services. This paper compares the goals and targets of UN MDGs and India Vision 2020 with the status of the slum dwellers of Varanasi city. The study is based on primary data, which includes survey of 150 households of five different slums of Varanasi city. The study reveals that more than 70% people are illiterate, less than 35% of population access government health facilities, only 36% household use tap water, etc. This comparison portrays a pathetic picture of slums and the inabilities to achieve both MDGs and national goals.Key words:MDGs, India Vision 2020, Slums, Poverty, Quality of life, Varanasi City, Indi

Effect of Solvent on of 4- Phenyl-7-Hydroxy Coumarin - P –Chloro Anilide

The absorption and fluorescence emission spectra of 4- phenyl-7-hydroxy Coumarin - p-chloroanilide have been reported in various polar and non polar solvents. The increase in solvent polarity shows the increase in absorption and emission wave length i.e ???* transition. This indicates the value of excited state dipole moments can’t be calculated using the solvatochromic data. The results are explained in terms of bathochromomic-shift, and possibility of hydrogen bonding

Sentiment Analysis on IMDb Movie Reviews Using Hybrid Feature Extraction Method

Social Networking sites have become popular and common places for sharing wide range of emotions through short texts. These emotions include happiness, sadness, anxiety, fear, etc. Analyzing short texts helps in identifying the sentiment expressed by the crowd. Sentiment Analysis on IMDb movie reviews identifies the overall sentiment or opinion expressed by a reviewer towards a movie. Many researchers are working on pruning the sentiment analysis model that clearly identifies and distinguishes between a positive review and a negative review. In the proposed work, we show that the use of Hybrid features obtained by concatenating Machine Learning features (TF, TF-IDF) with Lexicon features (Positive-Negative word count, Connotation) gives better results both in terms of accuracy and complexity when tested against classifiers like SVM, Naïve Bayes, KNN and Maximum Entropy. The proposed model clearly differentiates between a positive review and negative review. Since understanding the context of the reviews plays an important role in classification, using hybrid features helps in capturing the context of the movie reviews and hence increases the accuracy of classification

Assessing Knowledge, Attitude, and Practices towards Ivermectin Pre-exposure Prophylaxis for COVID-19 among Health Care Workers

Background: Health care workers (HCWs) are more likely to be at high risk of SARS-CoV-2 infection due to their direct and/or indirect participation in treatment facilities. Here, we aimed to evaluate the knowledge, attitude, and practices of ivermectin pre-exposure prophylaxis (PEP) in HCWs. Materials and Methods: In this observational study HCWs who were directly/indirectly involved in the medication of COVID-19 patients were selected. The study questionnaire included demographic data; knowledge, attitudes, practices, and associated adverse drug reactions (ADR) after using ivermectin as pre-exposure prophylaxis in COVID-19. Results: The mean age of the selected 306 participants was 34.41 {±standard deviation 4.08}. 66.66% of the participants were men. 15.69% of individuals had co-morbidities. HCWs were about COVID-19, and 94.12% of people know the role of ivermectin as PEP.  Additionally, 180 participants (58.82%) realized that ivermectin can cause adverse drug reactions (ADRs). 70.59% of the study-participants admitted that ivermectin has a protective effect on COVID-19, while 62.75% of the participants believed that the PEP benefits of using ivermectin outweigh the risks. 57.37% (n = 210) of HCW used ivermectin and completed the recommended medication process. Eighteen participants (8.57%) experienced adverse reactions and reported to the ADR monitoring center. Conclusion: 70.59% of the studied participants agreed that ivermectin has a protective effect on COVID-19, and 57.37% of the participants have taken ivermectin as PEP. However, 8.57% of the individuals reported ADR, but none of the participants were severe enough to discontinue the drug

Vacuum assisted closure-utilization as home based therapy in the management of complex diabetic extremity wounds

Objective: Vacuum assisted closure is a reported technique to manage complex wounds. We have utilized this technique by using simple locally available material in the management of our patients on outpatient basis. The objective of this study is to present our experience. Methods: This study was conducted from June 2011 to June 2013 at Dow University Hospital and Aga Khan University Hospital, Karachi. There were 38 patients managed with vacuum assisted closure. Mean age was 56±7.8 years. Twenty three patients presented with necrotizing fasciitis and 15 patients with gangrene. Lower limbs were involved in majority of the patients. Debridement or amputations were done. Vacuum dressing was changed twice weekly in outpatient department. Wounds were closed secondarily if possible or covered with split thickness skin graft in another admission. Results: All the wounds were successfully granulated at the end of vacuum therapy. Mean hospital stay was 7.5 days. Vacuum dressing was applied for a mean of 20 days. There was reduction in the size of the wound. Thirteen patients underwent secondary closure of the wound under local anesthesia, 18 patients required coverage with split thickness skin graft and 7 patients healed with secondary intention. Conclusion: Vacuum assisted closure appeared to be an effective method to manage complex diabetic wounds requiring sterile wound environment

Gamma Rays Bursts: A Viable Cosmological Probe?

In this work, our focus is on exploring the potential of current GRB measurements to provide reliable constraints on cosmological model parameters at high redshift. This work is divided into two parts. First, we calibrate the Amati relation in a model-independent way by using Hubble parameter measurements obtained from the differential ages of the galaxies. We further check if the Amati relation parameters evolve with the GRBs' redshift or not, using the data of Old Astrophysical Objects. The results indicate that GRBs do seem to evolve with redshift. In the second part, we test different cosmological models with the calibrated GRB data obtained by using constant and dynamical Amati relation. Our results indicate that the present quality of GRB data is not good enough to put tight constraints on the cosmological parameters. Hence we perform a joint analysis with the combined data of GRBs and Type Ia Supernovae (SNe) and find that this can considerably enhance cosmological constraints in contrast to solely relying on GRBs.Comment: 35 Pages, 14 Figures, 13 Tables. Accepted for publication in JCA