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Transcript: Pulsed wave Doppler measurement of blood flow velocity. 2 MHz transducers direct ultrasound wave at the basal blood vessels. Image reveals speed and direction of flow. Use bone windows to insonate: Transtemporal (MCA, ACA, PCA, ACoA, PCoA) Suboccipital (vertebrals, basilar) Transorbital (Ophthalmic, Carotid siphon) Submandibular (ICA) Locating arteries: Depth of the volume. Angle of the transducer. Direction of the blood flow relative to the transducer. Spatial relationship of one doppler signal to another. Traceability of an artery. Transducer remains on window, constant angle for each vessel Constant interface of signal intensity Arterial identification: Adjust angle to obtain proper artery Adjust depth to insonate from distal to proximal (depth measured based on head size) TCD Spectral Waveforms determine velocity at the artery: 1.Mean velocity 2. Pulsatility Index: Peak systolic-end diastolic ----------------------------------------- mean velocity After calculation: Compare velocity to norms to determine stenosis/spasm. Compare spectral waveforms from anterior and posterior circulations, left and right. Note any disease of extracranial carotid and vertebral arteries which may impact values. Note pulsatility index. Consider physiologic factors. Artery Mean velocity (cm/sec) MCA 62 ± 12 ACA 50 ± 11 T-ICA 39 ± 9 PCA 30 ± 10 Ophth 21 ± 5 Siphon 47 ±14 Vertebral 38 ± 10 Basilar 41 ± 10 ICA-sub 37 ± 9 Physiologic Factors AGE : lower velocities with increasing age H&H : hematocrit less than 30 increases velocity Hypoglycemia: increases bloodflow to increase delivery of glucose to brain, only a factor with glucose below 40 Hyperventilation: decreases velocity Hypoventilation: increases velocity Gender: Females have 3-5% increase in MCA velocity Fever: velocity increases by 10% for every degree of increase in temperature Heart rate: sweep speed has to be accomodated for bradycardia or tachycardia.) Normal TCD Criteria: 1. Normal Flow direction 2. Velocity/pulsatility symmetry (Left:Right difference is less than 30%) 3. Velocity hierarchy is respected. 4. Pulsatility Index is 0.6-1.1 in normotensive patients. (can be>1.2 in patients with hypertension) 5. Note any microemboli, shunt (if applicable) 6. Vasoreactivity response Microemboli detection: assessment of stroke risk or antiplatelet therapy. Emboli detection device 1. Doppler microembolic signals are transient; generally lasting less than 300 msec. Embolic signal duration depends on the time of passage through the Doppler sample volume, random throughout cardiac cycle.. 2. Microembolic signal amplitude is usually at least three decibels higher than that of the background blood flow signal. 3. Microembolic signals are unidirectional within the Doppler velocity spectrum 4. A microembolic signal audibly sounds like a snap, chirp, or moan BUBBLE STUDY Detects a right to left shunt via PFO or a pulmonary AVM. Protocol 1. Patient in the supine position. 2. Place an IV line in the antecubital vein using a 21-gauge needle on a butterfly with a plastic tube extension to a 3-way stopcock. 3. Monitor the bilateral middle cerebral arteries (MCA) using a headframe and two 2 MHz TCD probes fixated on the bilateral transtemporal window. ( MCA flow is toward the probe and monitored at a depth of 50 to 55 mm. 4. Record a five minute baseline. 5. Attach an empty 10mL syringe to the 3-way stopcock. 6. Fill a second 1OmL syringe with 9 mL of bacteriostatic saline and 1 mL of air. Place the second syringe on the 3-way stopcock. 7. Agitate the saline between the two syringes and when it becomes a frothy white color inject the saline as a bolus. 8. As soon as the bolus is finished, watch and listen for microembolic signals to pass through the MCA waveforms. 9. Wait another five minutes and repeat the protocol, this time having the patient perform a Valsalva maneuver just at the bolus is finished. The patient should hold the maneuver for ten seconds. Bubble Study Interpretation As the bolus is finished, listen for microembolic signals. Any amount of bubbles seen and heard within either MCA is suggestive of a right to left shunt. . The grading scale is as follows: Grade I 1 to 10 microembolic signals Grade II 11 to 30 microembolic signals Grade III 31 to 100 microembolic signals Grade IV 101 to 300 microembolic signals Grade V greater than 300 microembolic signals, many uncountable Vasoreactivity study: Assessment of Collateralization Vasomotor reserve is the ability of the cerebral vessels to maintain adequate blood perfusion in the brain regardless of changes in pressure gradients, body position, or blood pressure. This perfusion is maintained through vessel dilation and constriction. If this perfusion mechanism is abnormal, the patient has an elevated risk for stroke. This can be tested by breath holding induced vasodilation. Alternatively it can be tested by CO2 challenge. The breath-holding maneuver (TCD breath-holding test): 1. Normal breathing of room air for approximately 4

Tcd project

Transcript: Eoghan Táílliúir Nomura's Jellyfish Habitat Nomura's Jellyfish ca be found mainly in Asian waters between Japan, China and Korea. During the last decade, the Nomura's jellyfish population has risen sharply and they have become a serious plague in Japan. It is not known exactly what is causing the population of this animal to be out of control , but it is believed that the increase in the water temperature and the excessive fishing of their predators contribute to this phenomenon. Population increase Ventilation pattern and Mechanism Oxygen dissolved in the water will diffuse directly into the jellyfish's body. As the jellyfish swim, the oxygen is absorbed into their first layer of skin, called the epidermis. Then, it is stored in the mesoglea before being defused ito the jellyfish's cells. CO2 is released from the cells into the water. This is a uni-directional system Jellyfish anatomy Respiratory surface Jellyfish have no specific respiratory organs. Jellyfish have such thin tissue that they can get most of the oxygen they need from direct diffusion without any specific type of respiration. Oxygen is stored in the mesoglea of the jellyfish and is diffused directly into the jellyfish's cells and is taken in to the metabolic area of the jellyfish. The mesoglea also acts as an oxygen store for when they encounter an environement with low levels of oxygen. Jellyfish require very little oxygen and this allows the to thrive in low-oxygen waters Delivery system Image Nomara's Jellyfish (Nemopilema Nomurai) are the largest and heaviest species of jellyfish in the world. These jellyfish begin their life as polyps the size of a pinhead, but grow very fast and in less than a year reach a diameter of 2 metres and reach 200 kilograms. Introduction Image Diet They mainly feed on plankton and crustaceans such as crabs but due to their huge size they can consume bigger fish such as tuna,salmon and catfish.

TCD presentation

Transcript: Fig. 1: Structure of hcGAS (hcGAS) R376I, TM1788 a dgcZ (pdb: 5VDP, 4URS, 4H54) (red - bound cyclic dinucleotides, blue - Mg, green - Zn, pink - GTP) Aims of thesis Enzyme production Cyclic dinucleotides production prof. Ing. Martin Fusek, Ph.D. Mgr. Miroslav Hájek, Ph.D. Mgr. Gabriel Birkuš, Ph.D. Research group HBV-Cure, IOCB Fig. 5: SDS-PAGE electroforeogram: Samples collected in isolation by affinity chromatography hcGAS R376I, TM1788 and dgcZ. M - marker, 1 - before incubation with Ni-NTA, FT - flow trough, W1 - wash 1, W2 - wash 2, E - elution 12 5 3 4 3 dinucleotide cyclases Oligonucleotide directed mutagenesis 10 hcGAS R376I Lucie Černá dgcZ Commercial vector high substrate specificity human cGAS R376I 11 Izolation of genomic E. coli DNA 4. Test - DSF a EC dgcZ 15 4 TM1788 Yield CDN low affinity to STING protein Fig. 2: Dinucleotide cyclase vector 8 Fig. 3: Western blot: hcGAS (60 kDa), TM1788 (15,5 kDa), dgcZ (34,5 kDa). Antibodies: 6x-His Tag Monoclonal Antibody (1:1000), Mouse IgG HRP-conjugated Antibody (1:1000). 2. Analogues of dinucleotides 5 Conclusion 14 10 1 Yield G[3'′–5'′]pA[3′'–5'′]p 15 Future plans dgcZ (E. coli) 3. Large scale reaction 2 Fig. 7: Chromatogram of products: reaction 20µM dgcZ, GTP, 16hr incubation (UV detector LC-MS). dgcZ Yield: 3 hcGAS R376I cloning, expression and purification of dinucleotide cyclases characterization of substrate specificity preparation of cyclic dinucleotides (CDNs) characterization of biological activity CDNs in biochemical and cell based assays TM1788 16 hcGAS R376I Yield CDN 3. Izolation - IMAC TM1788 a dgcZ 3'3'-c-di-GMP 100 % 50 2 2. Bacterial production G[3'′–5'′]pG[3′'–5'′]p TM1788 1 9 6 hcGAS R376I 16 TM1788 Fig. 5: FPLC chromatogram hcGAS, TM1788 and dgcZ. Sepax SRT-10 SEC-300, Bio-Rad NGC™ Chromatography system, flow rate 5 ml/min, wave length 280 nm. G[3'′–5'′]pG[3′'–5'′]p 7 4. Purification - FPLC Fig. 6: Chromatogram of products: reaction 20µM hcGAS R376I, GTP, ATP, 16hr incubation (UV detector LC-MS). 13 Yield 2 3 cyclic dinucleotides hcGAS R376I study of PrRP-ghrelin analogues in vitro competetive binding experiments signalization Acknowledgment Fig. 9: Structures of GTP, 7-deaza-GTP a 6-thio-GTP used for production of cyclic dinucleotides 1. Enzymatic reaction 1. Vector TM1788 (T. maritima) Fig. 8: List of used NTPs and their conversion to CDNs by enzymes TM1788 a dgcZ dgcZ Identification of Dinucleotide Cyclases Suitable for Enzymatic Preparation of Analogues of Cyclic Dinucleotides AMP ADP ATP GDP GTP 3'3'-cGAMP 6 % 20 % 33 % 14 % 27 % <0,5 % Fig. 10: Diferential scanning fluorimetry (horizontal) and EC (vertical) of standards (blue) and prepared (black) CDNs with WT STING. 50 volume (ml) volume (ml) volume (ml) hcGAS R376I TM1788 dgcZ 0,68 mg/l 13,9 mg/l 2,9 mg/l n =1 H

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